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Validation of on-chip capture and imaging. (A) DB cells dual-labeled with Hoechst and anti-CD45-APC, and captured and imaged on-chip. Capture sites are butterfly-shaped, staggered, and customized for lymphocyte size-based capture. Scale bar: 25 µm. (B) Capture efficiency of DB and Daudi cells is greater than 90% when 10, 100, or 1,000 lymphoma cells (nominal numbers) were introduced to the chip. The counted cell number is displayed as mean ± s.d. from quadruplicate measurements. The nominal cell number is displaced as mean ± Poisson error. (C) Optimization of flow rate based on capture efficiency of DB cells. (D) Proposed workflow for clinical diagnosis using image analysis.
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thnov05p0796g002
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
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[
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[{'image_id': 'thnov05p0796g001', 'image_file_name': 'thnov05p0796g001.jpg', 'image_path': '../data/media_files/PMC4440438/thnov05p0796g001.jpg', 'caption': 'Process design. (A) Summary of overall scheme: paucicellular samples are harvested and captured on the chip without preprocessing. Following on-chip fixation, permeabilization, and immunostaining, the chip is imaged and cytometry is carried out with an in-house image process algorithm. (B) Photograph of lymphocyte capture chip attached to a microslide, showing inlet, debris filter, and capture area, which contains four arrays of 20 × 300 single-cell capture sites.', 'hash': '19485956525a446895c549896871819079b46b40f44b2b52f10c882a14948fbd'}, {'image_id': 'thnov05p0796g006', 'image_file_name': 'thnov05p0796g006.jpg', 'image_path': '../data/media_files/PMC4440438/thnov05p0796g006.jpg', 'caption': 'Theranostic on-chip imaging. BTK-positive Rec-1 cells or BTK-negative Jurkat T-cell leukemia cells using fluorescent BTK inhibitor (Ibrutinib-BFL), anti-CD20-APC, and Hoechst stain. Note the high drug uptake and binding in Rec-1 cells. Scale bar: 5 µm.', 'hash': 'e2db4151caf5ae5850c06231847e9b688201a8a29dea9a7608cee538422aba5b'}, {'image_id': 'thnov05p0796g003', 'image_file_name': 'thnov05p0796g003.jpg', 'image_path': '../data/media_files/PMC4440438/thnov05p0796g003.jpg', 'caption': "Antibody validation and cell line profiling by flow cytometry. Relative expression levels of B-cell antigens relevant to diagnosis and prognosis (rows) on several lymphoma cell lines (columns). Daudi is a Burkitt's lymphoma line; DB, SuDHL4, DOHH2, and Toledo are GCB-type DLBCL lines, RC-K8 is an ABC-type DLBCL line, Rec-1 is a mantle cell lymphoma line, and Hut-78 is a T-cell lymphoma control.", 'hash': '4b5a729fbce412b2a72003af7202244f463df3b3398b07adf2039c47783e6515'}, {'image_id': 'thnov05p0796g004', 'image_file_name': 'thnov05p0796g004.jpg', 'image_path': '../data/media_files/PMC4440438/thnov05p0796g004.jpg', 'caption': 'On-Chip Imaging. A 1:1 mixture of DB and Daudi cells were captured and stained on-chip using a cocktail of antibodies: anti-CD19-PE, anti-CD20-PE, anti-Kappa-Brilliant Violet 421, anti-Lambda-Alexa Fluor 647, and anti-Ki-67-Alexa Fluor 488. (A) Low-magnification image shows overall capture site layout and cell heterogeneity. Scale bar: 75 µm. (B) High-resolution images of differential expression of individual markers on the two cell lines. Scale bar: 5 µm.', 'hash': 'a9a8dcedd1aedc3d974f29b79f1005ab75d04a63818944ac744175a7124ad4b0'}, {'image_id': 'thnov05p0796g005', 'image_file_name': 'thnov05p0796g005.jpg', 'image_path': '../data/media_files/PMC4440438/thnov05p0796g005.jpg', 'caption': 'Cell profiling for kappa/lambda monoclonality by image analysis. (A) Sample image analysis using an in-house image processing algorithm. Thresholding in the PE channel (CD19, CD20) is used to select B cells, and size-based filtering removes non-cell debris (white arrow). Target channels are analyzed within masks created from PE channel gating. (B) Scatterplots of mean pixel intensities from target imaging channels show clear separation of populations based on kappa and lambda light chain expression; top, DB cells; middle, Daudi cells; bottom, 1:1 mixture of DB and Daudi cells.', 'hash': '65a731b8b64b1be2e98bd03bcd74aaa12559cb91bdce408f87c01762e98029fa'}, {'image_id': 'thnov05p0796g002', 'image_file_name': 'thnov05p0796g002.jpg', 'image_path': '../data/media_files/PMC4440438/thnov05p0796g002.jpg', 'caption': 'Validation of on-chip capture and imaging. (A) DB cells dual-labeled with Hoechst and anti-CD45-APC, and captured and imaged on-chip. Capture sites are butterfly-shaped, staggered, and customized for lymphocyte size-based capture. Scale bar: 25 µm. (B) Capture efficiency of DB and Daudi cells is greater than 90% when 10, 100, or 1,000 lymphoma cells (nominal numbers) were introduced to the chip. The counted cell number is displayed as mean ± s.d. from quadruplicate measurements. The nominal cell number is displaced as mean ± Poisson error. (C) Optimization of flow rate based on capture efficiency of DB cells. (D) Proposed workflow for clinical diagnosis using image analysis.', 'hash': '34ded2fba9f20546241df7ff0f7b108d6345cb110ad58b5375aa0603e748f6e1'}]
|
{'thnov05p0796g001': ['Fig. <xref ref-type="fig" rid="thnov05p0796g001">1</xref>A summarizes the procedure for lymphocyte detection and profiling. First, samples are harvested, typically in the range of 1-3 mL. The entire sample is then loaded onto the chip; individual cells are captured in sub-nanoliter traps and on-chip stained for fluorescent imaging. Acquired images are then analyzed with an automatic computational algorithm to generate cell characterization data. The 2 × 4 cmA summarizes the procedure for lymphocyte detection and profiling. First, samples are harvested, typically in the range of 1-3 mL. The entire sample is then loaded onto the chip; individual cells are captured in sub-nanoliter traps and on-chip stained for fluorescent imaging. Acquired images are then analyzed with an automatic computational algorithm to generate cell characterization data. The 2 × 4 cm2 chip contains 24,000 staggered, butterfly-shaped traps arranged in four bands of 20 × 300 (Fig. <xref ref-type="fig" rid="thnov05p0796g001">1</xref>B; additional details in B; additional details in Supplementary Material). The capture site architecture was optimized to trap a single lymphocyte, while a 4-µm gap between the butterfly “wings” was incorporated to allow smaller cells, such as erythrocytes, to pass through without being captured (Supplementary Material: Fig. S1). Each trap was self-limiting; once a cell blocks the gap, the structure presents high fluidic resistance, preventing further cell-trapping. To remove cellular debris and aggregates, we also incorporated a column-filter in the sample inlet (Supplementary Material: Fig. S1). The chips were fabricated via standard soft lithography and the estimated cost per chip is <$1. Containing a large number of capturing sites, the chip enables high-throughput analysis. For instance, with typical flow rates of 2-5 mL/hr, target cells could be captured and stained in <1 hour, important for processing clinical samples.'], 'thnov05p0796g002': ['We first characterized the device performance for cell capture. DB GCB-type DLBCL and the Daudi Burkitt lymphoma cell lines were stained for CD45 (an extracellular pan-lymphocyte marker) and nucleus, and samples were prepared with the nominal cell counts of 10, 100, or 1000 of the DB or Daudi cells. When these samples were processed by the chip (Fig. <xref ref-type="fig" rid="thnov05p0796g002">2</xref>A and A and <xref ref-type="fig" rid="thnov05p0796g002">2</xref>B), the observed capture efficiency was >90%; this contrasts with the 17-30% cell loss that occurs at each centrifugation step in traditional sample processing B), the observed capture efficiency was >90%; this contrasts with the 17-30% cell loss that occurs at each centrifugation step in traditional sample processing 21,22. The optimal flow rate for maximal capture yield was between 2-5 mL/hr (Fig. <xref ref-type="fig" rid="thnov05p0796g002">2</xref>C). At low flow rates (≤ 1 mL/hr), we observed that cells could switch to the flow stream that bypasses the capture site. When the flow speed increased, however, cells were quickly lodged into the capture site without diversion, which resulted in higher capture yield. We note that the capture yield was statistically identical at the flow rate of 2 and 5 mL/hr. For the subsequent cell experiment, we used the flow rate of 2 mL/hr to minimize potential cell lysis.C). At low flow rates (≤ 1 mL/hr), we observed that cells could switch to the flow stream that bypasses the capture site. When the flow speed increased, however, cells were quickly lodged into the capture site without diversion, which resulted in higher capture yield. We note that the capture yield was statistically identical at the flow rate of 2 and 5 mL/hr. For the subsequent cell experiment, we used the flow rate of 2 mL/hr to minimize potential cell lysis.', 'Captured cells could be analyzed on-chip through multi-color immuno-microscopy. As outlined in Fig. <xref ref-type="fig" rid="thnov05p0796g002">2</xref>D, three classifications can be performed: 1) the use of CD19 and/or CD20 to determine B cells; 2) the use of kappa or lambda light chains to identify clonal populations; and 3) additional phenotypic markers for subtyping and prognostic tasks. We validated these markers and their respective antibodies by profiling a panel of cell lines via flow cytometry (Figure D, three classifications can be performed: 1) the use of CD19 and/or CD20 to determine B cells; 2) the use of kappa or lambda light chains to identify clonal populations; and 3) additional phenotypic markers for subtyping and prognostic tasks. We validated these markers and their respective antibodies by profiling a panel of cell lines via flow cytometry (Figure <xref ref-type="fig" rid="thnov05p0796g003">3</xref>). Besides the B-cell lymphoma lines Daudi and DB, we also profiled SuDHL4, DOHH2, and Toledo GCB-type DLBCL lines, the RC-K8 ABC-type DLBCL line, and the Rec-1 mantle cell lymphoma line. Hut-78, a T-cell line, was used as a control. The profiling results showed the importance of including both CD19 and CD20 to identify B cells; not all B-cell lines were found to express both markers. This finding is also supported by other reports that showed decreased CD20 in lymphomas either due to the cancer cell-of-origin or anti-CD20 immunotherapy ). Besides the B-cell lymphoma lines Daudi and DB, we also profiled SuDHL4, DOHH2, and Toledo GCB-type DLBCL lines, the RC-K8 ABC-type DLBCL line, and the Rec-1 mantle cell lymphoma line. Hut-78, a T-cell line, was used as a control. The profiling results showed the importance of including both CD19 and CD20 to identify B cells; not all B-cell lines were found to express both markers. This finding is also supported by other reports that showed decreased CD20 in lymphomas either due to the cancer cell-of-origin or anti-CD20 immunotherapy 23-25. We also found the restricted expression of kappa or lambda light chain surface immunoglobulins, which are markers of clonality, across the cell lines.', 'As a proof-of-concept of lymphocyte analysis from clinical samples, we developed an image-processing algorithm for clonality assessment. Following the workflow described in Fig. <xref ref-type="fig" rid="thnov05p0796g002">2</xref>, we first made a mask around cells expressing CD19 and/or CD20, and then quantified the mean fluorescence intensity from our target channels in each individual cell (Fig. , we first made a mask around cells expressing CD19 and/or CD20, and then quantified the mean fluorescence intensity from our target channels in each individual cell (Fig. <xref ref-type="fig" rid="thnov05p0796g005">5</xref>A). A size filter was also included to exclude non-cell debris from analysis (Fig. A). A size filter was also included to exclude non-cell debris from analysis (Fig. <xref ref-type="fig" rid="thnov05p0796g005">5</xref>A, white arrow). We then analyzed images of a single cell-type population (either Daudi or DB; Fig. A, white arrow). We then analyzed images of a single cell-type population (either Daudi or DB; Fig. <xref ref-type="fig" rid="thnov05p0796g005">5</xref>B). From ~600 individual cell images, we determined the threshold (Th) values of mean fluorescence intensities to distinguish each cell type (Daudi, ThB). From ~600 individual cell images, we determined the threshold (Th) values of mean fluorescence intensities to distinguish each cell type (Daudi, Thkappa = 50; DB, Thlambda = 20). When these criteria were applied to another validation samples (>2,000 cells), we obtained high sensitivities (Daudi, 96%; DB, 99%) and specificities (Daudi, 98%; DB, 98%).'], 'thnov05p0796g004': ['We chose to use Daudi and DB cells as a model system for on-chip analysis, since they respectively highly express kappa and lambda light chain. To demonstrate both extracellular and intracellular antigen analysis, we performed on-chip staining of CD19/CD20, kappa/lambda, and Ki-67. We prepared samples by spiking known numbers of DB and Daudi lymphoma cells into artificial CSF (see Methods for details). The cells were then fixed and stained on the chip, and imaged in four channels (Fig. <xref ref-type="fig" rid="thnov05p0796g004">4</xref>; see ; see Supplementary Material: Table S2 for antibody clones and fluorochromes). Fig. <xref ref-type="fig" rid="thnov05p0796g004">4</xref>A shows the overlay of the four imaging channels after a 1:1 mixture of DB and Daudi cells were captured and stained on-chip. Fig. A shows the overlay of the four imaging channels after a 1:1 mixture of DB and Daudi cells were captured and stained on-chip. Fig. <xref ref-type="fig" rid="thnov05p0796g004">4</xref>B demonstrates high-resolution imaging of individual cells and markers. Although the cell populations appear to be heterogeneous, their restricted kappa/lambda expression can be seen at higher magnification.B demonstrates high-resolution imaging of individual cells and markers. Although the cell populations appear to be heterogeneous, their restricted kappa/lambda expression can be seen at higher magnification.'], 'thnov05p0796g006': ['We further performed drug sensitivity testing that would be clinically useful to guide intrathecal and/or systemic chemo- and targeted therapies. We used a companion imaging drugs that has recently been reported, Ibrutinib-BFL, an inhibitor of Bruton\'s Tyrosine Kinase (BTK) 28; other imaging drugs include fluorescent rituximab or caged methotrexate. Ibrutinib is approved for several B-cell malignancies, including mantle cell lymphoma, and the Rec-1 cell line has been shown to be sensitive to the drug 29,30. Imaging the Rec-1 cells with Ibrutinib-BFL on the chip shows not only the binding of Ibrutinib, but also their cell-to-cell heterogeneity due to differences in BTK inhibitor sensitivity and BTK protein turnover (Fig. <xref ref-type="fig" rid="thnov05p0796g006">6</xref>).).']}
|
On Chip Analysis of CNS Lymphoma in Cerebrospinal Fluid
|
[
"lymphoma",
"microfluidics",
"point-of-care",
"cerebrospinal fluid",
"drug testing"
] |
Theranostics
|
1429254000
|
None
| null |
other
|
PMC4440438
| null | null |
[
""
] |
Theranostics. 2015 Apr 17; 5(8):796-804
|
NO-CC CODE
|
|
Decreased inter-sister chromatid distances at DSB sites. (A) DT40 cells were fixed and stained with antibodies to γ-H2AX (red) and counterstained with DAPI following addition of TA. The distance between GFP spots (green), which mark the TetO array, was measured in metaphase mitotic chromosomes. The presence of a γ-H2AX signal at the TetO array indicated a DSB site. Scale bar, 10 μm. (B) Analysis of distances between GFP spots. Pooled data from three independent repeats of the experiment are shown as individual data points and as mean ± SEM. At each timepoint, inter-sister distances were significantly reduced in cells with a DSB (P < 0.0001, unpaired Student's t-test).
|
gkp684f5
|
2
|
2897185f59d9034f1b9e36e8257782f8bec34e1984f3bab44fd0f007463273bb
|
gkp684f5.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
480,
644
] |
[{'image_id': 'gkp684f4', 'image_file_name': 'gkp684f4.jpg', 'image_path': '../data/media_files/PMC2764452/gkp684f4.jpg', 'caption': 'Abnormal recruitment of Rad51 to DSBs in H2AX−/− and ATM−/− cells. (A) Micrograph showing localization of γ-H2AX (red), Rad51 (blue) and TetR-GFP (green) in DT40 cells, before and at the indicated times after addition of TA. Scale bar, 10 µm. (B) Graph comparing the kinetics of γ-H2AX and Rad51 recruitment to the array in wild type cells that stably express RFP-I-SceI-GR over time following TA addition. At least 20 cells per timepoint were scored for localization of γ-H2AX, Rad51 (red curves, left axis) or both (the percentage of γ-H2AX/array positive cells which also have Rad51; black curve, right axis) at the array. The experiment was repeated at least three times and the error bars show the SEM. (C) Micrograph showing localization of RFP-I-SceI-GR (blue), Rad51 (red) and TetR-GFP (green) in wild-type and Atm−/− DT40 cells, before and at the indicated times after addition of TA. WT cells were stably transfected with an RFP-I-SceI-GR expression vector and treated with TA for 4 h. Atm−/− cells were transiently transfected and 16 h post-transfection, treated with TA for 2 h. Scale bar, 10 μm. (D) Graph showing the kinetics of Rad51 localization at the array in RFP-I-SceI-GR-positive Atm−/− and H2ax−/− DT40 cells after transient transfection with RFP-I-SceI-GR. At least 20 cells that expressed RFP-I-SceI-GR, as determined by microscopy, were analysed per timepoint for each cell line. The experiment was repeated at least three times for each cell line and the error bars show the SEM. (E) Immunoblot showing the Rad51 levels in wild-type, Atm−/− and H2ax−/− cells. α-tubulin was used as a loading control.', 'hash': '2a8e144ad09758d5709c58da6802321206f2710a3d42e07303970ba660b655f9'}, {'image_id': 'gkp684f3', 'image_file_name': 'gkp684f3.jpg', 'image_path': '../data/media_files/PMC2764452/gkp684f3.jpg', 'caption': 'Abnormal recruitment of 53Bp1 to DSBs in H2AX−/− cells. (A) Micrograph showing localization of RFP-I-SceI-GR (blue), 53Bp1 (red) and TetR-GFP (green) in DT40 cells after addition of TA. WT cells were stably transfected with an RFP-I-SceI-GR expression vector and treated with TA for 2 h. Atm−/− and H2ax−/− cells were transiently transfected with the RFP-I-SceI-GR expression construct and 16 h post-transfection, treated with TA for 4 and 6 h, respectively. Scale bar, 10 μm. (B) Graph showing the kinetics of 53Bp1 localization at the array in RFP-I-SceI-GR-expressing DT40 cells of the indicated genotypes. At least 20 cells that expressed RFP-I-SceI-GR, as determined by microscopy, were analysed per timepoint for each cell line. The experiment was repeated at least three times for each cell line and the error bars show the SEM.', 'hash': 'e54625d764e28ab3f8f1c0be66d81bddcac61c673d02fdd78ba2a274e4fea9f7'}, {'image_id': 'gkp684f2', 'image_file_name': 'gkp684f2.jpg', 'image_path': '../data/media_files/PMC2764452/gkp684f2.jpg', 'caption': 'γ-H2AX recruitment to the TetO array after I-SceI induction. (A) Micrograph showing localization of RFP-I-SceI-GR (blue), γ-H2AX (red) and TetR-GFP (green) in DT40 cells of the indicated genotype, before and 30 min after addition of TA. Scale bar, 10 μm. (B) Graph showing the kinetics of γ-H2AX localization at the array in wild type, Ku70−/− and Atm−/− DT40 cells. At least 20 cells that stably expressed RFP-I-SceI-GR, as determined by microscopy, were analysed per timepoint for each cell line. The experiment was repeated at least three times for each cell line and the error bars show the SEM. (C) Immunoblot showing (i) γ-H2AX levels, and (ii) Chk1 levels in wild-type I-SceI site-containing cells that stably express RFP-I-SceI-GR over time following TA addition. Wild-type DT40 cells treated with 20 Gy IR 1 h before harvesting were used as positive control. Actin was used as a loading control. (D) Flow cytometry analysis of I-SceI site-containing wild-type cells that stably express RFP-I-SceI-GR at the indicated times after induction of I-SceI.', 'hash': '0e85017ff3edbd7ba5d95ac718276bafe5984bd4e741565ecb1ea1c4a08ad48e'}, {'image_id': 'gkp684f5', 'image_file_name': 'gkp684f5.jpg', 'image_path': '../data/media_files/PMC2764452/gkp684f5.jpg', 'caption': "Decreased inter-sister chromatid distances at DSB sites. (A) DT40 cells were fixed and stained with antibodies to γ-H2AX (red) and counterstained with DAPI following addition of TA. The distance between GFP spots (green), which mark the TetO array, was measured in metaphase mitotic chromosomes. The presence of a γ-H2AX signal at the TetO array indicated a DSB site. Scale bar, 10 μm. (B) Analysis of distances between GFP spots. Pooled data from three independent repeats of the experiment are shown as individual data points and as mean ± SEM. At each timepoint, inter-sister distances were significantly reduced in cells with a DSB (P < 0.0001, unpaired Student's t-test).", 'hash': '2897185f59d9034f1b9e36e8257782f8bec34e1984f3bab44fd0f007463273bb'}, {'image_id': 'gkp684f6', 'image_file_name': 'gkp684f6.jpg', 'image_path': '../data/media_files/PMC2764452/gkp684f6.jpg', 'caption': "Involvement of ATM in inter-sister chromatid distances at DSB sites. (A) Southern blot showing the targeting of Atm in the same clone used for the analysis in Figure 5. (B) DT40 cells were fixed and stained with antibodies to γ-H2AX (red) and counterstained with DAPI following addition of TA. The distance between GFP spots (green), which mark the TetO array, was measured in metaphase mitotic chromosomes. The presence of a γ-H2AX signal at the TetO array indicated a DSB site. Scale bar, 10 μm. (C) Analysis of distances between GFP spots. Pooled data from three independent repeats of the experiment are shown as individual data points and as mean ± SEM. Comparison of the mean distances using an unpaired Student's t-test showed no difference between wild-type and Atm−/− sister chromatid separation in the absence of DNA damage (data not shown), a highly-significant reduction in inter-sister distances in wild-type chromatids with a DSB (P < 0.0001) and a notably less significant reduction in inter-sister distances in Atm−/− chromatids after DSB induction (P = 0.0017). The inter-sister chromatid separation after DSB induction differed between wild-type and Atm−/− cells with a moderate level of statistical significance (P = 0.0470).", 'hash': '961c104824d124485d8de2d5fc023e4d51bc94709bfe22cc89c919028a50c115'}, {'image_id': 'gkp684f1', 'image_file_name': 'gkp684f1.jpg', 'image_path': '../data/media_files/PMC2764452/gkp684f1.jpg', 'caption': 'Generation of an inducible DSB in chicken DT40 cells and human U2OS cells. (A) Schematic representation of TetO/TetR array next to I-SceI restriction site. DSBs are induced by the addition of triamcinolone acetonide (TA), which results in the nuclear localization of the I-SceI endonuclease. (B) Southern blot analysis of in vitro I-SceI-digested genomic DNAs from DT40 and U2OS cells with randomly-integrated I-SceI sites, hybridized to the TetO array as probe. I-SceI cleaves within the genomic PstI fragments, but adjacent to the SacI site used in cloning the array. Line diagrams indicate the randomly integrated TetO array (TetO x112) and I-SceI restriction site (ISc) in the genomic DNA of both DT40 cells and U2OS cells. Locations for the SacI (S) and PstI (P) sites outside the integrated construct have been derived from the Southern blot data. The probe is shown in red. Numbers show fragment sizes in kilo basepairs. (C) Micrograph showing localization of RFP-I-SceI-GR (blue), γ-H2AX (red) and TetR-GFP (green) in DT40 cells, before and 30 min after addition of TA. Scale bar, 10 μm. (D) Diagram of the ligation-mediated PCR method used to detect a break following induction of I-SceI in vivo. (E) Ligation-mediated PCR. Genomic DNA was extracted from cells at the indicated time points following treatment with TA. Positive controls were genomic (‘Gen’) and plasmid (‘Plas’) DNA cut with recombinant I-SceI in vitro. Negative controls were genomic DNA cut with I-SceI in vitro in the absence of either the adaptor (‘-Ad’) or T4 DNA ligase (‘-Lig’). (F) Micrograph showing localization of DNA (blue), γ-H2AX (red) and TetR-GFP (green) in a U2OS cell after transient transfection of an RFP-I-SceI-GR expression construct. Scale bar, 10 μm.', 'hash': '65b64a141a4cdd045fdbe0f7cc16bcc0d19167543f2af4827a14c7b9c35f5fba'}]
|
{'gkp684f1': ['We aimed to generate a system for the visualization and analysis of single, chromosomal DSBs in vertebrate cells using the yeast homing endonuclease I-SceI. As shown in <xref ref-type="fig" rid="gkp684f1">Figure 1</xref>A, we cloned the 18-bp recognition site for I-SceI beside a 112-repeat tetracycline operator array (TetO x112) (A, we cloned the 18-bp recognition site for I-SceI beside a 112-repeat tetracycline operator array (TetO x112) (21) and co-transfected this construct into chicken DT40 and human U2OS cells along with an expression vector encoding a tetracycline repressor-GFP (TetR-GFP) fusion protein. We then selected for clones with either one or two GFP spots per cell, indicating TetR-GFP binding to the randomly integrated TetO array (data not shown). Genomic DNA from these clones was digested with various enzymes alone, or in combination with recombinant I-SceI, then Southern blotted and hybridized with the TetO array as probe (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>B). We saw a decrease in the band sizes detected with the TetO array whenever recombinant I-SceI was added to a restriction digest (B). We saw a decrease in the band sizes detected with the TetO array whenever recombinant I-SceI was added to a restriction digest (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>B and data not shown), indicating that the I-Sce site was chromosomally integrated and accessible to restriction digestion. In a control experiment, we saw no decrease in the SacI band size after SacI-I-SceI double digestion, as the SacI site is located very close to the I-SceI site in the construct. A line diagram indicates the random integration of the TetO array into the genomic DNA, the probe used and the location of the SacI/PstI restriction sites external to the integrated construct (B and data not shown), indicating that the I-Sce site was chromosomally integrated and accessible to restriction digestion. In a control experiment, we saw no decrease in the SacI band size after SacI-I-SceI double digestion, as the SacI site is located very close to the I-SceI site in the construct. A line diagram indicates the random integration of the TetO array into the genomic DNA, the probe used and the location of the SacI/PstI restriction sites external to the integrated construct (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>B).\nB).\nFigure 1.Generation of an inducible DSB in chicken DT40 cells and human U2OS cells. (A) Schematic representation of TetO/TetR array next to I-SceI restriction site. DSBs are induced by the addition of triamcinolone acetonide (TA), which results in the nuclear localization of the I-SceI endonuclease. (B) Southern blot analysis of in vitro I-SceI-digested genomic DNAs from DT40 and U2OS cells with randomly-integrated I-SceI sites, hybridized to the TetO array as probe. I-SceI cleaves within the genomic PstI fragments, but adjacent to the SacI site used in cloning the array. Line diagrams indicate the randomly integrated TetO array (TetO x112) and I-SceI restriction site (ISc) in the genomic DNA of both DT40 cells and U2OS cells. Locations for the SacI (S) and PstI (P) sites outside the integrated construct have been derived from the Southern blot data. The probe is shown in red. Numbers show fragment sizes in kilo basepairs. (C) Micrograph showing localization of RFP-I-SceI-GR (blue), γ-H2AX (red) and TetR-GFP (green) in DT40 cells, before and 30 min after addition of TA. Scale bar, 10 μm. (D) Diagram of the ligation-mediated PCR method used to detect a break following induction of I-SceI in vivo. (E) Ligation-mediated PCR. Genomic DNA was extracted from cells at the indicated time points following treatment with TA. Positive controls were genomic (‘Gen’) and plasmid (‘Plas’) DNA cut with recombinant I-SceI in vitro. Negative controls were genomic DNA cut with I-SceI in vitro in the absence of either the adaptor (‘-Ad’) or T4 DNA ligase (‘-Lig’). (F) Micrograph showing localization of DNA (blue), γ-H2AX (red) and TetR-GFP (green) in a U2OS cell after transient transfection of an RFP-I-SceI-GR expression construct. Scale bar, 10 μm.', 'Before or 30 min after addition of the activating TA, DT40 cells were fixed and stained with an antibody to the DNA damage response marker, γ-H2AX, then visualized using microscopy. As shown in <xref ref-type="fig" rid="gkp684f1">Figure 1</xref>C, the RFP-I-SceI-GR moved to the nucleus after TA activation. We saw co-localization of γ-H2AX with the TetR-GFP-marked array, indicating the formation of DSBs. To confirm the generation of these breaks, we used ligation-mediated PCR. Genomic DNA was extracted from DT40 cells with an integrated TetO array and expressing both TetR-GFP and RFP-I-SceI-GR. Cells had either been untreated or treated for various times with TA. A double-stranded oligonucleotide with an overhang specific for the I-SceI cut site was ligated to the genomic DNA and primers specific for the ligated oligonucleotide and the TetO array were used for PCR amplification (C, the RFP-I-SceI-GR moved to the nucleus after TA activation. We saw co-localization of γ-H2AX with the TetR-GFP-marked array, indicating the formation of DSBs. To confirm the generation of these breaks, we used ligation-mediated PCR. Genomic DNA was extracted from DT40 cells with an integrated TetO array and expressing both TetR-GFP and RFP-I-SceI-GR. Cells had either been untreated or treated for various times with TA. A double-stranded oligonucleotide with an overhang specific for the I-SceI cut site was ligated to the genomic DNA and primers specific for the ligated oligonucleotide and the TetO array were used for PCR amplification (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>D). As shown in D). As shown in <xref ref-type="fig" rid="gkp684f1">Figure 1</xref>E, the diagnostic 360 bp band was amplified from genomic DNA after I-SceI induction E, the diagnostic 360 bp band was amplified from genomic DNA after I-SceI induction in vivo or after in vitro I-SceI digestion. A positive control for the ligation-mediated PCR was the amplification of in vitro I-SceI-digested plasmid DNA. Notably, no amplification was seen in the absence of TA or in the absence of either the double stranded adaptor or T4 DNA ligase, confirming the specificity of the reaction and the reliability of the TA induction of the I-SceI.', 'This DSB induction system was also successfully introduced into U2OS cells. In a manner similar to that described for the DT40 cells we determined that the I-SceI sites was chromosomally integrated and accessible to restriction digestion by digestion of genomic DNA and Southern blotting (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>B). We confirmed the chromosomal integration of the TetO array and binding of the TetR-GFP protein by microscopy (B). We confirmed the chromosomal integration of the TetO array and binding of the TetR-GFP protein by microscopy (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>F). This cell line was then transiently transfected with the RFP-I-SceI-GR construct and treated with TA. Although Southern analysis did not demonstrate cleavage at the I-SceI site F). This cell line was then transiently transfected with the RFP-I-SceI-GR construct and treated with TA. Although Southern analysis did not demonstrate cleavage at the I-SceI site in vivo (data not shown), microscopy showed the colocalization of γ-H2AX with the TetR-GFP-marked array (<xref ref-type="fig" rid="gkp684f1">Figure 1</xref>F and F and Supplementary Figure 2), confirming DSB induction.'], 'gkp684f2': ['We used microscopy to monitor the timing and frequency of colocalization of γ-H2AX and the TetO array as an indicator of DSB induction after I-SceI activation in DT40 cells (C and 2A). We observed a time-dependent increase in the percentage of RFP-I-SceI-GR-positive cells that showed a co-localization between the γH2AX and the TetO array following the addition of the drug TA (<xref ref-type="fig" rid="gkp684f2">Figure 2</xref>B), with around 60% of cells having a DSB 4–6 h after induction. Higher levels of induction were reported in a similar experiment performed in mouse cells (B), with around 60% of cells having a DSB 4–6 h after induction. Higher levels of induction were reported in a similar experiment performed in mouse cells (20), where 80% of cells showed localization of γ-H2AX foci to a target array 30 min after drug addition. We do not have an explanation for this difference, which may relate to I-SceI expression levels or cell type- or species-specificity. We confirmed these microscopy data by immunoblot analysis of γ-H2AX induction (<xref ref-type="fig" rid="gkp684f2">Figure 2</xref>C). As Chk1 is phosphorylated following IR treatment through a mechanism involving ATM recruitment of ATR to DSBs and subsequent Chk1 activation (C). As Chk1 is phosphorylated following IR treatment through a mechanism involving ATM recruitment of ATR to DSBs and subsequent Chk1 activation (28,29), we also examined the levels of phospho-Chk1 to monitor the extent of checkpoint activation and found no major increase in Chk1 phosphorylation (<xref ref-type="fig" rid="gkp684f2">Figure 2</xref>C). We then used flow cytometry to test whether the DNA damage response impacted on the cell cycle. No difference in the cell cycle profile as determined by DNA labelling was observed following addition of TA (C). We then used flow cytometry to test whether the DNA damage response impacted on the cell cycle. No difference in the cell cycle profile as determined by DNA labelling was observed following addition of TA (<xref ref-type="fig" rid="gkp684f2">Figure 2</xref>D). We also monitored the G2 checkpoint after I-SceI induction by culturing cells in nocodazole and measuring the increase in mitotic index over 4 h. No difference in the mitotic percentages was observed in the presence or absence of TA (data not shown). These results suggest that an I-SceI-induced break at one locus did not cause significant checkpoint activation, as determined by phosphorylation of Chk1 or a G2 phase cell cycle arrest.\nD). We also monitored the G2 checkpoint after I-SceI induction by culturing cells in nocodazole and measuring the increase in mitotic index over 4 h. No difference in the mitotic percentages was observed in the presence or absence of TA (data not shown). These results suggest that an I-SceI-induced break at one locus did not cause significant checkpoint activation, as determined by phosphorylation of Chk1 or a G2 phase cell cycle arrest.\nFigure 2.γ-H2AX recruitment to the TetO array after I-SceI induction. (A) Micrograph showing localization of RFP-I-SceI-GR (blue), γ-H2AX (red) and TetR-GFP (green) in DT40 cells of the indicated genotype, before and 30 min after addition of TA. Scale bar, 10 μm. (B) Graph showing the kinetics of γ-H2AX localization at the array in wild type, Ku70−/− and Atm−/− DT40 cells. At least 20 cells that stably expressed RFP-I-SceI-GR, as determined by microscopy, were analysed per timepoint for each cell line. The experiment was repeated at least three times for each cell line and the error bars show the SEM. (C) Immunoblot showing (i) γ-H2AX levels, and (ii) Chk1 levels in wild-type I-SceI site-containing cells that stably express RFP-I-SceI-GR over time following TA addition. Wild-type DT40 cells treated with 20 Gy IR 1 h before harvesting were used as positive control. Actin was used as a loading control. (D) Flow cytometry analysis of I-SceI site-containing wild-type cells that stably express RFP-I-SceI-GR at the indicated times after induction of I-SceI.', 'To test whether the response to I-SceI-induced DSBs involves the ATM kinase or the non-homologous end-joining (NHEJ) pathway of DNA repair, we analysed the kinetics of γ-H2AX localization to the TetO array after I-SceI induction in Atm−/− and Ku70−/− DT40 cells. As shown in <xref ref-type="fig" rid="gkp684f2">Figure 2</xref>B, the kinetics of γH2AX/TetO array colocalization was very similar for wild type, ATM mutant and Ku70 mutant cells, indicating that neither ATM signalling nor NHEJ are required for the generation of the γ-H2AX signal seen after I-SceI digestion of chromosomal DNA. To analyse H2AX-dependent activities in response to enzymatic DSB induction, we also integrated this DSB system into B, the kinetics of γH2AX/TetO array colocalization was very similar for wild type, ATM mutant and Ku70 mutant cells, indicating that neither ATM signalling nor NHEJ are required for the generation of the γ-H2AX signal seen after I-SceI digestion of chromosomal DNA. To analyse H2AX-dependent activities in response to enzymatic DSB induction, we also integrated this DSB system into H2ax−/− DT40 cells (25). Following transient transfection of RFP-I-SceI-GR in Atm−/− and H2ax−/− mutant cells we followed the colocalization of the TetO array with the DNA damage response factor 53Bp1 (<xref ref-type="fig" rid="gkp684f3">Figure 3</xref>A). The kinetics of 53Bp1 localization to the induced DSB were very similar in A). The kinetics of 53Bp1 localization to the induced DSB were very similar in Atm−/− cells and in wild-type cells that stably expressed the RFP-I-SceI-GR, and closely reflected the kinetics of γ-H2AX localization. However, in H2AX-deficient cells, only a very small percentage showed co-localization between 53Bp1 and the array (<xref ref-type="fig" rid="gkp684f3">Figure 3</xref>B). This suggests that efficient 53Bp1 localization to I-SceI-induced DSBs requires H2AX.\nB). This suggests that efficient 53Bp1 localization to I-SceI-induced DSBs requires H2AX.\nFigure 3.Abnormal recruitment of 53Bp1 to DSBs in H2AX−/− cells. (A) Micrograph showing localization of RFP-I-SceI-GR (blue), 53Bp1 (red) and TetR-GFP (green) in DT40 cells after addition of TA. WT cells were stably transfected with an RFP-I-SceI-GR expression vector and treated with TA for 2 h. Atm−/− and H2ax−/− cells were transiently transfected with the RFP-I-SceI-GR expression construct and 16 h post-transfection, treated with TA for 4 and 6 h, respectively. Scale bar, 10 μm. (B) Graph showing the kinetics of 53Bp1 localization at the array in RFP-I-SceI-GR-expressing DT40 cells of the indicated genotypes. At least 20 cells that expressed RFP-I-SceI-GR, as determined by microscopy, were analysed per timepoint for each cell line. The experiment was repeated at least three times for each cell line and the error bars show the SEM.'], 'gkp684f4': ['To determine the relationship between DNA damage signalling and DNA repair of the I-SceI-induced DSB, we examined the colocalization of the Rad51 recombinase with the TetO array (<xref ref-type="fig" rid="gkp684f4">Figure 4</xref>A). The kinetics of localization of both γ-H2AX and Rad51 to the array in wild type cells were very similar, both peaking at 4 h after the addition of TA (A). The kinetics of localization of both γ-H2AX and Rad51 to the array in wild type cells were very similar, both peaking at 4 h after the addition of TA (<xref ref-type="fig" rid="gkp684f4">Figure 4</xref>B). The percentage of γ-H2AX positive cells that also had Rad51 at the array was recorded. This was maximal at 2 h after TA addition, consistent with the recruitment of Rad51 to γ-H2AX-containing chromatin (B). The percentage of γ-H2AX positive cells that also had Rad51 at the array was recorded. This was maximal at 2 h after TA addition, consistent with the recruitment of Rad51 to γ-H2AX-containing chromatin (30). We then investigated the localization of Rad51 at the array in H2ax−/− and Atm−/− DT40 cells. These mutant lines were transiently transfected with RFP-I-SceI-GR and then the percentage of cells showing colocalization between the array and Rad51 was recorded (<xref ref-type="fig" rid="gkp684f4">Figure 4</xref>C). Rad51 localization to the induced DSB was slow and inefficient in both ATM- and H2AX-deficient cells (C). Rad51 localization to the induced DSB was slow and inefficient in both ATM- and H2AX-deficient cells (<xref ref-type="fig" rid="gkp684f4">Figure 4</xref>D). Immunoblot analysis demonstrated that Rad51 protein levels were not affected by the loss of either ATM or H2AX (D). Immunoblot analysis demonstrated that Rad51 protein levels were not affected by the loss of either ATM or H2AX (<xref ref-type="fig" rid="gkp684f4">Figure 4</xref>E). These data indicate defective Rad51 mobilization to a single enzyme-induced DSB, consistent with previous observations of defective IR-induced Rad51 focus formation in E). These data indicate defective Rad51 mobilization to a single enzyme-induced DSB, consistent with previous observations of defective IR-induced Rad51 focus formation in Atm−/− and H2ax−/− cells (24,25).\nFigure 4.Abnormal recruitment of Rad51 to DSBs in H2AX−/− and ATM−/− cells. (A) Micrograph showing localization of γ-H2AX (red), Rad51 (blue) and TetR-GFP (green) in DT40 cells, before and at the indicated times after addition of TA. Scale bar, 10 µm. (B) Graph comparing the kinetics of γ-H2AX and Rad51 recruitment to the array in wild type cells that stably express RFP-I-SceI-GR over time following TA addition. At least 20 cells per timepoint were scored for localization of γ-H2AX, Rad51 (red curves, left axis) or both (the percentage of γ-H2AX/array positive cells which also have Rad51; black curve, right axis) at the array. The experiment was repeated at least three times and the error bars show the SEM. (C) Micrograph showing localization of RFP-I-SceI-GR (blue), Rad51 (red) and TetR-GFP (green) in wild-type and Atm−/− DT40 cells, before and at the indicated times after addition of TA. WT cells were stably transfected with an RFP-I-SceI-GR expression vector and treated with TA for 4 h. Atm−/− cells were transiently transfected and 16 h post-transfection, treated with TA for 2 h. Scale bar, 10 μm. (D) Graph showing the kinetics of Rad51 localization at the array in RFP-I-SceI-GR-positive Atm−/− and H2ax−/− DT40 cells after transient transfection with RFP-I-SceI-GR. At least 20 cells that expressed RFP-I-SceI-GR, as determined by microscopy, were analysed per timepoint for each cell line. The experiment was repeated at least three times for each cell line and the error bars show the SEM. (E) Immunoblot showing the Rad51 levels in wild-type, Atm−/− and H2ax−/− cells. α-tubulin was used as a loading control.'], 'gkp684f5': ['Several recent studies in yeast and vertebrate cells have shown a connection between the cohesin complex and DSB repair (31,32). Cohesin is recruited around DSBs in yeast (10,11,33,34) and human cells (9). To test whether such recruitment could impact on sister chromatid cohesion, we induced a single DSB in DT40 cells using the inducible I-SceI system and measured the distances between the TetR-GFP spots on opposite sister chromatids in mitotic cells. We used γ-H2AX localization to the array to determine whether a DSB had been generated in a given cell (<xref ref-type="fig" rid="gkp684f5">Figure 5</xref>A). Mitotic cells were analysed to ensure that two sisters were visible, as very close TetO arrays in G2 cells might have been scored as single foci in G1. Recent data have indicated that low-levels of DSB induction by IR do not arrest cells in G2 (A). Mitotic cells were analysed to ensure that two sisters were visible, as very close TetO arrays in G2 cells might have been scored as single foci in G1. Recent data have indicated that low-levels of DSB induction by IR do not arrest cells in G2 (35). Cells from the same experiment but without γ-H2AX at the TetO sites were used as a negative control and showed no difference in inter-sister distances from untreated cells (<xref ref-type="fig" rid="gkp684f5">Figure 5</xref>B). We found that inter-sister distances were significantly reduced where a DSB was induced (B). We found that inter-sister distances were significantly reduced where a DSB was induced (<xref ref-type="fig" rid="gkp684f5">Figure 5</xref>B), suggesting that one outcome of DSB-induced cohesin loading is an increased proximity of sister chromatids.\nB), suggesting that one outcome of DSB-induced cohesin loading is an increased proximity of sister chromatids.\nFigure 5.Decreased inter-sister chromatid distances at DSB sites. (A) DT40 cells were fixed and stained with antibodies to γ-H2AX (red) and counterstained with DAPI following addition of TA. The distance between GFP spots (green), which mark the TetO array, was measured in metaphase mitotic chromosomes. The presence of a γ-H2AX signal at the TetO array indicated a DSB site. Scale bar, 10 μm. (B) Analysis of distances between GFP spots. Pooled data from three independent repeats of the experiment are shown as individual data points and as mean ± SEM. At each timepoint, inter-sister distances were significantly reduced in cells with a DSB (P < 0.0001, unpaired Student\'s t-test).'], 'gkp684f6': ['We next wished to test whether this cohesion involved the cohesin complex. The tet-repressible Scc1 transgene precluded our using the conditional Scc1 knockout DT40 line (31), so we attempted to use RNAi of cohesin in the U2OS cell line. Unfortunately, in U2OS cells colocalization of the γ-H2AX signal with the TetO array was not visible above the high background in transfected mitotic cells (Supplementary Figure 2). However, recent data have indicated that the cohesin complex is an ATM target in the DNA damage response (36–38) so next, we tested whether the DSB signal through ATM mediates increased sister chromatid cohesion. We targeted the Atm locus in the same TetO-integrated, inducible I-SceI and TetR-GFP-expressing clone used for the cohesion analysis (<xref ref-type="fig" rid="gkp684f6">Figure 6</xref>A). Inducible I-SceI expression and DSB induction was maintained in A). Inducible I-SceI expression and DSB induction was maintained in Atm−/− clones (<xref ref-type="fig" rid="gkp684f6">Figure 6</xref>B). When we measured the inter-sister distances, we found that they were indistinguishable between wild-type and B). When we measured the inter-sister distances, we found that they were indistinguishable between wild-type and Atm−/− cells, but that the reduction in sister separation after DSB induction was significantly greater in wild-type than in Atm−/− cells (<xref ref-type="fig" rid="gkp684f6">Figure 6</xref>C). This observation suggests that the increased cohesion at a DSB involves ATM activity.\nC). This observation suggests that the increased cohesion at a DSB involves ATM activity.\nFigure 6.Involvement of ATM in inter-sister chromatid distances at DSB sites. (A) Southern blot showing the targeting of Atm in the same clone used for the analysis in <xref ref-type="fig" rid="gkp684f5">Figure 5</xref>. (. (B) DT40 cells were fixed and stained with antibodies to γ-H2AX (red) and counterstained with DAPI following addition of TA. The distance between GFP spots (green), which mark the TetO array, was measured in metaphase mitotic chromosomes. The presence of a γ-H2AX signal at the TetO array indicated a DSB site. Scale bar, 10 μm. (C) Analysis of distances between GFP spots. Pooled data from three independent repeats of the experiment are shown as individual data points and as mean ± SEM. Comparison of the mean distances using an unpaired Student\'s t-test showed no difference between wild-type and Atm−/− sister chromatid separation in the absence of DNA damage (data not shown), a highly-significant reduction in inter-sister distances in wild-type chromatids with a DSB (P < 0.0001) and a notably less significant reduction in inter-sister distances in Atm−/− chromatids after DSB induction (P = 0.0017). The inter-sister chromatid separation after DSB induction differed between wild-type and Atm−/− cells with a moderate level of statistical significance (P = 0.0470).']}
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Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells
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Nucleic Acids Res
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1256108400
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None
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other
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PMC2764452
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[
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Nucleic Acids Res. 2009 Oct 21; 37(18):6054-6063
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NO-CC CODE
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Localization and interactions of the ASCC complex.(a) and (b) Images of U2OS or U2OS cells expressing the indicated vectors after MMS treatment (n=3 biological replicates). (c) Silver staining of the Flag-HA-ASCC2 complex purified from HeLa-S nuclear extract separated on 4%−12% SDS-PAGE gel (n=1 independent experiment). (d) Tagged ASCC2 was purified with or without MMS and analyzed by mass spectrometry. Peptide numbers for identified proteins were plotted for each condition. Expanded view is shown on the right (n=1 independent experiment). (e) and (f) Immunofluorescence analysis of U2OS or HA-ASCC2 expressing U2OS cells upon exposure to MMS (n=3 biological replicates). (g) U2OS cells were treated with MMS, and processed for immunofluorescence with or without initial incubation with RNase A (50 nM). Numbers indicate the percent of cells expressing five or more ASCC3 foci (n=3 biological replicates; mean ± S.D.). (h) Biotinylated RNAs (20mer, 35mer, or 50mer) were immobilized and tested for binding to recombinant His-NΔ-ASCC3 (n=2 independent experiments). Scale bars, 10 μm.
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nihms-910979-f0007
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2
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91f394672af6b9eb6461f2ad721aa921a59c4addc450653257dc36ff97dedac1
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nihms-910979-f0007.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
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881
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[{'image_id': 'nihms-910979-f0011', 'image_file_name': 'nihms-910979-f0011.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0011.jpg', 'caption': 'ASCC2 coordinates ASCC-ALKBH3 complex recruitment during alkylation damage.(a) Whole cell lysates from Extended Data Figure 6i (left) Figure 3e (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (b) Immunoprecipitation of HA-ASCC2 or HA-ASCC2 L506A was performed and analyzed by Western blot as shown (n=2 biological replicates). (c) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to full-length (FL) His-ASCC3. (d) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to N-terminally deleted His-ASCC3 (His-ASCC3-ΔN) (n=2 independent experiments). (e) Flag-ALKBH3 was immobilized and tested for binding to His-ASCC2, with His-ASCC3-C (C-terminus of ASCC3) serving as a positive control (n=2 independent experiments). (f) ASCC-ALKBH3 complex model.', 'hash': 'c6803223ad4ee38da12fc28721ed2ab3a779dd788d70c19f1ede6eec8d7d774d'}, {'image_id': 'nihms-910979-f0009', 'image_file_name': 'nihms-910979-f0009.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0009.jpg', 'caption': 'ASCC2 binds specifically to K63-linked ubiquitin chains.(a) and (b) His-ASCC2 or the indicated His-ASCC2 deletions were immobilized on Ni-NTA and assessed for binding to K63-Ub2–7\n(a) or K48-Ub2–7\n(b) (n=3 independent experiments). (c) Schematic of ASCC2 or different ASCC2 deletions and their observed respective binding towards K63-Ub2–7 or K48-Ub2–7. N.D., not determined. (d) Sequence alignment and conservation of residues 373–415 of human ASCC2. (e) Interaction model between ubiquitin and the CUE domain of ASCC2 (PDB ID: 2DI0). The positions of four residues (L478, L479, P498, and L506) are shown. (f) Binding assays were performed with K63-Ub2–7 using WT or the mutants of His-ASCC2 (n=3 independent experiments).', 'hash': '5d592cefb399728c903ede93bc6868bb072619263a46ad8d0d92fa35f2bccd67'}, {'image_id': 'nihms-910979-f0007', 'image_file_name': 'nihms-910979-f0007.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0007.jpg', 'caption': 'Localization and interactions of the ASCC complex.(a) and (b) Images of U2OS or U2OS cells expressing the indicated vectors after MMS treatment (n=3 biological replicates). (c) Silver staining of the Flag-HA-ASCC2 complex purified from HeLa-S nuclear extract separated on 4%−12% SDS-PAGE gel (n=1 independent experiment). (d) Tagged ASCC2 was purified with or without MMS and analyzed by mass spectrometry. Peptide numbers for identified proteins were plotted for each condition. Expanded view is shown on the right (n=1 independent experiment). (e) and (f) Immunofluorescence analysis of U2OS or HA-ASCC2 expressing U2OS cells upon exposure to MMS (n=3 biological replicates). (g) U2OS cells were treated with MMS, and processed for immunofluorescence with or without initial incubation with RNase A (50 nM). Numbers indicate the percent of cells expressing five or more ASCC3 foci (n=3 biological replicates; mean ± S.D.). (h) Biotinylated RNAs (20mer, 35mer, or 50mer) were immobilized and tested for binding to recombinant His-NΔ-ASCC3 (n=2 independent experiments). Scale bars, 10 μm.', 'hash': '91f394672af6b9eb6461f2ad721aa921a59c4addc450653257dc36ff97dedac1'}, {'image_id': 'nihms-910979-f0010', 'image_file_name': 'nihms-910979-f0010.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0010.jpg', 'caption': 'Characterization of ASCC2 KO cells.(a) ASCC2 gene knockouts in U2OS and PC-3 cells were generated using CRISPR/Cas9 technology and verified by deep sequencing. Whole cell lysates of the parental and KO cells were analyzed by Western blotting as shown (n=2 independent experiments). (b) Flow cytometry of WT and ASCC2-KO U2OS cells after MMS treatment to determine cell cycle distribution. (c) Immunofluorescence analysis of HA-ALKBH2 expressing cells after MMS. Numbers indicate the percent of cells expressing five or more HA-ALKBH2 foci (n=3 biological replicates; mean ± S.D.). (d) MMS sensitivity of WT or ASCC2 KO cells using MTS assay (mean ± S.D.; n=5 biological replicates). (e-f) Sensitivity of WT and ASCC2 KO cells to MMS (e) or camptothecin (f) was assessed by clonogenic survival assay (n=4 biological replicates; mean ± S.D.). (g-h) WT PC-3 and ASCC2-KO cell sensitivity to camptothecin (g) or bleomycin (h) using the MTS assay (n=5 biological replicates; mean ± S.D.). (i) Images of WT or ASCC2 KO cells expressing the indicated vectors after MMS exposure. (j) Quantitation of (i) (n=2 independent experiments; mean ± S.D.). (k) WT or ASCC2-KO cells expressing indicated vectors were assessed for sensitivity to MMS using the MTS assay (n=5 technical replicates; mean ±S.D.). Scale bar, 10 μm.', 'hash': '7b671aef7f458a6ec23a6f39a26503458a1881f8ddb7d56506425b8e668d16f2'}, {'image_id': 'nihms-910979-f0006', 'image_file_name': 'nihms-910979-f0006.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0006.jpg', 'caption': 'Subcellular localization of ASCC2 and other alkylation repair factors.(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from Figure 1c (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': '372e42172ba563db000ac08103cdc6febe655893f8aa73dfbeba51afb7d89661'}, {'image_id': 'nihms-910979-f0001', 'image_file_name': 'nihms-910979-f0001.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0001.jpg', 'caption': 'The ASCC complex forms foci upon alkylation damage.(a) Images of ASCC3 and pH2A.X immunofluorescence after treatment with damaging agents. (b) ASCC3 foci quantitation (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (c) PLA images in control or MMS-treated cells using 1meA and ASCC3 antibodies (n=3 biological replicates). (d) Immunofluorescence of HA-ASCC2 expressing cells treated with MMS. (e) Quantitation of MMS-induced co-localizations of HA-ASCC2 foci (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': '516a069547a57fdefbf32b3c80bc37033962e13b423ecb6ea3b96de99b0072d1'}, {'image_id': 'nihms-910979-f0008', 'image_file_name': 'nihms-910979-f0008.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0008.jpg', 'caption': 'Functional interactions of the ASCC complex with other signaling pathways.Immunofluorescence images of U2OS cells treated with MMS in the presence of spliceosomal inhibitor PLA-B (a) (100 nM; n=3 biological replicates; mean ± S.D.), the RNA Pol II inhibitor DRB (b) (100 μM; n=3 biological replicates; mean ± S.D.), or the indicated damage signaling kinase inhibitor (c) (n=2 biological replicates; mean). Numbers indicate the percent of cells expressing five or more ASCC3 foci. (d) Immunofluorescence of HA-ASCC2 and FK2 in cells after MMS (n=3 biological replicates; mean ± S.D.). (e) His-ASCC2 was purified on Ni-NTA, separated on a 10% SDS-PAGE gel, and analyzed by Coomassie blue staining (n=2 independent experiments). (f) Immunofluorescence of HA-ASCC2 cells and K63-ubiquitin (top) or K48-ubiquitin (bottom) after MMS treatment (n=2 independent experiments). Scale bars, 10 μm.', 'hash': 'ab8a3da80ed2ae0cf9f5f752fc8278b450947af0dfa81cf374790e1ad70c556b'}, {'image_id': 'nihms-910979-f0002', 'image_file_name': 'nihms-910979-f0002.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0002.jpg', 'caption': 'ASCC2 binds to K63-linked ubiquitin chains via its CUE domain.(a) ASCC2 sequence alignment. (b) Structure of the ASCC2 CUE domain (PDB ID: 2DI0; grey) overlayed with the Vps9 CUE:ubiquitin complex (PDB ID: 1P3Q). (c) His-ASCC2 was immobilized and assessed for binding to K48-Ub2–7 (left) or K63-Ub2–7. ALKBH3 and gp78-CUE served as controls. Bound material was analyzed by Western blot or Coomassie Blue (CBB) (n=3 independent experiments). (d) ITC was performed with K63-Ub2 and His-ASCC2 or the L506A mutant (n=1 independent experiment; mean ± S.E.). (g) Immunofluorescence images of MMS-induced foci in cells expressing various forms of HA-ASCC2. Numbers indicate the percentage of cells expressing ten or more HA-ASCC2 foci (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': 'f8963fcf41cec6681ac9ed0f2e46cee047cb861b8485f3be04bb41fa2ccf5515'}, {'image_id': 'nihms-910979-f0005', 'image_file_name': 'nihms-910979-f0005.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0005.jpg', 'caption': 'The ASCC complex forms foci upon alkylation damage.(a) ASCC3 KO cells were generated using CRISPR/Cas9 technology. Lysates were analyzed by Western blotting (n=2 independent experiments). Clone #10 was verified to be a knockout by deep sequencing. (b) Images of U2OS parental cells or ASCC3-KO cells after MMS (n=3 biological replicates). (c) Immunofluorescence of U2OS cells after exposure to γ-irradiation (IR; 5 Gy) or UV (25 J/m2) (n=3 biological replicates). (d) Images of U2OS cells after treatment with the alkylating agents busulfan (4 mM), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; 100 μM), or temozolomide (TMZ; 1.0 mM) (n=2 biological replicates). Numbers indicate the mean percent of cells expressing five or more foci. (e) Immunofluorescence of HA-ASCC2 expressing cells after exposure to the indicated damaging agents (n=3 biological replicates). Scale bars, 10 μm. For gel source data, see Supplementary Figure 1.', 'hash': '1ecf6abce6d27cff30f2f6823337e325fb811bda1e074cfa9d780911a020a0bc'}, {'image_id': 'nihms-910979-f0014', 'image_file_name': 'nihms-910979-f0014.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0014.jpg', 'caption': 'Functional characterization of RNF113A.(a) Schematic of human RNF113A and its domain structure. The three deletion constructs used for localization analysis are also shown. (b) Images of cells expressing WT or the indicated HA-RNF113A deletion constructs. Scale bar, 10 μm. Quantitation of co-localization between each RNF113A construct and PRP8 is shown on the right (n=3 biological replicates; mean ± S.D.). (c) 293T cells expressing His-ubiquitin were transduced with control or RNF113A-targeting shRNAs and treated with MMS. Ubiquitinated proteins were isolated by Ni-NTA under denaturing conditions and Western blotted as shown. Input lysates were also analyzed as indicated. SF3B3, another ubiquitinated spliceosomal protein, was used as a control (n=3 independent experiments). (d) Cells expressing the indicated HA-vectors were treated with MMS as in (c). Lysates were then used for ubiquitin pulldown assays using GST-ASCC2, then blotted as shown. Input lysates were also analyzed as indicated (n=2 independent experiments). (e) His-NΔ-BRR2 was purified from Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (left). This was then used as a substrate for ubiquitination assays using HA-Ub and wildtype (WT) or a RING-deletion (ΔRING) RNF113A (n=2 independent experiments). (f) Western blot analysis of U2OS cells expressing the indicated shRNAs used for immunofluorescence analysis in Figure 4F (n=2 independent experiments). (g) Quantitation of Figure 4F (n=3 biological replicates; mean ± S.D.; two-tailed t-test, # = p < 0.001). (h) MMS sensitivity of PC-3 cells expressing the indicated shRNAs was determined by MTS assay (n=5 technical replicates; mean ± S.D.).', 'hash': '912acb951e1fe59686b4784725aff4d2c96e3c8b8572d1b4e9d00408b9735d22'}, {'image_id': 'nihms-910979-f0013', 'image_file_name': 'nihms-910979-f0013.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0013.jpg', 'caption': 'Characterization of the E3 ubiquitin ligase activity of RNF113A and TTDN1.(a) TAP-RNF113A and the I264A RING finger mutant were stably expressed in HeLa-S cells and purified using anti-Flag resin. The eluted proteins were then analyzed by silver staining after SDS-PAGE (n=3 independent experiments). (b) Ubiquitin ligase assays using E1, E2 (UbcH5c plus Ubc13/MMS2; 50 nM each), and wildtype or I264A RNF113A. Reactions were analyzed by Western blot (n=3 independent experiments). (c) MMS sensitivity of lymphoblasts from two X-TTD patients in comparison to an unaffected individual (n=5 biological replicates; mean ± S.D.). (d) U2OS cells expressing the indicated combination of shRNA and RNF113A rescue vector were assessed for MMS sensitivity using MTS assay (n=5 technical replicates; mean ± S.D.). (e) Whole cell lysates of control or X-TTD lymphoblasts expressing indicated vectors after selection (n=2 independent experiments). (f) Immunofluorescence analysis of U2OS cells expressing the indicated shRNAs after MMS treatment. Western blot (n=2 independent experiments) from the same cells is shown on the bottom, as is the quantification of ASCC3 foci (n=3 biological replicates; mean ± S.D.).', 'hash': 'df5349c6a10293efaff3516b5f0a3ac01a852fd9a9e8e9ec523d3eb3b22989dd'}, {'image_id': 'nihms-910979-f0004', 'image_file_name': 'nihms-910979-f0004.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0004.jpg', 'caption': 'RNF113A ubiquitination recruits the ASCC complex.(a) MMS-induced foci in U2OS cells expressing indicated shRNAs (n=3 technical replicates; mean). (b) Ubiquitin ligase assays using E1, E2 (UBC13/MMS2; 250 nM), and Flag-RNF113A. K63R ubiquitin was substituted as shown (n=2 independent experiments). (c) Images of X-TTD or control lymphoblasts expressing the indicated vectors after MMS (n=3 technical replicates; mean). (d). ASCC2 interactome analysis. UBC13 substrates were previously described19. (e) HA-RNF113A deletions were immunoprecipitated to analyze BRR2 interaction. (n=3 independent experiments). (f) Images of U2OS cells expressing indicated shRNAs. Numbers indicate the percentage of cells expressing at least five (c) or ten (a) foci. Scale bars, 10 μm.', 'hash': '69239f011df44323addea37d6fc7933ee1710a20298dda42b5f751ffc1e92d13'}, {'image_id': 'nihms-910979-f0003', 'image_file_name': 'nihms-910979-f0003.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0003.jpg', 'caption': 'ASCC2 is critical for ASCC3-ALKBH3 recruitment and alkylation resistance.(a) MMS-induced ASCC3 foci were assessed in WT and ASCC2-KO cells. (b) Quantitation of (a) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (c) HA-ALKBH3 foci were assessed as in (a). Numbers indicate the percentage of cells expressing five or more foci (n=2 biological replicates; mean). (d) 1meA quantitation in WT or ASCC2-KO cells after MMS treatment (n=3 biological replicates; mean ± S.D.). (e) Images of WT or ASCC2-KO cells expressing indicated vectors upon MMS. (f) Quantitation of (e) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001, # = p < 0.05). Scale bars, 10 μm.', 'hash': '3599bd784d2637a3063f11f072e100d4fec53adbfe7740ae7824ab60ba608bf7'}, {'image_id': 'nihms-910979-f0012', 'image_file_name': 'nihms-910979-f0012.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0012.jpg', 'caption': 'Identification of the RNF113A E3 ligase.(a) Whole cell lysates of U2OS cells infected with the indicated shRNAs were analyzed by Western blot. SHPRH was used as a loading control (n=1 independent experiment). (b) Immunofluorescence images of MMS-induced HA-ASCC2 foci in cells expressing the indicated shRNAs. (c) HA-ASCC2 foci quantitation from (b) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (d) Compilation of E3 ligase shRNA screen results. For each candidate, U2OS cells were transduced with HA-ASCC2 and an E3 targeting shRNA. MMS-induced HA-ASCC2 foci formation was analyzed by immunofluorescence. Results were normalized to a scrambled shRNA (normalized score = 100). UBC13 denotes the positive control (purple). Results of three different shRNA to RNF113A are indicated in red (n=1 independent experiment for each shRNA). (e) Whole cell lysates of U2OS cells infected with the indicated shRNAs were analyzed by Western blot. Asterisk (*) indicates a non-specific band in the RNF113A blot (n=2 independent experiments). (f) Localization of Flag-ASCC2 and HA-RNF113A after MMS treatment (n=3 biological replicates). (g) Immunofluorescence of cells expressing Flag-RNF113A without MMS treatment (n=3 biological replicates).', 'hash': 'b570592437196db6baef164d931603c1d57ea5d1392567174cd2ca280e39d210'}]
|
{'nihms-910979-f0001': ['Previous studies established that the dealkylating enzyme, ALKBH3, functions in concert with the ASCC helicase complex7. We tested the subcellular localization of the catalytic subunit, ASCC3 upon exposure to various DNA damaging agents. Endogenous ASCC3 formed nuclear foci upon treatment of U2OS cells with the alkylating agent, methyl methanesulphonate (MMS; <xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1A</xref>). Knockout of ASCC3 abrogated these foci (). Knockout of ASCC3 abrogated these foci (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1A and 1B</xref>). Strikingly, other types of DNA damaging agents did not significantly induce ASCC3 foci (). Strikingly, other types of DNA damaging agents did not significantly induce ASCC3 foci (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1A and 1B</xref>; ; <xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1C</xref>), although these genotoxins induced pH2A.X foci, indicative of DNA damage. ASCC3 foci were also observed with other alkylating agents used clinically in the treatment of various tumors), although these genotoxins induced pH2A.X foci, indicative of DNA damage. ASCC3 foci were also observed with other alkylating agents used clinically in the treatment of various tumors8 (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1D</xref>). The ASCC complex subunit ASCC2 also formed foci specifically after treatment with MMS (). The ASCC complex subunit ASCC2 also formed foci specifically after treatment with MMS (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1E</xref>). These foci were largely limited to G1/early S-phase of the cell cycle (). These foci were largely limited to G1/early S-phase of the cell cycle (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2A</xref>). Consistent with their known physical association). Consistent with their known physical association7,9, HA-ASCC2 co-localized with ASCC3 upon MMS treatment, as did the dealkylase ALKBH3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2B</xref>).).', 'To ascertain that the ASCC complex is recruited to regions of the nucleus that have alkylation damage, we performed a proximity ligation assay (PLA). We found that a specific nuclear PLA signal between 1-methyladenosine (1-meA) and ASCC3 is induced upon MMS damage (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1C</xref> and and <xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2C</xref>). The dealkylase ALKBH2 also formed foci that co-localized partially with ASCC3 (). The dealkylase ALKBH2 also formed foci that co-localized partially with ASCC3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2D and 2E</xref>). Conversely, two other alkylation repair factors, methylguanine methyltransferase (MGMT) and alkyladenine glycosylase (AAG), showed minimal co-localization with ASCC3 (). Conversely, two other alkylation repair factors, methylguanine methyltransferase (MGMT) and alkyladenine glycosylase (AAG), showed minimal co-localization with ASCC3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2D and 2E</xref>))', '(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from <xref rid="nihms-910979-f0001" ref-type="fig">Figure 1c</xref> (n=3 biological replicates; mean ± S.D.; two-tailed (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.'], 'nihms-910979-f0007': ['ASCC foci did not co-localize with pH2A.X or 53BP1, demonstrating that they are distinct from double-stranded break (DSB)-induced foci (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3A</xref>). These foci were also distinct from GFP-PCNA or BMI-1 (). These foci were also distinct from GFP-PCNA or BMI-1 (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3B</xref>). We took an unbiased proteomic approach to identify the factors associated with ASCC foci in response to alkylation damage using tandem affinity purification (TAP) (). We took an unbiased proteomic approach to identify the factors associated with ASCC foci in response to alkylation damage using tandem affinity purification (TAP) (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3C</xref>). Mass spectrometric analysis of ASCC2-associated proteins revealed the constitutive association of ASCC3 and ASCC1 (). Mass spectrometric analysis of ASCC2-associated proteins revealed the constitutive association of ASCC3 and ASCC1 (Supplementary Table 1). ASCC2 also associated with many spliceosome components and basal transcription factors (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3D</xref> and and Supplementary Table 1). These factors, including BRR2, PRP8, and TFII-I had 2–3 fold higher total peptide numbers from cells exposed to MMS, suggesting an increased association with the ASCC complex in response to alkylation-induced damage. Focused immunofluorescence studies revealed that ASCC components co-localized with BRR2 and PRP8 upon alkylation damage (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1D-E</xref>). Furthermore, ASCC foci co-localized with elongating (Ser2 phosphorylated) RNA polymerase II, but not other transcription-associated nuclear bodies, such as paraspeckles (). Furthermore, ASCC foci co-localized with elongating (Ser2 phosphorylated) RNA polymerase II, but not other transcription-associated nuclear bodies, such as paraspeckles (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3E-F</xref>). Consistently, RNase treatment prior to processing for immunofluorescence significantly reduced ASCC3 foci formation (). Consistently, RNase treatment prior to processing for immunofluorescence significantly reduced ASCC3 foci formation (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3G</xref>). Purified ASCC3 bound to ssRNA ). Purified ASCC3 bound to ssRNA in vitro (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3H</xref>). Chemical inhibition of transcription or splicing during alkylation damage also reduced ASCC3 foci (). Chemical inhibition of transcription or splicing during alkylation damage also reduced ASCC3 foci (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4A-B</xref>).).', 'To uncover the relevant RNF113A substrate, we combined our initial proteomics screen (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3D</xref>) with a second screen for proteins that interact preferentially with WT ASCC2 relative to the L506A mutant () with a second screen for proteins that interact preferentially with WT ASCC2 relative to the L506A mutant (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4D</xref> and and Supplementary Table 3). Of the remaining putative ubiquitinated substrates, eight have been shown to be ubiquitinated by UBC1315. Of these, BRR2 was the most obvious candidate, as it co-localized with RNF113A and ASCC components. Indeed, BRR2 co-immunoprecipitated with RNF113A in a manner dependent upon the N-terminal domain of RNF113A (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4E</xref>). Deletion analysis revealed that the RNF113A N-terminus was also critical for its co-localization with PRP8 (). Deletion analysis revealed that the RNF113A N-terminus was also critical for its co-localization with PRP8 (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10A-B</xref>). Ubiquitin conjugation of BRR2 was significantly reduced upon loss of RNF113A (). Ubiquitin conjugation of BRR2 was significantly reduced upon loss of RNF113A (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10C</xref>). Furthermore, RNF113A promoted BRR2 binding to ASCC2, which was dependent on its RING domain (). Furthermore, RNF113A promoted BRR2 binding to ASCC2, which was dependent on its RING domain (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10D</xref>). Recombinant BRR2 was ubiquitinated ). Recombinant BRR2 was ubiquitinated in vitro by RNF113A, also in a manner dependent on its RING domain (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10E</xref>). Knockdown of BRR2, or its partner PRP8). Knockdown of BRR2, or its partner PRP816, significantly reduced ASCC3 foci formation upon MMS damage (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10F-G</xref> and and <xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4F</xref>). Consistently, loss of BRR2 increased sensitivity to MMS (). Consistently, loss of BRR2 increased sensitivity to MMS (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10H</xref>). Thus, BRR2 likely represents at least one physiologic substrate for RNF113A in this alkylation repair pathway.). Thus, BRR2 likely represents at least one physiologic substrate for RNF113A in this alkylation repair pathway.'], 'nihms-910979-f0008': ['While recruitment of certain repair complexes is dependent on specific upstream signaling kinases1–3, inhibition of ATM moderately increased ASCC3 foci formation, and ATR inhibition had no impact (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4C</xref>). We found that HA-ASCC2 foci co-localized with polyubiquitin, suggesting that ubiquitin signaling may recruit this repair complex (). We found that HA-ASCC2 foci co-localized with polyubiquitin, suggesting that ubiquitin signaling may recruit this repair complex (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4D</xref>). Analysis of the ASCC2 protein sequence revealed a highly conserved CUE domain (residues 467–509), which belongs to the ubiquitin binding domain superfamily). Analysis of the ASCC2 protein sequence revealed a highly conserved CUE domain (residues 467–509), which belongs to the ubiquitin binding domain superfamily10 (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2A</xref>). A deposited but unpublished NMR structure of the ASCC2 CUE domain (PDB ID: 2DI0) was used to model its interaction with ubiquitin in comparison to another CUE domain from Vps9 (). A deposited but unpublished NMR structure of the ASCC2 CUE domain (PDB ID: 2DI0) was used to model its interaction with ubiquitin in comparison to another CUE domain from Vps9 (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2B</xref>). While Vps9 CUE binds to ubiquitin as a dimer). While Vps9 CUE binds to ubiquitin as a dimer11, our model predicts ubiquitin binding by a monomeric form of the ASCC2 CUE. His-tagged ASCC2 (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4E</xref>) bound K63- but not K48-linked ubiquitin chains () bound K63- but not K48-linked ubiquitin chains (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2C</xref>). Furthermore, ASCC2 co-localized with K63- but not K48-linked ubiquitin foci upon MMS damage (). Furthermore, ASCC2 co-localized with K63- but not K48-linked ubiquitin foci upon MMS damage (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4F</xref>). The minimal domain of ASCC2 for ubiquitin binding ). The minimal domain of ASCC2 for ubiquitin binding in vitro was comprised of residues 457–525 (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5A-5D</xref>). However, the presence of an additional conserved region adjacent to the CUE domain was necessary for specific binding to K63-linked ubiquitin (). However, the presence of an additional conserved region adjacent to the CUE domain was necessary for specific binding to K63-linked ubiquitin (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5A-5D</xref>).).'], 'nihms-910979-f0009': ['We introduced point mutations in the ASCC2 CUE domain at residues predicted to be critical for ubiquitin recognition (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5E</xref>). The mutations L506A and LL478–9AA abrogated ubiquitin binding ). The mutations L506A and LL478–9AA abrogated ubiquitin binding in vitro, while another, P498A, bound to K63-Ub similar to wild type (WT) ASCC2 (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5F</xref>). Isothermal titration calorimetry (ITC) experiments demonstrated that WT ASCC2 bound K63-linked di-ubiquitin chains with a ). Isothermal titration calorimetry (ITC) experiments demonstrated that WT ASCC2 bound K63-linked di-ubiquitin chains with a Kd of 10.1 μM, which is similar to other CUE domains12. In contrast, the L506A mutant showed no detectable binding (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2D</xref>). Notably, ASCC2 mutants that abrogate ubiquitin binding showed significantly reduced foci formation upon MMS treatment (). Notably, ASCC2 mutants that abrogate ubiquitin binding showed significantly reduced foci formation upon MMS treatment (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2E</xref>).).'], 'nihms-910979-f0010': ['We reasoned that ASCC2 acts as an intermediary subunit to recruit other components of the ASCC-ALKBH3 complex. Thus, we generated ASCC2 knockout cells using CRISPR/Cas9 (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6A</xref>). Two independent ASCC2 knockout clones showed a significant reduction in ASCC3 foci formation upon MMS treatment (). Two independent ASCC2 knockout clones showed a significant reduction in ASCC3 foci formation upon MMS treatment (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3A-B</xref>). This reduction was not due to a change in the population of cells in G1 (). This reduction was not due to a change in the population of cells in G1 (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6B</xref>). HA-ALKBH3 and HA-ALKBH2 foci were also diminished in the mutant cells, albeit more modestly for HA-ALKBH2 (). HA-ALKBH3 and HA-ALKBH2 foci were also diminished in the mutant cells, albeit more modestly for HA-ALKBH2 (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3C</xref> and and <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6C</xref>). Consistent with a role in the recruitment of these factors, ASCC2-deficient PC-3 cells were hypersensitive to MMS, but not to camptothecin or bleomycin. (Extended Data Fig. D-H). DNA alkylated lesion repair kinetics was also significantly slower in ASCC2 knockout cells (). Consistent with a role in the recruitment of these factors, ASCC2-deficient PC-3 cells were hypersensitive to MMS, but not to camptothecin or bleomycin. (Extended Data Fig. D-H). DNA alkylated lesion repair kinetics was also significantly slower in ASCC2 knockout cells (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3D</xref>).).', '(a) Whole cell lysates from <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Figure 6i</xref> (left) (left) <xref rid="nihms-910979-f0003" ref-type="fig">Figure 3e</xref> (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (b) Immunoprecipitation of HA-ASCC2 or HA-ASCC2 L506A was performed and analyzed by Western blot as shown (n=2 biological replicates). (c) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to full-length (FL) His-ASCC3. (d) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to N-terminally deleted His-ASCC3 (His-ASCC3-ΔN) (n=2 independent experiments). (e) Flag-ALKBH3 was immobilized and tested for binding to His-ASCC2, with His-ASCC3-C (C-terminus of ASCC3) serving as a positive control (n=2 independent experiments). (f) ASCC-ALKBH3 complex model.'], 'nihms-910979-f0003': ['Next, we reconstituted ASCC2-KO cells with WT and mutant versions of ASCC2. WT ASCC2, but not the L506A CUE mutant, restored MMS-induced ASCC3 and HA-ALKBH3 foci formation (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3E-F</xref> and and <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6I-J</xref> and and <xref rid="nihms-910979-f0011" ref-type="fig">7A</xref>). Similarly, WT, but not L506A ASCC2, rescued MMS sensitivity of ASCC2 knockout cells (). Similarly, WT, but not L506A ASCC2, rescued MMS sensitivity of ASCC2 knockout cells (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6K</xref>). WT and L506A ASCC2 equally co-immunoprecipitated ASCC3 (). WT and L506A ASCC2 equally co-immunoprecipitated ASCC3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7B</xref>). Indeed His-ASCC3 bound to immobilized Flag-ASCC2 and Flag-ALKBH3, although binding with ALKBH3 appeared weaker (). Indeed His-ASCC3 bound to immobilized Flag-ASCC2 and Flag-ALKBH3, although binding with ALKBH3 appeared weaker (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7C</xref>). Deletion of the ASCC3 N-terminus abrogated its ability to bind ASCC2, while retaining ALKBH3 binding (). Deletion of the ASCC3 N-terminus abrogated its ability to bind ASCC2, while retaining ALKBH3 binding (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7D</xref>). Conversely, ASCC2 did not interact with recombinant ALKBH3 (). Conversely, ASCC2 did not interact with recombinant ALKBH3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7E</xref>). ASCC2 therefore appears to bridge ASCC3 and K63-linked ubiquitin chains, while ALKBH3 is indirectly recruited by ASCC2 through its interaction with ASCC3 (). ASCC2 therefore appears to bridge ASCC3 and K63-linked ubiquitin chains, while ALKBH3 is indirectly recruited by ASCC2 through its interaction with ASCC3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7F</xref>).).'], 'nihms-910979-f0012': ['UBC13, a major E2 ubiquitin ligase responsible for formation of K63-linked ubiquitin chains, has been implicated in DNA damage response pathways13–15. Knockdown of UBC13 reduced 53BP1 foci, and also attenuated MMS-induced HA-ASCC2 foci (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8A-8C</xref>). However, knockdown of RNF8 or RNF168, two E3 ligases involved in the double-stranded break repair). However, knockdown of RNF8 or RNF168, two E3 ligases involved in the double-stranded break repair1, did not affect HA-ASCC2 foci formation (data not shown), suggesting that a distinct E3 ligase functions in the alkylation pathway. To identify this E3 ligase, we performed a screen using a custom library of shRNAs that target UBC13-interacting E3 ligases or other ligases implicated in DNA repair (Supplementary Table 2). The screen identified RNF113A as a potential candidate, with three distinct shRNAs reducing HA-ASCC2 foci to UBC13 knockdown levels (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8D</xref>). We confirmed that these shRNAs attenuated both RNF113A protein levels and HA-ASCC2 foci formation (). We confirmed that these shRNAs attenuated both RNF113A protein levels and HA-ASCC2 foci formation (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8E</xref> and and <xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4A</xref>). Importantly, MMS-induced ASCC2 foci co-localized with RNF113A (). Importantly, MMS-induced ASCC2 foci co-localized with RNF113A (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8F</xref>). In the absence of damage, RNF113A co-localized with PRP8 and BRR2 (). In the absence of damage, RNF113A co-localized with PRP8 and BRR2 (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8G</xref>), consistent with previous studies suggesting it nominally serves as a spliceosome component), consistent with previous studies suggesting it nominally serves as a spliceosome component16.'], 'nihms-910979-f0013': ['We purified Flag-tagged RNF113A from HeLa-S cells to analyze its E3 ligase activity (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9A</xref>). RNF113A exhibited robust E3 activity ). RNF113A exhibited robust E3 activity in vitro, which was significantly reduced with a RING-finger point mutation (I264A; <xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9B</xref>). Use of K63R ubiquitin abrogated chain elongation, suggesting that RNF113A may function to promote the E2 activity of UBC13 (). Use of K63R ubiquitin abrogated chain elongation, suggesting that RNF113A may function to promote the E2 activity of UBC13 (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4B</xref>). A recent study identified a nonsense mutation (Q301*) in RNF113A in two related individuals suffering from X-linked trichothiodystrophy). A recent study identified a nonsense mutation (Q301*) in RNF113A in two related individuals suffering from X-linked trichothiodystrophy17 (X-TTD). While most TTD patient cells are hypersensitive to UV damage, X-TTD cells do not have this phenotype17. Lymphoblastoid cell lines obtained from these two patients were hypersensitive to MMS, as was an RNF113A knockdown cell line (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9C-D</xref>). Strikingly, X-TTD cells had significantly reduced ASCC3 foci formation (). Strikingly, X-TTD cells had significantly reduced ASCC3 foci formation (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4C</xref> and and <xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9E</xref>). Reconstitution of these patient cells with WT RNF113A rescued ASCC3 foci formation, while the I264A mutant gave a partial rescue, possibly due to the small degree of remaining E3 ligase activity. Loss of TTDN1, another TTD-associated gene). Reconstitution of these patient cells with WT RNF113A rescued ASCC3 foci formation, while the I264A mutant gave a partial rescue, possibly due to the small degree of remaining E3 ligase activity. Loss of TTDN1, another TTD-associated gene18, also reduced ASCC3 foci formation (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9F</xref>).).'], 'nihms-910979-f0002': ['PyMOL (The PyMOL Molecular Graphics System, Version 1.8.0.5 Schrödinger, LLC.) was used to align the structure of ASCC2 residues 463–525 (PDB ID: 2DI0) with the VSP9 CUE:ubiquitin complex structure (PDB ID: 1P3Q). <xref rid="nihms-910979-f0002" ref-type="fig">Figure 2C</xref> and and <xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Figure 3E</xref> were generated using PyMOL. were generated using PyMOL.'], 'nihms-910979-f0004': ['(a) Schematic of human RNF113A and its domain structure. The three deletion constructs used for localization analysis are also shown. (b) Images of cells expressing WT or the indicated HA-RNF113A deletion constructs. Scale bar, 10 μm. Quantitation of co-localization between each RNF113A construct and PRP8 is shown on the right (n=3 biological replicates; mean ± S.D.). (c) 293T cells expressing His-ubiquitin were transduced with control or RNF113A-targeting shRNAs and treated with MMS. Ubiquitinated proteins were isolated by Ni-NTA under denaturing conditions and Western blotted as shown. Input lysates were also analyzed as indicated. SF3B3, another ubiquitinated spliceosomal protein, was used as a control (n=3 independent experiments). (d) Cells expressing the indicated HA-vectors were treated with MMS as in (c). Lysates were then used for ubiquitin pulldown assays using GST-ASCC2, then blotted as shown. Input lysates were also analyzed as indicated (n=2 independent experiments). (e) His-NΔ-BRR2 was purified from Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (left). This was then used as a substrate for ubiquitination assays using HA-Ub and wildtype (WT) or a RING-deletion (ΔRING) RNF113A (n=2 independent experiments). (f) Western blot analysis of U2OS cells expressing the indicated shRNAs used for immunofluorescence analysis in <xref rid="nihms-910979-f0004" ref-type="fig">Figure 4F</xref> (n=2 independent experiments). (n=2 independent experiments). (g) Quantitation of <xref rid="nihms-910979-f0004" ref-type="fig">Figure 4F</xref> (n=3 biological replicates; mean ± S.D.; two-tailed (n=3 biological replicates; mean ± S.D.; two-tailed t-test, # = p < 0.001). (h) MMS sensitivity of PC-3 cells expressing the indicated shRNAs was determined by MTS assay (n=5 technical replicates; mean ± S.D.).']}
|
A ubiquitin-dependent signaling axis specific for ALKBH-mediated DNA dealkylation repair
| null |
Nature
|
1510819200
|
Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.
|
[
"Animals",
"Chromatography, Liquid",
"Cross-Linking Reagents",
"Humans",
"Pancreatic Elastase",
"Peptides",
"Proteolysis",
"Sf9 Cells",
"Spodoptera",
"Succinimides",
"TATA-Binding Protein Associated Factors",
"Tandem Mass Spectrometry",
"Transcription Factor TFIID",
"Trypsin"
] |
other
|
PMC6458054
| null |
37
|
[
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Nature. 2017 Nov 16; 551(7680):389-393
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Subcellular localization of ASCC2 and other alkylation repair factors.(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from Figure 1c (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.
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[{'image_id': 'nihms-910979-f0011', 'image_file_name': 'nihms-910979-f0011.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0011.jpg', 'caption': 'ASCC2 coordinates ASCC-ALKBH3 complex recruitment during alkylation damage.(a) Whole cell lysates from Extended Data Figure 6i (left) Figure 3e (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (b) Immunoprecipitation of HA-ASCC2 or HA-ASCC2 L506A was performed and analyzed by Western blot as shown (n=2 biological replicates). (c) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to full-length (FL) His-ASCC3. (d) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to N-terminally deleted His-ASCC3 (His-ASCC3-ΔN) (n=2 independent experiments). (e) Flag-ALKBH3 was immobilized and tested for binding to His-ASCC2, with His-ASCC3-C (C-terminus of ASCC3) serving as a positive control (n=2 independent experiments). (f) ASCC-ALKBH3 complex model.', 'hash': 'c6803223ad4ee38da12fc28721ed2ab3a779dd788d70c19f1ede6eec8d7d774d'}, {'image_id': 'nihms-910979-f0009', 'image_file_name': 'nihms-910979-f0009.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0009.jpg', 'caption': 'ASCC2 binds specifically to K63-linked ubiquitin chains.(a) and (b) His-ASCC2 or the indicated His-ASCC2 deletions were immobilized on Ni-NTA and assessed for binding to K63-Ub2–7\n(a) or K48-Ub2–7\n(b) (n=3 independent experiments). (c) Schematic of ASCC2 or different ASCC2 deletions and their observed respective binding towards K63-Ub2–7 or K48-Ub2–7. N.D., not determined. (d) Sequence alignment and conservation of residues 373–415 of human ASCC2. (e) Interaction model between ubiquitin and the CUE domain of ASCC2 (PDB ID: 2DI0). The positions of four residues (L478, L479, P498, and L506) are shown. (f) Binding assays were performed with K63-Ub2–7 using WT or the mutants of His-ASCC2 (n=3 independent experiments).', 'hash': '5d592cefb399728c903ede93bc6868bb072619263a46ad8d0d92fa35f2bccd67'}, {'image_id': 'nihms-910979-f0007', 'image_file_name': 'nihms-910979-f0007.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0007.jpg', 'caption': 'Localization and interactions of the ASCC complex.(a) and (b) Images of U2OS or U2OS cells expressing the indicated vectors after MMS treatment (n=3 biological replicates). (c) Silver staining of the Flag-HA-ASCC2 complex purified from HeLa-S nuclear extract separated on 4%−12% SDS-PAGE gel (n=1 independent experiment). (d) Tagged ASCC2 was purified with or without MMS and analyzed by mass spectrometry. Peptide numbers for identified proteins were plotted for each condition. Expanded view is shown on the right (n=1 independent experiment). (e) and (f) Immunofluorescence analysis of U2OS or HA-ASCC2 expressing U2OS cells upon exposure to MMS (n=3 biological replicates). (g) U2OS cells were treated with MMS, and processed for immunofluorescence with or without initial incubation with RNase A (50 nM). Numbers indicate the percent of cells expressing five or more ASCC3 foci (n=3 biological replicates; mean ± S.D.). (h) Biotinylated RNAs (20mer, 35mer, or 50mer) were immobilized and tested for binding to recombinant His-NΔ-ASCC3 (n=2 independent experiments). Scale bars, 10 μm.', 'hash': '91f394672af6b9eb6461f2ad721aa921a59c4addc450653257dc36ff97dedac1'}, {'image_id': 'nihms-910979-f0010', 'image_file_name': 'nihms-910979-f0010.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0010.jpg', 'caption': 'Characterization of ASCC2 KO cells.(a) ASCC2 gene knockouts in U2OS and PC-3 cells were generated using CRISPR/Cas9 technology and verified by deep sequencing. Whole cell lysates of the parental and KO cells were analyzed by Western blotting as shown (n=2 independent experiments). (b) Flow cytometry of WT and ASCC2-KO U2OS cells after MMS treatment to determine cell cycle distribution. (c) Immunofluorescence analysis of HA-ALKBH2 expressing cells after MMS. Numbers indicate the percent of cells expressing five or more HA-ALKBH2 foci (n=3 biological replicates; mean ± S.D.). (d) MMS sensitivity of WT or ASCC2 KO cells using MTS assay (mean ± S.D.; n=5 biological replicates). (e-f) Sensitivity of WT and ASCC2 KO cells to MMS (e) or camptothecin (f) was assessed by clonogenic survival assay (n=4 biological replicates; mean ± S.D.). (g-h) WT PC-3 and ASCC2-KO cell sensitivity to camptothecin (g) or bleomycin (h) using the MTS assay (n=5 biological replicates; mean ± S.D.). (i) Images of WT or ASCC2 KO cells expressing the indicated vectors after MMS exposure. (j) Quantitation of (i) (n=2 independent experiments; mean ± S.D.). (k) WT or ASCC2-KO cells expressing indicated vectors were assessed for sensitivity to MMS using the MTS assay (n=5 technical replicates; mean ±S.D.). Scale bar, 10 μm.', 'hash': '7b671aef7f458a6ec23a6f39a26503458a1881f8ddb7d56506425b8e668d16f2'}, {'image_id': 'nihms-910979-f0006', 'image_file_name': 'nihms-910979-f0006.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0006.jpg', 'caption': 'Subcellular localization of ASCC2 and other alkylation repair factors.(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from Figure 1c (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': '372e42172ba563db000ac08103cdc6febe655893f8aa73dfbeba51afb7d89661'}, {'image_id': 'nihms-910979-f0001', 'image_file_name': 'nihms-910979-f0001.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0001.jpg', 'caption': 'The ASCC complex forms foci upon alkylation damage.(a) Images of ASCC3 and pH2A.X immunofluorescence after treatment with damaging agents. (b) ASCC3 foci quantitation (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (c) PLA images in control or MMS-treated cells using 1meA and ASCC3 antibodies (n=3 biological replicates). (d) Immunofluorescence of HA-ASCC2 expressing cells treated with MMS. (e) Quantitation of MMS-induced co-localizations of HA-ASCC2 foci (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': '516a069547a57fdefbf32b3c80bc37033962e13b423ecb6ea3b96de99b0072d1'}, {'image_id': 'nihms-910979-f0008', 'image_file_name': 'nihms-910979-f0008.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0008.jpg', 'caption': 'Functional interactions of the ASCC complex with other signaling pathways.Immunofluorescence images of U2OS cells treated with MMS in the presence of spliceosomal inhibitor PLA-B (a) (100 nM; n=3 biological replicates; mean ± S.D.), the RNA Pol II inhibitor DRB (b) (100 μM; n=3 biological replicates; mean ± S.D.), or the indicated damage signaling kinase inhibitor (c) (n=2 biological replicates; mean). Numbers indicate the percent of cells expressing five or more ASCC3 foci. (d) Immunofluorescence of HA-ASCC2 and FK2 in cells after MMS (n=3 biological replicates; mean ± S.D.). (e) His-ASCC2 was purified on Ni-NTA, separated on a 10% SDS-PAGE gel, and analyzed by Coomassie blue staining (n=2 independent experiments). (f) Immunofluorescence of HA-ASCC2 cells and K63-ubiquitin (top) or K48-ubiquitin (bottom) after MMS treatment (n=2 independent experiments). Scale bars, 10 μm.', 'hash': 'ab8a3da80ed2ae0cf9f5f752fc8278b450947af0dfa81cf374790e1ad70c556b'}, {'image_id': 'nihms-910979-f0002', 'image_file_name': 'nihms-910979-f0002.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0002.jpg', 'caption': 'ASCC2 binds to K63-linked ubiquitin chains via its CUE domain.(a) ASCC2 sequence alignment. (b) Structure of the ASCC2 CUE domain (PDB ID: 2DI0; grey) overlayed with the Vps9 CUE:ubiquitin complex (PDB ID: 1P3Q). (c) His-ASCC2 was immobilized and assessed for binding to K48-Ub2–7 (left) or K63-Ub2–7. ALKBH3 and gp78-CUE served as controls. Bound material was analyzed by Western blot or Coomassie Blue (CBB) (n=3 independent experiments). (d) ITC was performed with K63-Ub2 and His-ASCC2 or the L506A mutant (n=1 independent experiment; mean ± S.E.). (g) Immunofluorescence images of MMS-induced foci in cells expressing various forms of HA-ASCC2. Numbers indicate the percentage of cells expressing ten or more HA-ASCC2 foci (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': 'f8963fcf41cec6681ac9ed0f2e46cee047cb861b8485f3be04bb41fa2ccf5515'}, {'image_id': 'nihms-910979-f0005', 'image_file_name': 'nihms-910979-f0005.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0005.jpg', 'caption': 'The ASCC complex forms foci upon alkylation damage.(a) ASCC3 KO cells were generated using CRISPR/Cas9 technology. Lysates were analyzed by Western blotting (n=2 independent experiments). Clone #10 was verified to be a knockout by deep sequencing. (b) Images of U2OS parental cells or ASCC3-KO cells after MMS (n=3 biological replicates). (c) Immunofluorescence of U2OS cells after exposure to γ-irradiation (IR; 5 Gy) or UV (25 J/m2) (n=3 biological replicates). (d) Images of U2OS cells after treatment with the alkylating agents busulfan (4 mM), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; 100 μM), or temozolomide (TMZ; 1.0 mM) (n=2 biological replicates). Numbers indicate the mean percent of cells expressing five or more foci. (e) Immunofluorescence of HA-ASCC2 expressing cells after exposure to the indicated damaging agents (n=3 biological replicates). Scale bars, 10 μm. For gel source data, see Supplementary Figure 1.', 'hash': '1ecf6abce6d27cff30f2f6823337e325fb811bda1e074cfa9d780911a020a0bc'}, {'image_id': 'nihms-910979-f0014', 'image_file_name': 'nihms-910979-f0014.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0014.jpg', 'caption': 'Functional characterization of RNF113A.(a) Schematic of human RNF113A and its domain structure. The three deletion constructs used for localization analysis are also shown. (b) Images of cells expressing WT or the indicated HA-RNF113A deletion constructs. Scale bar, 10 μm. Quantitation of co-localization between each RNF113A construct and PRP8 is shown on the right (n=3 biological replicates; mean ± S.D.). (c) 293T cells expressing His-ubiquitin were transduced with control or RNF113A-targeting shRNAs and treated with MMS. Ubiquitinated proteins were isolated by Ni-NTA under denaturing conditions and Western blotted as shown. Input lysates were also analyzed as indicated. SF3B3, another ubiquitinated spliceosomal protein, was used as a control (n=3 independent experiments). (d) Cells expressing the indicated HA-vectors were treated with MMS as in (c). Lysates were then used for ubiquitin pulldown assays using GST-ASCC2, then blotted as shown. Input lysates were also analyzed as indicated (n=2 independent experiments). (e) His-NΔ-BRR2 was purified from Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (left). This was then used as a substrate for ubiquitination assays using HA-Ub and wildtype (WT) or a RING-deletion (ΔRING) RNF113A (n=2 independent experiments). (f) Western blot analysis of U2OS cells expressing the indicated shRNAs used for immunofluorescence analysis in Figure 4F (n=2 independent experiments). (g) Quantitation of Figure 4F (n=3 biological replicates; mean ± S.D.; two-tailed t-test, # = p < 0.001). (h) MMS sensitivity of PC-3 cells expressing the indicated shRNAs was determined by MTS assay (n=5 technical replicates; mean ± S.D.).', 'hash': '912acb951e1fe59686b4784725aff4d2c96e3c8b8572d1b4e9d00408b9735d22'}, {'image_id': 'nihms-910979-f0013', 'image_file_name': 'nihms-910979-f0013.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0013.jpg', 'caption': 'Characterization of the E3 ubiquitin ligase activity of RNF113A and TTDN1.(a) TAP-RNF113A and the I264A RING finger mutant were stably expressed in HeLa-S cells and purified using anti-Flag resin. The eluted proteins were then analyzed by silver staining after SDS-PAGE (n=3 independent experiments). (b) Ubiquitin ligase assays using E1, E2 (UbcH5c plus Ubc13/MMS2; 50 nM each), and wildtype or I264A RNF113A. Reactions were analyzed by Western blot (n=3 independent experiments). (c) MMS sensitivity of lymphoblasts from two X-TTD patients in comparison to an unaffected individual (n=5 biological replicates; mean ± S.D.). (d) U2OS cells expressing the indicated combination of shRNA and RNF113A rescue vector were assessed for MMS sensitivity using MTS assay (n=5 technical replicates; mean ± S.D.). (e) Whole cell lysates of control or X-TTD lymphoblasts expressing indicated vectors after selection (n=2 independent experiments). (f) Immunofluorescence analysis of U2OS cells expressing the indicated shRNAs after MMS treatment. Western blot (n=2 independent experiments) from the same cells is shown on the bottom, as is the quantification of ASCC3 foci (n=3 biological replicates; mean ± S.D.).', 'hash': 'df5349c6a10293efaff3516b5f0a3ac01a852fd9a9e8e9ec523d3eb3b22989dd'}, {'image_id': 'nihms-910979-f0004', 'image_file_name': 'nihms-910979-f0004.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0004.jpg', 'caption': 'RNF113A ubiquitination recruits the ASCC complex.(a) MMS-induced foci in U2OS cells expressing indicated shRNAs (n=3 technical replicates; mean). (b) Ubiquitin ligase assays using E1, E2 (UBC13/MMS2; 250 nM), and Flag-RNF113A. K63R ubiquitin was substituted as shown (n=2 independent experiments). (c) Images of X-TTD or control lymphoblasts expressing the indicated vectors after MMS (n=3 technical replicates; mean). (d). ASCC2 interactome analysis. UBC13 substrates were previously described19. (e) HA-RNF113A deletions were immunoprecipitated to analyze BRR2 interaction. (n=3 independent experiments). (f) Images of U2OS cells expressing indicated shRNAs. Numbers indicate the percentage of cells expressing at least five (c) or ten (a) foci. Scale bars, 10 μm.', 'hash': '69239f011df44323addea37d6fc7933ee1710a20298dda42b5f751ffc1e92d13'}, {'image_id': 'nihms-910979-f0003', 'image_file_name': 'nihms-910979-f0003.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0003.jpg', 'caption': 'ASCC2 is critical for ASCC3-ALKBH3 recruitment and alkylation resistance.(a) MMS-induced ASCC3 foci were assessed in WT and ASCC2-KO cells. (b) Quantitation of (a) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (c) HA-ALKBH3 foci were assessed as in (a). Numbers indicate the percentage of cells expressing five or more foci (n=2 biological replicates; mean). (d) 1meA quantitation in WT or ASCC2-KO cells after MMS treatment (n=3 biological replicates; mean ± S.D.). (e) Images of WT or ASCC2-KO cells expressing indicated vectors upon MMS. (f) Quantitation of (e) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001, # = p < 0.05). Scale bars, 10 μm.', 'hash': '3599bd784d2637a3063f11f072e100d4fec53adbfe7740ae7824ab60ba608bf7'}, {'image_id': 'nihms-910979-f0012', 'image_file_name': 'nihms-910979-f0012.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0012.jpg', 'caption': 'Identification of the RNF113A E3 ligase.(a) Whole cell lysates of U2OS cells infected with the indicated shRNAs were analyzed by Western blot. SHPRH was used as a loading control (n=1 independent experiment). (b) Immunofluorescence images of MMS-induced HA-ASCC2 foci in cells expressing the indicated shRNAs. (c) HA-ASCC2 foci quantitation from (b) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (d) Compilation of E3 ligase shRNA screen results. For each candidate, U2OS cells were transduced with HA-ASCC2 and an E3 targeting shRNA. MMS-induced HA-ASCC2 foci formation was analyzed by immunofluorescence. Results were normalized to a scrambled shRNA (normalized score = 100). UBC13 denotes the positive control (purple). Results of three different shRNA to RNF113A are indicated in red (n=1 independent experiment for each shRNA). (e) Whole cell lysates of U2OS cells infected with the indicated shRNAs were analyzed by Western blot. Asterisk (*) indicates a non-specific band in the RNF113A blot (n=2 independent experiments). (f) Localization of Flag-ASCC2 and HA-RNF113A after MMS treatment (n=3 biological replicates). (g) Immunofluorescence of cells expressing Flag-RNF113A without MMS treatment (n=3 biological replicates).', 'hash': 'b570592437196db6baef164d931603c1d57ea5d1392567174cd2ca280e39d210'}]
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{'nihms-910979-f0001': ['Previous studies established that the dealkylating enzyme, ALKBH3, functions in concert with the ASCC helicase complex7. We tested the subcellular localization of the catalytic subunit, ASCC3 upon exposure to various DNA damaging agents. Endogenous ASCC3 formed nuclear foci upon treatment of U2OS cells with the alkylating agent, methyl methanesulphonate (MMS; <xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1A</xref>). Knockout of ASCC3 abrogated these foci (). Knockout of ASCC3 abrogated these foci (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1A and 1B</xref>). Strikingly, other types of DNA damaging agents did not significantly induce ASCC3 foci (). Strikingly, other types of DNA damaging agents did not significantly induce ASCC3 foci (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1A and 1B</xref>; ; <xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1C</xref>), although these genotoxins induced pH2A.X foci, indicative of DNA damage. ASCC3 foci were also observed with other alkylating agents used clinically in the treatment of various tumors), although these genotoxins induced pH2A.X foci, indicative of DNA damage. ASCC3 foci were also observed with other alkylating agents used clinically in the treatment of various tumors8 (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1D</xref>). The ASCC complex subunit ASCC2 also formed foci specifically after treatment with MMS (). The ASCC complex subunit ASCC2 also formed foci specifically after treatment with MMS (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1E</xref>). These foci were largely limited to G1/early S-phase of the cell cycle (). These foci were largely limited to G1/early S-phase of the cell cycle (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2A</xref>). Consistent with their known physical association). Consistent with their known physical association7,9, HA-ASCC2 co-localized with ASCC3 upon MMS treatment, as did the dealkylase ALKBH3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2B</xref>).).', 'To ascertain that the ASCC complex is recruited to regions of the nucleus that have alkylation damage, we performed a proximity ligation assay (PLA). We found that a specific nuclear PLA signal between 1-methyladenosine (1-meA) and ASCC3 is induced upon MMS damage (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1C</xref> and and <xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2C</xref>). The dealkylase ALKBH2 also formed foci that co-localized partially with ASCC3 (). The dealkylase ALKBH2 also formed foci that co-localized partially with ASCC3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2D and 2E</xref>). Conversely, two other alkylation repair factors, methylguanine methyltransferase (MGMT) and alkyladenine glycosylase (AAG), showed minimal co-localization with ASCC3 (). Conversely, two other alkylation repair factors, methylguanine methyltransferase (MGMT) and alkyladenine glycosylase (AAG), showed minimal co-localization with ASCC3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2D and 2E</xref>))', '(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from <xref rid="nihms-910979-f0001" ref-type="fig">Figure 1c</xref> (n=3 biological replicates; mean ± S.D.; two-tailed (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.'], 'nihms-910979-f0007': ['ASCC foci did not co-localize with pH2A.X or 53BP1, demonstrating that they are distinct from double-stranded break (DSB)-induced foci (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3A</xref>). These foci were also distinct from GFP-PCNA or BMI-1 (). These foci were also distinct from GFP-PCNA or BMI-1 (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3B</xref>). We took an unbiased proteomic approach to identify the factors associated with ASCC foci in response to alkylation damage using tandem affinity purification (TAP) (). We took an unbiased proteomic approach to identify the factors associated with ASCC foci in response to alkylation damage using tandem affinity purification (TAP) (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3C</xref>). Mass spectrometric analysis of ASCC2-associated proteins revealed the constitutive association of ASCC3 and ASCC1 (). Mass spectrometric analysis of ASCC2-associated proteins revealed the constitutive association of ASCC3 and ASCC1 (Supplementary Table 1). ASCC2 also associated with many spliceosome components and basal transcription factors (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3D</xref> and and Supplementary Table 1). These factors, including BRR2, PRP8, and TFII-I had 2–3 fold higher total peptide numbers from cells exposed to MMS, suggesting an increased association with the ASCC complex in response to alkylation-induced damage. Focused immunofluorescence studies revealed that ASCC components co-localized with BRR2 and PRP8 upon alkylation damage (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1D-E</xref>). Furthermore, ASCC foci co-localized with elongating (Ser2 phosphorylated) RNA polymerase II, but not other transcription-associated nuclear bodies, such as paraspeckles (). Furthermore, ASCC foci co-localized with elongating (Ser2 phosphorylated) RNA polymerase II, but not other transcription-associated nuclear bodies, such as paraspeckles (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3E-F</xref>). Consistently, RNase treatment prior to processing for immunofluorescence significantly reduced ASCC3 foci formation (). Consistently, RNase treatment prior to processing for immunofluorescence significantly reduced ASCC3 foci formation (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3G</xref>). Purified ASCC3 bound to ssRNA ). Purified ASCC3 bound to ssRNA in vitro (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3H</xref>). Chemical inhibition of transcription or splicing during alkylation damage also reduced ASCC3 foci (). Chemical inhibition of transcription or splicing during alkylation damage also reduced ASCC3 foci (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4A-B</xref>).).', 'To uncover the relevant RNF113A substrate, we combined our initial proteomics screen (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3D</xref>) with a second screen for proteins that interact preferentially with WT ASCC2 relative to the L506A mutant () with a second screen for proteins that interact preferentially with WT ASCC2 relative to the L506A mutant (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4D</xref> and and Supplementary Table 3). Of the remaining putative ubiquitinated substrates, eight have been shown to be ubiquitinated by UBC1315. Of these, BRR2 was the most obvious candidate, as it co-localized with RNF113A and ASCC components. Indeed, BRR2 co-immunoprecipitated with RNF113A in a manner dependent upon the N-terminal domain of RNF113A (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4E</xref>). Deletion analysis revealed that the RNF113A N-terminus was also critical for its co-localization with PRP8 (). Deletion analysis revealed that the RNF113A N-terminus was also critical for its co-localization with PRP8 (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10A-B</xref>). Ubiquitin conjugation of BRR2 was significantly reduced upon loss of RNF113A (). Ubiquitin conjugation of BRR2 was significantly reduced upon loss of RNF113A (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10C</xref>). Furthermore, RNF113A promoted BRR2 binding to ASCC2, which was dependent on its RING domain (). Furthermore, RNF113A promoted BRR2 binding to ASCC2, which was dependent on its RING domain (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10D</xref>). Recombinant BRR2 was ubiquitinated ). Recombinant BRR2 was ubiquitinated in vitro by RNF113A, also in a manner dependent on its RING domain (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10E</xref>). Knockdown of BRR2, or its partner PRP8). Knockdown of BRR2, or its partner PRP816, significantly reduced ASCC3 foci formation upon MMS damage (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10F-G</xref> and and <xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4F</xref>). Consistently, loss of BRR2 increased sensitivity to MMS (). Consistently, loss of BRR2 increased sensitivity to MMS (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10H</xref>). Thus, BRR2 likely represents at least one physiologic substrate for RNF113A in this alkylation repair pathway.). Thus, BRR2 likely represents at least one physiologic substrate for RNF113A in this alkylation repair pathway.'], 'nihms-910979-f0008': ['While recruitment of certain repair complexes is dependent on specific upstream signaling kinases1–3, inhibition of ATM moderately increased ASCC3 foci formation, and ATR inhibition had no impact (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4C</xref>). We found that HA-ASCC2 foci co-localized with polyubiquitin, suggesting that ubiquitin signaling may recruit this repair complex (). We found that HA-ASCC2 foci co-localized with polyubiquitin, suggesting that ubiquitin signaling may recruit this repair complex (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4D</xref>). Analysis of the ASCC2 protein sequence revealed a highly conserved CUE domain (residues 467–509), which belongs to the ubiquitin binding domain superfamily). Analysis of the ASCC2 protein sequence revealed a highly conserved CUE domain (residues 467–509), which belongs to the ubiquitin binding domain superfamily10 (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2A</xref>). A deposited but unpublished NMR structure of the ASCC2 CUE domain (PDB ID: 2DI0) was used to model its interaction with ubiquitin in comparison to another CUE domain from Vps9 (). A deposited but unpublished NMR structure of the ASCC2 CUE domain (PDB ID: 2DI0) was used to model its interaction with ubiquitin in comparison to another CUE domain from Vps9 (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2B</xref>). While Vps9 CUE binds to ubiquitin as a dimer). While Vps9 CUE binds to ubiquitin as a dimer11, our model predicts ubiquitin binding by a monomeric form of the ASCC2 CUE. His-tagged ASCC2 (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4E</xref>) bound K63- but not K48-linked ubiquitin chains () bound K63- but not K48-linked ubiquitin chains (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2C</xref>). Furthermore, ASCC2 co-localized with K63- but not K48-linked ubiquitin foci upon MMS damage (). Furthermore, ASCC2 co-localized with K63- but not K48-linked ubiquitin foci upon MMS damage (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4F</xref>). The minimal domain of ASCC2 for ubiquitin binding ). The minimal domain of ASCC2 for ubiquitin binding in vitro was comprised of residues 457–525 (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5A-5D</xref>). However, the presence of an additional conserved region adjacent to the CUE domain was necessary for specific binding to K63-linked ubiquitin (). However, the presence of an additional conserved region adjacent to the CUE domain was necessary for specific binding to K63-linked ubiquitin (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5A-5D</xref>).).'], 'nihms-910979-f0009': ['We introduced point mutations in the ASCC2 CUE domain at residues predicted to be critical for ubiquitin recognition (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5E</xref>). The mutations L506A and LL478–9AA abrogated ubiquitin binding ). The mutations L506A and LL478–9AA abrogated ubiquitin binding in vitro, while another, P498A, bound to K63-Ub similar to wild type (WT) ASCC2 (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5F</xref>). Isothermal titration calorimetry (ITC) experiments demonstrated that WT ASCC2 bound K63-linked di-ubiquitin chains with a ). Isothermal titration calorimetry (ITC) experiments demonstrated that WT ASCC2 bound K63-linked di-ubiquitin chains with a Kd of 10.1 μM, which is similar to other CUE domains12. In contrast, the L506A mutant showed no detectable binding (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2D</xref>). Notably, ASCC2 mutants that abrogate ubiquitin binding showed significantly reduced foci formation upon MMS treatment (). Notably, ASCC2 mutants that abrogate ubiquitin binding showed significantly reduced foci formation upon MMS treatment (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2E</xref>).).'], 'nihms-910979-f0010': ['We reasoned that ASCC2 acts as an intermediary subunit to recruit other components of the ASCC-ALKBH3 complex. Thus, we generated ASCC2 knockout cells using CRISPR/Cas9 (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6A</xref>). Two independent ASCC2 knockout clones showed a significant reduction in ASCC3 foci formation upon MMS treatment (). Two independent ASCC2 knockout clones showed a significant reduction in ASCC3 foci formation upon MMS treatment (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3A-B</xref>). This reduction was not due to a change in the population of cells in G1 (). This reduction was not due to a change in the population of cells in G1 (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6B</xref>). HA-ALKBH3 and HA-ALKBH2 foci were also diminished in the mutant cells, albeit more modestly for HA-ALKBH2 (). HA-ALKBH3 and HA-ALKBH2 foci were also diminished in the mutant cells, albeit more modestly for HA-ALKBH2 (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3C</xref> and and <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6C</xref>). Consistent with a role in the recruitment of these factors, ASCC2-deficient PC-3 cells were hypersensitive to MMS, but not to camptothecin or bleomycin. (Extended Data Fig. D-H). DNA alkylated lesion repair kinetics was also significantly slower in ASCC2 knockout cells (). Consistent with a role in the recruitment of these factors, ASCC2-deficient PC-3 cells were hypersensitive to MMS, but not to camptothecin or bleomycin. (Extended Data Fig. D-H). DNA alkylated lesion repair kinetics was also significantly slower in ASCC2 knockout cells (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3D</xref>).).', '(a) Whole cell lysates from <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Figure 6i</xref> (left) (left) <xref rid="nihms-910979-f0003" ref-type="fig">Figure 3e</xref> (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (b) Immunoprecipitation of HA-ASCC2 or HA-ASCC2 L506A was performed and analyzed by Western blot as shown (n=2 biological replicates). (c) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to full-length (FL) His-ASCC3. (d) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to N-terminally deleted His-ASCC3 (His-ASCC3-ΔN) (n=2 independent experiments). (e) Flag-ALKBH3 was immobilized and tested for binding to His-ASCC2, with His-ASCC3-C (C-terminus of ASCC3) serving as a positive control (n=2 independent experiments). (f) ASCC-ALKBH3 complex model.'], 'nihms-910979-f0003': ['Next, we reconstituted ASCC2-KO cells with WT and mutant versions of ASCC2. WT ASCC2, but not the L506A CUE mutant, restored MMS-induced ASCC3 and HA-ALKBH3 foci formation (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3E-F</xref> and and <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6I-J</xref> and and <xref rid="nihms-910979-f0011" ref-type="fig">7A</xref>). Similarly, WT, but not L506A ASCC2, rescued MMS sensitivity of ASCC2 knockout cells (). Similarly, WT, but not L506A ASCC2, rescued MMS sensitivity of ASCC2 knockout cells (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6K</xref>). WT and L506A ASCC2 equally co-immunoprecipitated ASCC3 (). WT and L506A ASCC2 equally co-immunoprecipitated ASCC3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7B</xref>). Indeed His-ASCC3 bound to immobilized Flag-ASCC2 and Flag-ALKBH3, although binding with ALKBH3 appeared weaker (). Indeed His-ASCC3 bound to immobilized Flag-ASCC2 and Flag-ALKBH3, although binding with ALKBH3 appeared weaker (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7C</xref>). Deletion of the ASCC3 N-terminus abrogated its ability to bind ASCC2, while retaining ALKBH3 binding (). Deletion of the ASCC3 N-terminus abrogated its ability to bind ASCC2, while retaining ALKBH3 binding (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7D</xref>). Conversely, ASCC2 did not interact with recombinant ALKBH3 (). Conversely, ASCC2 did not interact with recombinant ALKBH3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7E</xref>). ASCC2 therefore appears to bridge ASCC3 and K63-linked ubiquitin chains, while ALKBH3 is indirectly recruited by ASCC2 through its interaction with ASCC3 (). ASCC2 therefore appears to bridge ASCC3 and K63-linked ubiquitin chains, while ALKBH3 is indirectly recruited by ASCC2 through its interaction with ASCC3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7F</xref>).).'], 'nihms-910979-f0012': ['UBC13, a major E2 ubiquitin ligase responsible for formation of K63-linked ubiquitin chains, has been implicated in DNA damage response pathways13–15. Knockdown of UBC13 reduced 53BP1 foci, and also attenuated MMS-induced HA-ASCC2 foci (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8A-8C</xref>). However, knockdown of RNF8 or RNF168, two E3 ligases involved in the double-stranded break repair). However, knockdown of RNF8 or RNF168, two E3 ligases involved in the double-stranded break repair1, did not affect HA-ASCC2 foci formation (data not shown), suggesting that a distinct E3 ligase functions in the alkylation pathway. To identify this E3 ligase, we performed a screen using a custom library of shRNAs that target UBC13-interacting E3 ligases or other ligases implicated in DNA repair (Supplementary Table 2). The screen identified RNF113A as a potential candidate, with three distinct shRNAs reducing HA-ASCC2 foci to UBC13 knockdown levels (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8D</xref>). We confirmed that these shRNAs attenuated both RNF113A protein levels and HA-ASCC2 foci formation (). We confirmed that these shRNAs attenuated both RNF113A protein levels and HA-ASCC2 foci formation (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8E</xref> and and <xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4A</xref>). Importantly, MMS-induced ASCC2 foci co-localized with RNF113A (). Importantly, MMS-induced ASCC2 foci co-localized with RNF113A (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8F</xref>). In the absence of damage, RNF113A co-localized with PRP8 and BRR2 (). In the absence of damage, RNF113A co-localized with PRP8 and BRR2 (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8G</xref>), consistent with previous studies suggesting it nominally serves as a spliceosome component), consistent with previous studies suggesting it nominally serves as a spliceosome component16.'], 'nihms-910979-f0013': ['We purified Flag-tagged RNF113A from HeLa-S cells to analyze its E3 ligase activity (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9A</xref>). RNF113A exhibited robust E3 activity ). RNF113A exhibited robust E3 activity in vitro, which was significantly reduced with a RING-finger point mutation (I264A; <xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9B</xref>). Use of K63R ubiquitin abrogated chain elongation, suggesting that RNF113A may function to promote the E2 activity of UBC13 (). Use of K63R ubiquitin abrogated chain elongation, suggesting that RNF113A may function to promote the E2 activity of UBC13 (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4B</xref>). A recent study identified a nonsense mutation (Q301*) in RNF113A in two related individuals suffering from X-linked trichothiodystrophy). A recent study identified a nonsense mutation (Q301*) in RNF113A in two related individuals suffering from X-linked trichothiodystrophy17 (X-TTD). While most TTD patient cells are hypersensitive to UV damage, X-TTD cells do not have this phenotype17. Lymphoblastoid cell lines obtained from these two patients were hypersensitive to MMS, as was an RNF113A knockdown cell line (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9C-D</xref>). Strikingly, X-TTD cells had significantly reduced ASCC3 foci formation (). Strikingly, X-TTD cells had significantly reduced ASCC3 foci formation (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4C</xref> and and <xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9E</xref>). Reconstitution of these patient cells with WT RNF113A rescued ASCC3 foci formation, while the I264A mutant gave a partial rescue, possibly due to the small degree of remaining E3 ligase activity. Loss of TTDN1, another TTD-associated gene). Reconstitution of these patient cells with WT RNF113A rescued ASCC3 foci formation, while the I264A mutant gave a partial rescue, possibly due to the small degree of remaining E3 ligase activity. Loss of TTDN1, another TTD-associated gene18, also reduced ASCC3 foci formation (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9F</xref>).).'], 'nihms-910979-f0002': ['PyMOL (The PyMOL Molecular Graphics System, Version 1.8.0.5 Schrödinger, LLC.) was used to align the structure of ASCC2 residues 463–525 (PDB ID: 2DI0) with the VSP9 CUE:ubiquitin complex structure (PDB ID: 1P3Q). <xref rid="nihms-910979-f0002" ref-type="fig">Figure 2C</xref> and and <xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Figure 3E</xref> were generated using PyMOL. were generated using PyMOL.'], 'nihms-910979-f0004': ['(a) Schematic of human RNF113A and its domain structure. The three deletion constructs used for localization analysis are also shown. (b) Images of cells expressing WT or the indicated HA-RNF113A deletion constructs. Scale bar, 10 μm. Quantitation of co-localization between each RNF113A construct and PRP8 is shown on the right (n=3 biological replicates; mean ± S.D.). (c) 293T cells expressing His-ubiquitin were transduced with control or RNF113A-targeting shRNAs and treated with MMS. Ubiquitinated proteins were isolated by Ni-NTA under denaturing conditions and Western blotted as shown. Input lysates were also analyzed as indicated. SF3B3, another ubiquitinated spliceosomal protein, was used as a control (n=3 independent experiments). (d) Cells expressing the indicated HA-vectors were treated with MMS as in (c). Lysates were then used for ubiquitin pulldown assays using GST-ASCC2, then blotted as shown. Input lysates were also analyzed as indicated (n=2 independent experiments). (e) His-NΔ-BRR2 was purified from Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (left). This was then used as a substrate for ubiquitination assays using HA-Ub and wildtype (WT) or a RING-deletion (ΔRING) RNF113A (n=2 independent experiments). (f) Western blot analysis of U2OS cells expressing the indicated shRNAs used for immunofluorescence analysis in <xref rid="nihms-910979-f0004" ref-type="fig">Figure 4F</xref> (n=2 independent experiments). (n=2 independent experiments). (g) Quantitation of <xref rid="nihms-910979-f0004" ref-type="fig">Figure 4F</xref> (n=3 biological replicates; mean ± S.D.; two-tailed (n=3 biological replicates; mean ± S.D.; two-tailed t-test, # = p < 0.001). (h) MMS sensitivity of PC-3 cells expressing the indicated shRNAs was determined by MTS assay (n=5 technical replicates; mean ± S.D.).']}
|
A ubiquitin-dependent signaling axis specific for ALKBH-mediated DNA dealkylation repair
| null |
Nature
|
1510819200
|
Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.
|
[
"Animals",
"Chromatography, Liquid",
"Cross-Linking Reagents",
"Humans",
"Pancreatic Elastase",
"Peptides",
"Proteolysis",
"Sf9 Cells",
"Spodoptera",
"Succinimides",
"TATA-Binding Protein Associated Factors",
"Tandem Mass Spectrometry",
"Transcription Factor TFIID",
"Trypsin"
] |
other
|
PMC6458054
| null |
37
|
[
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] |
Nature. 2017 Nov 16; 551(7680):389-393
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NO-CC CODE
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RNF113A ubiquitination recruits the ASCC complex.(a) MMS-induced foci in U2OS cells expressing indicated shRNAs (n=3 technical replicates; mean). (b) Ubiquitin ligase assays using E1, E2 (UBC13/MMS2; 250 nM), and Flag-RNF113A. K63R ubiquitin was substituted as shown (n=2 independent experiments). (c) Images of X-TTD or control lymphoblasts expressing the indicated vectors after MMS (n=3 technical replicates; mean). (d). ASCC2 interactome analysis. UBC13 substrates were previously described19. (e) HA-RNF113A deletions were immunoprecipitated to analyze BRR2 interaction. (n=3 independent experiments). (f) Images of U2OS cells expressing indicated shRNAs. Numbers indicate the percentage of cells expressing at least five (c) or ten (a) foci. Scale bars, 10 μm.
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nihms-910979-f0004
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69239f011df44323addea37d6fc7933ee1710a20298dda42b5f751ffc1e92d13
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
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[{'image_id': 'nihms-910979-f0011', 'image_file_name': 'nihms-910979-f0011.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0011.jpg', 'caption': 'ASCC2 coordinates ASCC-ALKBH3 complex recruitment during alkylation damage.(a) Whole cell lysates from Extended Data Figure 6i (left) Figure 3e (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (b) Immunoprecipitation of HA-ASCC2 or HA-ASCC2 L506A was performed and analyzed by Western blot as shown (n=2 biological replicates). (c) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to full-length (FL) His-ASCC3. (d) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to N-terminally deleted His-ASCC3 (His-ASCC3-ΔN) (n=2 independent experiments). (e) Flag-ALKBH3 was immobilized and tested for binding to His-ASCC2, with His-ASCC3-C (C-terminus of ASCC3) serving as a positive control (n=2 independent experiments). (f) ASCC-ALKBH3 complex model.', 'hash': 'c6803223ad4ee38da12fc28721ed2ab3a779dd788d70c19f1ede6eec8d7d774d'}, {'image_id': 'nihms-910979-f0009', 'image_file_name': 'nihms-910979-f0009.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0009.jpg', 'caption': 'ASCC2 binds specifically to K63-linked ubiquitin chains.(a) and (b) His-ASCC2 or the indicated His-ASCC2 deletions were immobilized on Ni-NTA and assessed for binding to K63-Ub2–7\n(a) or K48-Ub2–7\n(b) (n=3 independent experiments). (c) Schematic of ASCC2 or different ASCC2 deletions and their observed respective binding towards K63-Ub2–7 or K48-Ub2–7. N.D., not determined. (d) Sequence alignment and conservation of residues 373–415 of human ASCC2. (e) Interaction model between ubiquitin and the CUE domain of ASCC2 (PDB ID: 2DI0). The positions of four residues (L478, L479, P498, and L506) are shown. (f) Binding assays were performed with K63-Ub2–7 using WT or the mutants of His-ASCC2 (n=3 independent experiments).', 'hash': '5d592cefb399728c903ede93bc6868bb072619263a46ad8d0d92fa35f2bccd67'}, {'image_id': 'nihms-910979-f0007', 'image_file_name': 'nihms-910979-f0007.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0007.jpg', 'caption': 'Localization and interactions of the ASCC complex.(a) and (b) Images of U2OS or U2OS cells expressing the indicated vectors after MMS treatment (n=3 biological replicates). (c) Silver staining of the Flag-HA-ASCC2 complex purified from HeLa-S nuclear extract separated on 4%−12% SDS-PAGE gel (n=1 independent experiment). (d) Tagged ASCC2 was purified with or without MMS and analyzed by mass spectrometry. Peptide numbers for identified proteins were plotted for each condition. Expanded view is shown on the right (n=1 independent experiment). (e) and (f) Immunofluorescence analysis of U2OS or HA-ASCC2 expressing U2OS cells upon exposure to MMS (n=3 biological replicates). (g) U2OS cells were treated with MMS, and processed for immunofluorescence with or without initial incubation with RNase A (50 nM). Numbers indicate the percent of cells expressing five or more ASCC3 foci (n=3 biological replicates; mean ± S.D.). (h) Biotinylated RNAs (20mer, 35mer, or 50mer) were immobilized and tested for binding to recombinant His-NΔ-ASCC3 (n=2 independent experiments). Scale bars, 10 μm.', 'hash': '91f394672af6b9eb6461f2ad721aa921a59c4addc450653257dc36ff97dedac1'}, {'image_id': 'nihms-910979-f0010', 'image_file_name': 'nihms-910979-f0010.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0010.jpg', 'caption': 'Characterization of ASCC2 KO cells.(a) ASCC2 gene knockouts in U2OS and PC-3 cells were generated using CRISPR/Cas9 technology and verified by deep sequencing. Whole cell lysates of the parental and KO cells were analyzed by Western blotting as shown (n=2 independent experiments). (b) Flow cytometry of WT and ASCC2-KO U2OS cells after MMS treatment to determine cell cycle distribution. (c) Immunofluorescence analysis of HA-ALKBH2 expressing cells after MMS. Numbers indicate the percent of cells expressing five or more HA-ALKBH2 foci (n=3 biological replicates; mean ± S.D.). (d) MMS sensitivity of WT or ASCC2 KO cells using MTS assay (mean ± S.D.; n=5 biological replicates). (e-f) Sensitivity of WT and ASCC2 KO cells to MMS (e) or camptothecin (f) was assessed by clonogenic survival assay (n=4 biological replicates; mean ± S.D.). (g-h) WT PC-3 and ASCC2-KO cell sensitivity to camptothecin (g) or bleomycin (h) using the MTS assay (n=5 biological replicates; mean ± S.D.). (i) Images of WT or ASCC2 KO cells expressing the indicated vectors after MMS exposure. (j) Quantitation of (i) (n=2 independent experiments; mean ± S.D.). (k) WT or ASCC2-KO cells expressing indicated vectors were assessed for sensitivity to MMS using the MTS assay (n=5 technical replicates; mean ±S.D.). Scale bar, 10 μm.', 'hash': '7b671aef7f458a6ec23a6f39a26503458a1881f8ddb7d56506425b8e668d16f2'}, {'image_id': 'nihms-910979-f0006', 'image_file_name': 'nihms-910979-f0006.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0006.jpg', 'caption': 'Subcellular localization of ASCC2 and other alkylation repair factors.(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from Figure 1c (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': '372e42172ba563db000ac08103cdc6febe655893f8aa73dfbeba51afb7d89661'}, {'image_id': 'nihms-910979-f0001', 'image_file_name': 'nihms-910979-f0001.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0001.jpg', 'caption': 'The ASCC complex forms foci upon alkylation damage.(a) Images of ASCC3 and pH2A.X immunofluorescence after treatment with damaging agents. (b) ASCC3 foci quantitation (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (c) PLA images in control or MMS-treated cells using 1meA and ASCC3 antibodies (n=3 biological replicates). (d) Immunofluorescence of HA-ASCC2 expressing cells treated with MMS. (e) Quantitation of MMS-induced co-localizations of HA-ASCC2 foci (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': '516a069547a57fdefbf32b3c80bc37033962e13b423ecb6ea3b96de99b0072d1'}, {'image_id': 'nihms-910979-f0008', 'image_file_name': 'nihms-910979-f0008.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0008.jpg', 'caption': 'Functional interactions of the ASCC complex with other signaling pathways.Immunofluorescence images of U2OS cells treated with MMS in the presence of spliceosomal inhibitor PLA-B (a) (100 nM; n=3 biological replicates; mean ± S.D.), the RNA Pol II inhibitor DRB (b) (100 μM; n=3 biological replicates; mean ± S.D.), or the indicated damage signaling kinase inhibitor (c) (n=2 biological replicates; mean). Numbers indicate the percent of cells expressing five or more ASCC3 foci. (d) Immunofluorescence of HA-ASCC2 and FK2 in cells after MMS (n=3 biological replicates; mean ± S.D.). (e) His-ASCC2 was purified on Ni-NTA, separated on a 10% SDS-PAGE gel, and analyzed by Coomassie blue staining (n=2 independent experiments). (f) Immunofluorescence of HA-ASCC2 cells and K63-ubiquitin (top) or K48-ubiquitin (bottom) after MMS treatment (n=2 independent experiments). Scale bars, 10 μm.', 'hash': 'ab8a3da80ed2ae0cf9f5f752fc8278b450947af0dfa81cf374790e1ad70c556b'}, {'image_id': 'nihms-910979-f0002', 'image_file_name': 'nihms-910979-f0002.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0002.jpg', 'caption': 'ASCC2 binds to K63-linked ubiquitin chains via its CUE domain.(a) ASCC2 sequence alignment. (b) Structure of the ASCC2 CUE domain (PDB ID: 2DI0; grey) overlayed with the Vps9 CUE:ubiquitin complex (PDB ID: 1P3Q). (c) His-ASCC2 was immobilized and assessed for binding to K48-Ub2–7 (left) or K63-Ub2–7. ALKBH3 and gp78-CUE served as controls. Bound material was analyzed by Western blot or Coomassie Blue (CBB) (n=3 independent experiments). (d) ITC was performed with K63-Ub2 and His-ASCC2 or the L506A mutant (n=1 independent experiment; mean ± S.E.). (g) Immunofluorescence images of MMS-induced foci in cells expressing various forms of HA-ASCC2. Numbers indicate the percentage of cells expressing ten or more HA-ASCC2 foci (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.', 'hash': 'f8963fcf41cec6681ac9ed0f2e46cee047cb861b8485f3be04bb41fa2ccf5515'}, {'image_id': 'nihms-910979-f0005', 'image_file_name': 'nihms-910979-f0005.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0005.jpg', 'caption': 'The ASCC complex forms foci upon alkylation damage.(a) ASCC3 KO cells were generated using CRISPR/Cas9 technology. Lysates were analyzed by Western blotting (n=2 independent experiments). Clone #10 was verified to be a knockout by deep sequencing. (b) Images of U2OS parental cells or ASCC3-KO cells after MMS (n=3 biological replicates). (c) Immunofluorescence of U2OS cells after exposure to γ-irradiation (IR; 5 Gy) or UV (25 J/m2) (n=3 biological replicates). (d) Images of U2OS cells after treatment with the alkylating agents busulfan (4 mM), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; 100 μM), or temozolomide (TMZ; 1.0 mM) (n=2 biological replicates). Numbers indicate the mean percent of cells expressing five or more foci. (e) Immunofluorescence of HA-ASCC2 expressing cells after exposure to the indicated damaging agents (n=3 biological replicates). Scale bars, 10 μm. For gel source data, see Supplementary Figure 1.', 'hash': '1ecf6abce6d27cff30f2f6823337e325fb811bda1e074cfa9d780911a020a0bc'}, {'image_id': 'nihms-910979-f0014', 'image_file_name': 'nihms-910979-f0014.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0014.jpg', 'caption': 'Functional characterization of RNF113A.(a) Schematic of human RNF113A and its domain structure. The three deletion constructs used for localization analysis are also shown. (b) Images of cells expressing WT or the indicated HA-RNF113A deletion constructs. Scale bar, 10 μm. Quantitation of co-localization between each RNF113A construct and PRP8 is shown on the right (n=3 biological replicates; mean ± S.D.). (c) 293T cells expressing His-ubiquitin were transduced with control or RNF113A-targeting shRNAs and treated with MMS. Ubiquitinated proteins were isolated by Ni-NTA under denaturing conditions and Western blotted as shown. Input lysates were also analyzed as indicated. SF3B3, another ubiquitinated spliceosomal protein, was used as a control (n=3 independent experiments). (d) Cells expressing the indicated HA-vectors were treated with MMS as in (c). Lysates were then used for ubiquitin pulldown assays using GST-ASCC2, then blotted as shown. Input lysates were also analyzed as indicated (n=2 independent experiments). (e) His-NΔ-BRR2 was purified from Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (left). This was then used as a substrate for ubiquitination assays using HA-Ub and wildtype (WT) or a RING-deletion (ΔRING) RNF113A (n=2 independent experiments). (f) Western blot analysis of U2OS cells expressing the indicated shRNAs used for immunofluorescence analysis in Figure 4F (n=2 independent experiments). (g) Quantitation of Figure 4F (n=3 biological replicates; mean ± S.D.; two-tailed t-test, # = p < 0.001). (h) MMS sensitivity of PC-3 cells expressing the indicated shRNAs was determined by MTS assay (n=5 technical replicates; mean ± S.D.).', 'hash': '912acb951e1fe59686b4784725aff4d2c96e3c8b8572d1b4e9d00408b9735d22'}, {'image_id': 'nihms-910979-f0013', 'image_file_name': 'nihms-910979-f0013.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0013.jpg', 'caption': 'Characterization of the E3 ubiquitin ligase activity of RNF113A and TTDN1.(a) TAP-RNF113A and the I264A RING finger mutant were stably expressed in HeLa-S cells and purified using anti-Flag resin. The eluted proteins were then analyzed by silver staining after SDS-PAGE (n=3 independent experiments). (b) Ubiquitin ligase assays using E1, E2 (UbcH5c plus Ubc13/MMS2; 50 nM each), and wildtype or I264A RNF113A. Reactions were analyzed by Western blot (n=3 independent experiments). (c) MMS sensitivity of lymphoblasts from two X-TTD patients in comparison to an unaffected individual (n=5 biological replicates; mean ± S.D.). (d) U2OS cells expressing the indicated combination of shRNA and RNF113A rescue vector were assessed for MMS sensitivity using MTS assay (n=5 technical replicates; mean ± S.D.). (e) Whole cell lysates of control or X-TTD lymphoblasts expressing indicated vectors after selection (n=2 independent experiments). (f) Immunofluorescence analysis of U2OS cells expressing the indicated shRNAs after MMS treatment. Western blot (n=2 independent experiments) from the same cells is shown on the bottom, as is the quantification of ASCC3 foci (n=3 biological replicates; mean ± S.D.).', 'hash': 'df5349c6a10293efaff3516b5f0a3ac01a852fd9a9e8e9ec523d3eb3b22989dd'}, {'image_id': 'nihms-910979-f0004', 'image_file_name': 'nihms-910979-f0004.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0004.jpg', 'caption': 'RNF113A ubiquitination recruits the ASCC complex.(a) MMS-induced foci in U2OS cells expressing indicated shRNAs (n=3 technical replicates; mean). (b) Ubiquitin ligase assays using E1, E2 (UBC13/MMS2; 250 nM), and Flag-RNF113A. K63R ubiquitin was substituted as shown (n=2 independent experiments). (c) Images of X-TTD or control lymphoblasts expressing the indicated vectors after MMS (n=3 technical replicates; mean). (d). ASCC2 interactome analysis. UBC13 substrates were previously described19. (e) HA-RNF113A deletions were immunoprecipitated to analyze BRR2 interaction. (n=3 independent experiments). (f) Images of U2OS cells expressing indicated shRNAs. Numbers indicate the percentage of cells expressing at least five (c) or ten (a) foci. Scale bars, 10 μm.', 'hash': '69239f011df44323addea37d6fc7933ee1710a20298dda42b5f751ffc1e92d13'}, {'image_id': 'nihms-910979-f0003', 'image_file_name': 'nihms-910979-f0003.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0003.jpg', 'caption': 'ASCC2 is critical for ASCC3-ALKBH3 recruitment and alkylation resistance.(a) MMS-induced ASCC3 foci were assessed in WT and ASCC2-KO cells. (b) Quantitation of (a) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (c) HA-ALKBH3 foci were assessed as in (a). Numbers indicate the percentage of cells expressing five or more foci (n=2 biological replicates; mean). (d) 1meA quantitation in WT or ASCC2-KO cells after MMS treatment (n=3 biological replicates; mean ± S.D.). (e) Images of WT or ASCC2-KO cells expressing indicated vectors upon MMS. (f) Quantitation of (e) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001, # = p < 0.05). Scale bars, 10 μm.', 'hash': '3599bd784d2637a3063f11f072e100d4fec53adbfe7740ae7824ab60ba608bf7'}, {'image_id': 'nihms-910979-f0012', 'image_file_name': 'nihms-910979-f0012.jpg', 'image_path': '../data/media_files/PMC6458054/nihms-910979-f0012.jpg', 'caption': 'Identification of the RNF113A E3 ligase.(a) Whole cell lysates of U2OS cells infected with the indicated shRNAs were analyzed by Western blot. SHPRH was used as a loading control (n=1 independent experiment). (b) Immunofluorescence images of MMS-induced HA-ASCC2 foci in cells expressing the indicated shRNAs. (c) HA-ASCC2 foci quantitation from (b) (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.001). (d) Compilation of E3 ligase shRNA screen results. For each candidate, U2OS cells were transduced with HA-ASCC2 and an E3 targeting shRNA. MMS-induced HA-ASCC2 foci formation was analyzed by immunofluorescence. Results were normalized to a scrambled shRNA (normalized score = 100). UBC13 denotes the positive control (purple). Results of three different shRNA to RNF113A are indicated in red (n=1 independent experiment for each shRNA). (e) Whole cell lysates of U2OS cells infected with the indicated shRNAs were analyzed by Western blot. Asterisk (*) indicates a non-specific band in the RNF113A blot (n=2 independent experiments). (f) Localization of Flag-ASCC2 and HA-RNF113A after MMS treatment (n=3 biological replicates). (g) Immunofluorescence of cells expressing Flag-RNF113A without MMS treatment (n=3 biological replicates).', 'hash': 'b570592437196db6baef164d931603c1d57ea5d1392567174cd2ca280e39d210'}]
|
{'nihms-910979-f0001': ['Previous studies established that the dealkylating enzyme, ALKBH3, functions in concert with the ASCC helicase complex7. We tested the subcellular localization of the catalytic subunit, ASCC3 upon exposure to various DNA damaging agents. Endogenous ASCC3 formed nuclear foci upon treatment of U2OS cells with the alkylating agent, methyl methanesulphonate (MMS; <xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1A</xref>). Knockout of ASCC3 abrogated these foci (). Knockout of ASCC3 abrogated these foci (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1A and 1B</xref>). Strikingly, other types of DNA damaging agents did not significantly induce ASCC3 foci (). Strikingly, other types of DNA damaging agents did not significantly induce ASCC3 foci (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1A and 1B</xref>; ; <xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1C</xref>), although these genotoxins induced pH2A.X foci, indicative of DNA damage. ASCC3 foci were also observed with other alkylating agents used clinically in the treatment of various tumors), although these genotoxins induced pH2A.X foci, indicative of DNA damage. ASCC3 foci were also observed with other alkylating agents used clinically in the treatment of various tumors8 (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1D</xref>). The ASCC complex subunit ASCC2 also formed foci specifically after treatment with MMS (). The ASCC complex subunit ASCC2 also formed foci specifically after treatment with MMS (<xref rid="nihms-910979-f0005" ref-type="fig">Extended Data Fig. 1E</xref>). These foci were largely limited to G1/early S-phase of the cell cycle (). These foci were largely limited to G1/early S-phase of the cell cycle (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2A</xref>). Consistent with their known physical association). Consistent with their known physical association7,9, HA-ASCC2 co-localized with ASCC3 upon MMS treatment, as did the dealkylase ALKBH3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2B</xref>).).', 'To ascertain that the ASCC complex is recruited to regions of the nucleus that have alkylation damage, we performed a proximity ligation assay (PLA). We found that a specific nuclear PLA signal between 1-methyladenosine (1-meA) and ASCC3 is induced upon MMS damage (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1C</xref> and and <xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2C</xref>). The dealkylase ALKBH2 also formed foci that co-localized partially with ASCC3 (). The dealkylase ALKBH2 also formed foci that co-localized partially with ASCC3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2D and 2E</xref>). Conversely, two other alkylation repair factors, methylguanine methyltransferase (MGMT) and alkyladenine glycosylase (AAG), showed minimal co-localization with ASCC3 (). Conversely, two other alkylation repair factors, methylguanine methyltransferase (MGMT) and alkyladenine glycosylase (AAG), showed minimal co-localization with ASCC3 (<xref rid="nihms-910979-f0006" ref-type="fig">Extended Data Fig. 2D and 2E</xref>))', '(a) Flow cytometry analysis of Flag-ASCC2 expressing cells after MMS treatment and Triton X-100 extraction. Numbers indicate the percent of total cells in each quadrant (n=2 independent experiments). (b) Images of cells expressing HA-ASCC2 or HA-ALKBH3 after MMS treatment (n=2 independent experiments). (c) PLA quantitation from <xref rid="nihms-910979-f0001" ref-type="fig">Figure 1c</xref> (n=3 biological replicates; mean ± S.D.; two-tailed (n=3 biological replicates; mean ± S.D.; two-tailed t-test, * = p < 0.005). (d) Immunofluorescence of cells expressing HA-ALKBH2, HA-MGMT, or HA-AAG upon MMS treatment. (e) Quantitation of ASCC3 co-localization from (d) (n=3 biological replicates; mean ± S.D.). Scale bars, 10 μm.'], 'nihms-910979-f0007': ['ASCC foci did not co-localize with pH2A.X or 53BP1, demonstrating that they are distinct from double-stranded break (DSB)-induced foci (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3A</xref>). These foci were also distinct from GFP-PCNA or BMI-1 (). These foci were also distinct from GFP-PCNA or BMI-1 (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3B</xref>). We took an unbiased proteomic approach to identify the factors associated with ASCC foci in response to alkylation damage using tandem affinity purification (TAP) (). We took an unbiased proteomic approach to identify the factors associated with ASCC foci in response to alkylation damage using tandem affinity purification (TAP) (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3C</xref>). Mass spectrometric analysis of ASCC2-associated proteins revealed the constitutive association of ASCC3 and ASCC1 (). Mass spectrometric analysis of ASCC2-associated proteins revealed the constitutive association of ASCC3 and ASCC1 (Supplementary Table 1). ASCC2 also associated with many spliceosome components and basal transcription factors (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3D</xref> and and Supplementary Table 1). These factors, including BRR2, PRP8, and TFII-I had 2–3 fold higher total peptide numbers from cells exposed to MMS, suggesting an increased association with the ASCC complex in response to alkylation-induced damage. Focused immunofluorescence studies revealed that ASCC components co-localized with BRR2 and PRP8 upon alkylation damage (<xref rid="nihms-910979-f0001" ref-type="fig">Fig. 1D-E</xref>). Furthermore, ASCC foci co-localized with elongating (Ser2 phosphorylated) RNA polymerase II, but not other transcription-associated nuclear bodies, such as paraspeckles (). Furthermore, ASCC foci co-localized with elongating (Ser2 phosphorylated) RNA polymerase II, but not other transcription-associated nuclear bodies, such as paraspeckles (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3E-F</xref>). Consistently, RNase treatment prior to processing for immunofluorescence significantly reduced ASCC3 foci formation (). Consistently, RNase treatment prior to processing for immunofluorescence significantly reduced ASCC3 foci formation (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3G</xref>). Purified ASCC3 bound to ssRNA ). Purified ASCC3 bound to ssRNA in vitro (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3H</xref>). Chemical inhibition of transcription or splicing during alkylation damage also reduced ASCC3 foci (). Chemical inhibition of transcription or splicing during alkylation damage also reduced ASCC3 foci (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4A-B</xref>).).', 'To uncover the relevant RNF113A substrate, we combined our initial proteomics screen (<xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Fig. 3D</xref>) with a second screen for proteins that interact preferentially with WT ASCC2 relative to the L506A mutant () with a second screen for proteins that interact preferentially with WT ASCC2 relative to the L506A mutant (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4D</xref> and and Supplementary Table 3). Of the remaining putative ubiquitinated substrates, eight have been shown to be ubiquitinated by UBC1315. Of these, BRR2 was the most obvious candidate, as it co-localized with RNF113A and ASCC components. Indeed, BRR2 co-immunoprecipitated with RNF113A in a manner dependent upon the N-terminal domain of RNF113A (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4E</xref>). Deletion analysis revealed that the RNF113A N-terminus was also critical for its co-localization with PRP8 (). Deletion analysis revealed that the RNF113A N-terminus was also critical for its co-localization with PRP8 (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10A-B</xref>). Ubiquitin conjugation of BRR2 was significantly reduced upon loss of RNF113A (). Ubiquitin conjugation of BRR2 was significantly reduced upon loss of RNF113A (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10C</xref>). Furthermore, RNF113A promoted BRR2 binding to ASCC2, which was dependent on its RING domain (). Furthermore, RNF113A promoted BRR2 binding to ASCC2, which was dependent on its RING domain (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10D</xref>). Recombinant BRR2 was ubiquitinated ). Recombinant BRR2 was ubiquitinated in vitro by RNF113A, also in a manner dependent on its RING domain (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10E</xref>). Knockdown of BRR2, or its partner PRP8). Knockdown of BRR2, or its partner PRP816, significantly reduced ASCC3 foci formation upon MMS damage (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10F-G</xref> and and <xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4F</xref>). Consistently, loss of BRR2 increased sensitivity to MMS (). Consistently, loss of BRR2 increased sensitivity to MMS (<xref rid="nihms-910979-f0014" ref-type="fig">Extended Data Fig. 10H</xref>). Thus, BRR2 likely represents at least one physiologic substrate for RNF113A in this alkylation repair pathway.). Thus, BRR2 likely represents at least one physiologic substrate for RNF113A in this alkylation repair pathway.'], 'nihms-910979-f0008': ['While recruitment of certain repair complexes is dependent on specific upstream signaling kinases1–3, inhibition of ATM moderately increased ASCC3 foci formation, and ATR inhibition had no impact (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4C</xref>). We found that HA-ASCC2 foci co-localized with polyubiquitin, suggesting that ubiquitin signaling may recruit this repair complex (). We found that HA-ASCC2 foci co-localized with polyubiquitin, suggesting that ubiquitin signaling may recruit this repair complex (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4D</xref>). Analysis of the ASCC2 protein sequence revealed a highly conserved CUE domain (residues 467–509), which belongs to the ubiquitin binding domain superfamily). Analysis of the ASCC2 protein sequence revealed a highly conserved CUE domain (residues 467–509), which belongs to the ubiquitin binding domain superfamily10 (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2A</xref>). A deposited but unpublished NMR structure of the ASCC2 CUE domain (PDB ID: 2DI0) was used to model its interaction with ubiquitin in comparison to another CUE domain from Vps9 (). A deposited but unpublished NMR structure of the ASCC2 CUE domain (PDB ID: 2DI0) was used to model its interaction with ubiquitin in comparison to another CUE domain from Vps9 (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2B</xref>). While Vps9 CUE binds to ubiquitin as a dimer). While Vps9 CUE binds to ubiquitin as a dimer11, our model predicts ubiquitin binding by a monomeric form of the ASCC2 CUE. His-tagged ASCC2 (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4E</xref>) bound K63- but not K48-linked ubiquitin chains () bound K63- but not K48-linked ubiquitin chains (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2C</xref>). Furthermore, ASCC2 co-localized with K63- but not K48-linked ubiquitin foci upon MMS damage (). Furthermore, ASCC2 co-localized with K63- but not K48-linked ubiquitin foci upon MMS damage (<xref rid="nihms-910979-f0008" ref-type="fig">Extended Data Fig. 4F</xref>). The minimal domain of ASCC2 for ubiquitin binding ). The minimal domain of ASCC2 for ubiquitin binding in vitro was comprised of residues 457–525 (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5A-5D</xref>). However, the presence of an additional conserved region adjacent to the CUE domain was necessary for specific binding to K63-linked ubiquitin (). However, the presence of an additional conserved region adjacent to the CUE domain was necessary for specific binding to K63-linked ubiquitin (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5A-5D</xref>).).'], 'nihms-910979-f0009': ['We introduced point mutations in the ASCC2 CUE domain at residues predicted to be critical for ubiquitin recognition (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5E</xref>). The mutations L506A and LL478–9AA abrogated ubiquitin binding ). The mutations L506A and LL478–9AA abrogated ubiquitin binding in vitro, while another, P498A, bound to K63-Ub similar to wild type (WT) ASCC2 (<xref rid="nihms-910979-f0009" ref-type="fig">Extended Data Fig. 5F</xref>). Isothermal titration calorimetry (ITC) experiments demonstrated that WT ASCC2 bound K63-linked di-ubiquitin chains with a ). Isothermal titration calorimetry (ITC) experiments demonstrated that WT ASCC2 bound K63-linked di-ubiquitin chains with a Kd of 10.1 μM, which is similar to other CUE domains12. In contrast, the L506A mutant showed no detectable binding (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2D</xref>). Notably, ASCC2 mutants that abrogate ubiquitin binding showed significantly reduced foci formation upon MMS treatment (). Notably, ASCC2 mutants that abrogate ubiquitin binding showed significantly reduced foci formation upon MMS treatment (<xref rid="nihms-910979-f0002" ref-type="fig">Fig. 2E</xref>).).'], 'nihms-910979-f0010': ['We reasoned that ASCC2 acts as an intermediary subunit to recruit other components of the ASCC-ALKBH3 complex. Thus, we generated ASCC2 knockout cells using CRISPR/Cas9 (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6A</xref>). Two independent ASCC2 knockout clones showed a significant reduction in ASCC3 foci formation upon MMS treatment (). Two independent ASCC2 knockout clones showed a significant reduction in ASCC3 foci formation upon MMS treatment (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3A-B</xref>). This reduction was not due to a change in the population of cells in G1 (). This reduction was not due to a change in the population of cells in G1 (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6B</xref>). HA-ALKBH3 and HA-ALKBH2 foci were also diminished in the mutant cells, albeit more modestly for HA-ALKBH2 (). HA-ALKBH3 and HA-ALKBH2 foci were also diminished in the mutant cells, albeit more modestly for HA-ALKBH2 (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3C</xref> and and <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6C</xref>). Consistent with a role in the recruitment of these factors, ASCC2-deficient PC-3 cells were hypersensitive to MMS, but not to camptothecin or bleomycin. (Extended Data Fig. D-H). DNA alkylated lesion repair kinetics was also significantly slower in ASCC2 knockout cells (). Consistent with a role in the recruitment of these factors, ASCC2-deficient PC-3 cells were hypersensitive to MMS, but not to camptothecin or bleomycin. (Extended Data Fig. D-H). DNA alkylated lesion repair kinetics was also significantly slower in ASCC2 knockout cells (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3D</xref>).).', '(a) Whole cell lysates from <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Figure 6i</xref> (left) (left) <xref rid="nihms-910979-f0003" ref-type="fig">Figure 3e</xref> (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (right) and (right) were collected and expression was analyzed by Western blotting (n=2 independent experiments). (b) Immunoprecipitation of HA-ASCC2 or HA-ASCC2 L506A was performed and analyzed by Western blot as shown (n=2 biological replicates). (c) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to full-length (FL) His-ASCC3. (d) Flag-ASCC2 or Flag-ALKBH3 were immobilized and tested for binding to N-terminally deleted His-ASCC3 (His-ASCC3-ΔN) (n=2 independent experiments). (e) Flag-ALKBH3 was immobilized and tested for binding to His-ASCC2, with His-ASCC3-C (C-terminus of ASCC3) serving as a positive control (n=2 independent experiments). (f) ASCC-ALKBH3 complex model.'], 'nihms-910979-f0003': ['Next, we reconstituted ASCC2-KO cells with WT and mutant versions of ASCC2. WT ASCC2, but not the L506A CUE mutant, restored MMS-induced ASCC3 and HA-ALKBH3 foci formation (<xref rid="nihms-910979-f0003" ref-type="fig">Fig. 3E-F</xref> and and <xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6I-J</xref> and and <xref rid="nihms-910979-f0011" ref-type="fig">7A</xref>). Similarly, WT, but not L506A ASCC2, rescued MMS sensitivity of ASCC2 knockout cells (). Similarly, WT, but not L506A ASCC2, rescued MMS sensitivity of ASCC2 knockout cells (<xref rid="nihms-910979-f0010" ref-type="fig">Extended Data Fig. 6K</xref>). WT and L506A ASCC2 equally co-immunoprecipitated ASCC3 (). WT and L506A ASCC2 equally co-immunoprecipitated ASCC3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7B</xref>). Indeed His-ASCC3 bound to immobilized Flag-ASCC2 and Flag-ALKBH3, although binding with ALKBH3 appeared weaker (). Indeed His-ASCC3 bound to immobilized Flag-ASCC2 and Flag-ALKBH3, although binding with ALKBH3 appeared weaker (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7C</xref>). Deletion of the ASCC3 N-terminus abrogated its ability to bind ASCC2, while retaining ALKBH3 binding (). Deletion of the ASCC3 N-terminus abrogated its ability to bind ASCC2, while retaining ALKBH3 binding (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7D</xref>). Conversely, ASCC2 did not interact with recombinant ALKBH3 (). Conversely, ASCC2 did not interact with recombinant ALKBH3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7E</xref>). ASCC2 therefore appears to bridge ASCC3 and K63-linked ubiquitin chains, while ALKBH3 is indirectly recruited by ASCC2 through its interaction with ASCC3 (). ASCC2 therefore appears to bridge ASCC3 and K63-linked ubiquitin chains, while ALKBH3 is indirectly recruited by ASCC2 through its interaction with ASCC3 (<xref rid="nihms-910979-f0011" ref-type="fig">Extended Data Fig. 7F</xref>).).'], 'nihms-910979-f0012': ['UBC13, a major E2 ubiquitin ligase responsible for formation of K63-linked ubiquitin chains, has been implicated in DNA damage response pathways13–15. Knockdown of UBC13 reduced 53BP1 foci, and also attenuated MMS-induced HA-ASCC2 foci (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8A-8C</xref>). However, knockdown of RNF8 or RNF168, two E3 ligases involved in the double-stranded break repair). However, knockdown of RNF8 or RNF168, two E3 ligases involved in the double-stranded break repair1, did not affect HA-ASCC2 foci formation (data not shown), suggesting that a distinct E3 ligase functions in the alkylation pathway. To identify this E3 ligase, we performed a screen using a custom library of shRNAs that target UBC13-interacting E3 ligases or other ligases implicated in DNA repair (Supplementary Table 2). The screen identified RNF113A as a potential candidate, with three distinct shRNAs reducing HA-ASCC2 foci to UBC13 knockdown levels (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8D</xref>). We confirmed that these shRNAs attenuated both RNF113A protein levels and HA-ASCC2 foci formation (). We confirmed that these shRNAs attenuated both RNF113A protein levels and HA-ASCC2 foci formation (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8E</xref> and and <xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4A</xref>). Importantly, MMS-induced ASCC2 foci co-localized with RNF113A (). Importantly, MMS-induced ASCC2 foci co-localized with RNF113A (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8F</xref>). In the absence of damage, RNF113A co-localized with PRP8 and BRR2 (). In the absence of damage, RNF113A co-localized with PRP8 and BRR2 (<xref rid="nihms-910979-f0012" ref-type="fig">Extended Data Fig. 8G</xref>), consistent with previous studies suggesting it nominally serves as a spliceosome component), consistent with previous studies suggesting it nominally serves as a spliceosome component16.'], 'nihms-910979-f0013': ['We purified Flag-tagged RNF113A from HeLa-S cells to analyze its E3 ligase activity (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9A</xref>). RNF113A exhibited robust E3 activity ). RNF113A exhibited robust E3 activity in vitro, which was significantly reduced with a RING-finger point mutation (I264A; <xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9B</xref>). Use of K63R ubiquitin abrogated chain elongation, suggesting that RNF113A may function to promote the E2 activity of UBC13 (). Use of K63R ubiquitin abrogated chain elongation, suggesting that RNF113A may function to promote the E2 activity of UBC13 (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4B</xref>). A recent study identified a nonsense mutation (Q301*) in RNF113A in two related individuals suffering from X-linked trichothiodystrophy). A recent study identified a nonsense mutation (Q301*) in RNF113A in two related individuals suffering from X-linked trichothiodystrophy17 (X-TTD). While most TTD patient cells are hypersensitive to UV damage, X-TTD cells do not have this phenotype17. Lymphoblastoid cell lines obtained from these two patients were hypersensitive to MMS, as was an RNF113A knockdown cell line (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9C-D</xref>). Strikingly, X-TTD cells had significantly reduced ASCC3 foci formation (). Strikingly, X-TTD cells had significantly reduced ASCC3 foci formation (<xref rid="nihms-910979-f0004" ref-type="fig">Fig. 4C</xref> and and <xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9E</xref>). Reconstitution of these patient cells with WT RNF113A rescued ASCC3 foci formation, while the I264A mutant gave a partial rescue, possibly due to the small degree of remaining E3 ligase activity. Loss of TTDN1, another TTD-associated gene). Reconstitution of these patient cells with WT RNF113A rescued ASCC3 foci formation, while the I264A mutant gave a partial rescue, possibly due to the small degree of remaining E3 ligase activity. Loss of TTDN1, another TTD-associated gene18, also reduced ASCC3 foci formation (<xref rid="nihms-910979-f0013" ref-type="fig">Extended Data Fig. 9F</xref>).).'], 'nihms-910979-f0002': ['PyMOL (The PyMOL Molecular Graphics System, Version 1.8.0.5 Schrödinger, LLC.) was used to align the structure of ASCC2 residues 463–525 (PDB ID: 2DI0) with the VSP9 CUE:ubiquitin complex structure (PDB ID: 1P3Q). <xref rid="nihms-910979-f0002" ref-type="fig">Figure 2C</xref> and and <xref rid="nihms-910979-f0007" ref-type="fig">Extended Data Figure 3E</xref> were generated using PyMOL. were generated using PyMOL.'], 'nihms-910979-f0004': ['(a) Schematic of human RNF113A and its domain structure. The three deletion constructs used for localization analysis are also shown. (b) Images of cells expressing WT or the indicated HA-RNF113A deletion constructs. Scale bar, 10 μm. Quantitation of co-localization between each RNF113A construct and PRP8 is shown on the right (n=3 biological replicates; mean ± S.D.). (c) 293T cells expressing His-ubiquitin were transduced with control or RNF113A-targeting shRNAs and treated with MMS. Ubiquitinated proteins were isolated by Ni-NTA under denaturing conditions and Western blotted as shown. Input lysates were also analyzed as indicated. SF3B3, another ubiquitinated spliceosomal protein, was used as a control (n=3 independent experiments). (d) Cells expressing the indicated HA-vectors were treated with MMS as in (c). Lysates were then used for ubiquitin pulldown assays using GST-ASCC2, then blotted as shown. Input lysates were also analyzed as indicated (n=2 independent experiments). (e) His-NΔ-BRR2 was purified from Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (left). This was then used as a substrate for ubiquitination assays using HA-Ub and wildtype (WT) or a RING-deletion (ΔRING) RNF113A (n=2 independent experiments). (f) Western blot analysis of U2OS cells expressing the indicated shRNAs used for immunofluorescence analysis in <xref rid="nihms-910979-f0004" ref-type="fig">Figure 4F</xref> (n=2 independent experiments). (n=2 independent experiments). (g) Quantitation of <xref rid="nihms-910979-f0004" ref-type="fig">Figure 4F</xref> (n=3 biological replicates; mean ± S.D.; two-tailed (n=3 biological replicates; mean ± S.D.; two-tailed t-test, # = p < 0.001). (h) MMS sensitivity of PC-3 cells expressing the indicated shRNAs was determined by MTS assay (n=5 technical replicates; mean ± S.D.).']}
|
A ubiquitin-dependent signaling axis specific for ALKBH-mediated DNA dealkylation repair
| null |
Nature
|
1510819200
|
Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.
|
[
"Animals",
"Chromatography, Liquid",
"Cross-Linking Reagents",
"Humans",
"Pancreatic Elastase",
"Peptides",
"Proteolysis",
"Sf9 Cells",
"Spodoptera",
"Succinimides",
"TATA-Binding Protein Associated Factors",
"Tandem Mass Spectrometry",
"Transcription Factor TFIID",
"Trypsin"
] |
other
|
PMC6458054
| null |
37
|
[
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] |
Nature. 2017 Nov 16; 551(7680):389-393
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Rab1 colocalizes with ILP2 granules and is required for IPC development and ILP2 transport. (A) IPC neurite structure and nuclei were labeled with Ilp2 > mCD8-GFP and Ilp2 > nls-RFP in third instar larval brains. (Top) The white arrows indicate the dendrites of control IPCs (Ilp2-Gal4 > yw), which were missing in Ilp2-Gal4 > Rab1-DN brains. (B) Quantification of IPC cell numbers (based on Ilp2 > nls-RFP-labeled nuclei) indicated a reduction (five to six) in Ilp2-Gal4 > Rab1-DN brains (n = 9 brains; for controls, n = 10). (C and D) ILP2 fluorescence intensities in the brain IPC cell bodies were compared between control (tubGal80ts, Ilp2 > yw, n = 10 brains) and Rab1 inhibition (n = 14 brains) in IPCs for 24 hr (tubGal80ts, Ilp2 > Rab1-DN). The images were taken with the same laser settings and manipulated in the same way. (E) Labeling of Rab1 (Ilp2 > Rab1-YFP), a Golgi marker (Ilp2 > Grasp65-GFP), or an endosome marker (Ilp2 > FYVE-GFP) in combination with ILP2 staining. The left column (bottom magnification) shows the localization of GFP/YFP-tagged proteins as a projection of z-stacks. Rab1- and FYVE-labeled endosomes were localized almost exclusively in the cell bodies, while Grasp65-labeled cis-Golgi distributed in both the IPC cell bodies and the axons. Boxed regions are shown in higher magnification in the right columns. Rab1-YFP, Grasp-GFP, FYVE-GFP, and ILP2 all had punctate localization patterns in IPCs. For examination of colocalization between vesicles/granules, single z optical sections were shown. Bars, 50 μm except in the zoomed-in images in E, where the bar represents 10 μm. Error bars: SEM, **P < 0.01.
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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"fluorescence microscopy",
"bar plot",
"immunoblot"
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[{'image_id': '175fig7', 'image_file_name': '175fig7.jpg', 'image_path': '../data/media_files/PMC4012477/175fig7.jpg', 'caption': 'Rab1 colocalizes with ILP2 granules and is required for IPC development and ILP2 transport. (A) IPC neurite structure and nuclei were labeled with Ilp2 > mCD8-GFP and Ilp2 > nls-RFP in third instar larval brains. (Top) The white arrows indicate the dendrites of control IPCs (Ilp2-Gal4 > yw), which were missing in Ilp2-Gal4 > Rab1-DN brains. (B) Quantification of IPC cell numbers (based on Ilp2 > nls-RFP-labeled nuclei) indicated a reduction (five to six) in Ilp2-Gal4 > Rab1-DN brains (n = 9 brains; for controls, n = 10). (C and D) ILP2 fluorescence intensities in the brain IPC cell bodies were compared between control (tubGal80ts, Ilp2 > yw, n = 10 brains) and Rab1 inhibition (n = 14 brains) in IPCs for 24 hr (tubGal80ts, Ilp2 > Rab1-DN). The images were taken with the same laser settings and manipulated in the same way. (E) Labeling of Rab1 (Ilp2 > Rab1-YFP), a Golgi marker (Ilp2 > Grasp65-GFP), or an endosome marker (Ilp2 > FYVE-GFP) in combination with ILP2 staining. The left column (bottom magnification) shows the localization of GFP/YFP-tagged proteins as a projection of z-stacks. Rab1- and FYVE-labeled endosomes were localized almost exclusively in the cell bodies, while Grasp65-labeled cis-Golgi distributed in both the IPC cell bodies and the axons. Boxed regions are shown in higher magnification in the right columns. Rab1-YFP, Grasp-GFP, FYVE-GFP, and ILP2 all had punctate localization patterns in IPCs. For examination of colocalization between vesicles/granules, single z optical sections were shown. Bars, 50 μm except in the zoomed-in images in E, where the bar represents 10 μm. Error bars: SEM, **P < 0.01.', 'hash': '7d0e9563d0d2d098a4598256969cc42e73cbaab70bdaaf9536ef1bbf88228d37'}, {'image_id': '175fig1', 'image_file_name': '175fig1.jpg', 'image_path': '../data/media_files/PMC4012477/175fig1.jpg', 'caption': 'IPC morphology changes during development. IPC neuronal structures were followed from early larval to adult stages. Developmental time points after embryo deposition are indicated. Green: Ilp2 > mCD8-GFP; red: Ilp2 > nuclear-RFP; blue: DAPI. up: anterior; down: posterior. Shown are maximum z-projections of confocal images taken at 1-μm step size. Flies were raised at 25° throughout development. Bar, 50 μm.', 'hash': 'd27bd66f17bf262b737afc71ca891e73f229208d6a1d35a1b7d52b05ae446157'}, {'image_id': '175fig6', 'image_file_name': '175fig6.jpg', 'image_path': '../data/media_files/PMC4012477/175fig6.jpg', 'caption': 'Rab1 is required in IPCs for normal animal growth and development. (A and B) Adult weights and pupal volume were compared between control flies (Ilp2-Gal4 > yw, n = 153 flies, n = 5 pupae) and flies expressing Rab1-DN in IPCs (Ilp2 > Rab1-DN, n = 138 flies, n = 6 pupae). (C) In a single confocal optical section, IPCs were labeled with anti-ILP2 antibody, and Rab1-expressed cells were labeled with Rab1 > Lamin-GFP. The other IPCs that were not displayed in this optical section also expressed Rab1. Bar, 20 μm. (D and E) Developmental time points of the onset of metamorphosis and adult eclosion were compared between control flies and flies expressing Rab1-DN in IPCs. (F) Combined trehalose and glucose level in the third instar larval hemolymph was compared between control (n = 12 pooled hemolymph samples) and Ilp2-Gal4 > Rab1-DN flies (n = 10 pooled hemolymph samples). Data were represented as mean ± SEM, **P < 0.01.', 'hash': 'a3efca012d039320a71750463f755e21544f8d0929852ce6fa5e730ce4b6ce9f'}, {'image_id': '175fig5', 'image_file_name': '175fig5.jpg', 'image_path': '../data/media_files/PMC4012477/175fig5.jpg', 'caption': 'Unc-104 is localized to axons of IPCs and is required for IPC development and ILP2 secretion. (A) Control (Ilp2 > w1118, VDRC GD line control, #60000) and Ilp2 > unc-104 RNAi (VDRC line #23465) third instar brains were dissected. Ilp2 > mCD8-GFP (green) indicates the whole IPC neural structure. (B and C) ILP2 fluorescence intensities in the brain IPC cell bodies were compared between control (tubGal80ts, Ilp2 > w1118, VDRC GD line control, #60000, n = 9 brains) and unc-104 knockdown (n = 9 brains) in IPCs for 24 hr (tubGal80ts, Ilp2 > unc-104 RNAi). The images were taken with the same laser settings and manipulated in the same way. IPC cell bodies are outlined in the circle in control. (D and E) Localization of Unc-104 in IPCs was examined by expressing exogenous Unc-104-mCherry or Unc-104-GFP. Ilp2 > mCD8-GFP (green) indicates the whole IPC neural structure. Bars, 50 μm. Error bars: SEM, **P < 0.01.', 'hash': 'a93c282c12c9b260a845de7604a2c3a38843444f8660155b67cafd1a24a51578'}, {'image_id': '175fig2', 'image_file_name': '175fig2.jpg', 'image_path': '../data/media_files/PMC4012477/175fig2.jpg', 'caption': 'Capture of IPCs through laser microdissection. (A) Drosophila IPCs viewed by confocal microscopy. Green depicts IPC neuronal processes and cell bodies, and IPC cell nuclei are in red. (B) Schematic describing the cryosections that were chosen for IPC laser capture. (C) GFP-labeled IPCs before and after laser capture. Red boxes outline where IPCs are located and insets show higher magnifications of those regions. Dotted lines demarcate the boundary of the brain. Bars, 50 μm.', 'hash': 'cb32f3d96ee984ba99c7d44c72a2d469fb9033607863aae436c7a146a9396f55'}, {'image_id': '175fig3', 'image_file_name': '175fig3.jpg', 'image_path': '../data/media_files/PMC4012477/175fig3.jpg', 'caption': 'mRNA sequencing of IPCs and control neurons. (A) A 3′ bias of mRNA sequencing reads. Distribution of mRNA-seq reads along Ilp3 mRNA as seen in the UCSC Genome Browser (Karolchik et al. 2003). RefSeq Gene track shows Ilp3 mRNA structure. Wide blue bar: exons; narrow blue bar: 5′ and 3′ untranslated region; blue line: intron. Distribution of mRNA reads from IPC1, IPC2, control 1, and control 2 samples is shown as black bars in individual tracks. (B) RT-PCR validation of laser-captured IPCs. Ilp2 and Ilp5 mRNA levels were assayed in amplified mRNAs from captured IPCs and control neural tissues. The housekeeping gene Ribosomal protein L32 (RpL32) was used as control. (C) Scatter plot of the mRNA-Seq expression data of IPCs and controls. Both the x- and the y-axis represent RPKM in log10 ratio. Red dots highlight IPC-enriched genes. A pseudo-count of 0.01 was added to RPKMs of all genes to avoid errors in log transformation of true. Thus, zero expression genes were represented as dots with expression of −2 in log10 on the plot. (D) Histograms of RPKM levels of genes expressed in controls and IPCs. The x-axis is log10 scale of gene expression measured in RPKM; the y-axis is gene counts in log10 scale. A pseudo-count of 0.01 was added to RPKMs of all genes to avoid errors in log transformation of true. The dashed red line represents RPKM = 5.', 'hash': 'e94f59f03ea44c7a912c06291332d2e7938fed3963cd337d9b73227662ccf178'}, {'image_id': '175fig4', 'image_file_name': '175fig4.jpg', 'image_path': '../data/media_files/PMC4012477/175fig4.jpg', 'caption': 'ILP2 is transported in DCVs along the axonal bundles in IPCs. (A) IPC neuronal structure from a third instar larval brain was labeled with Ilp2->mCD8-GFP. ILP2 localizes to some IPC neuronal projections (white arrow) but not to others (yellow arrows). (B–D) Localization of axonal (Tau) and dendritic (Khc::nod) markers in IPCs. In both figures, anterior (labeled with “A”) is toward the top and posterior (“P”) is toward the bottom. Arrowhead in B labels the portion of IPC axon bundles extending posteriorly from the cell bodies, and white arrow indicates, after the turn, IPC axons projecting anteriorly. Yellow arrows in C indicate the dendritic branches of IPCs. (D) Schematic of IPC polarity. (E) Localization of DCV in IPCs was examined by expressing exogenous ANF-GFP. Higher magnification of boxed region is shown for both ANF-GFP and ILP2 (single z optical section). Bars: 50 μm except in the zoomed-in image in E, where the bar represents 10 μm.', 'hash': '5dbd5ea79888284d85b4568a48ca88406362323c0da9dcc9ad3dc5219138d956'}]
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{'175fig1': ['In preparation for laser dissection, we examined the developmental steps in the formation of IPCs to determine optimal conditions and timing for the dissection. Specification of fly brain IPCs during late embryogenesis has been characterized by several studies (Wang et al. 2007; Miguel-Aliaga et al. 2008; Hwang and Rulifson 2011), while post-specification developmental events such as neuronal morphogenesis of IPCs have been little studied. We followed the development of IPC morphology using Ilp2-Gal4-driven membrane (CD8) GFP (<xref ref-type="fig" rid="175fig1">Figure 1</xref> and and File S1, File S2, File S3, and File S4). At early larval stages, IPCs exist as two symmetrical groups consisting of seven cells in each of the brain hemispheres. Their neuronal processes extend laterally and posteriorly within the brains, with some ending outside the brain, potentially on the aorta and the corpora cardiac compartment of the ring gland (Rulifson et al. 2002) (<xref ref-type="fig" rid="175fig1">Figure 1</xref> and and File S2). At later larval stages, the cell bodies increase in size and their projections extend over greater distances. During pupal stages, the processes that had extended laterally from IPCs during larval stages are gradually dismantled. The processes that initially extended posteriorly from IPCs lengthen and eventually converge into one bundle (<xref ref-type="fig" rid="175fig1">Figure 1</xref> and and File S3). At later pupal stages, the two IPC clusters converge to form one cell group near the midline. During adulthood new processes beneath IPCs are formed. These extend laterally with extensive arborizations. The posterior projection bundle becomes thickened, with extensive arborization at the terminals (<xref ref-type="fig" rid="175fig1">Figure 1</xref> and and File S4). Larval IPCs are necessary for growth control and sugar homeostasis (Rulifson et al. 2002; Haselton and Fridell 2010). The adult IPCs, like larval IPCs, project to the corpora cardiac and to the aorta for ILP release (Rulifson et al. 2002; Kim and Rulifson 2004; Tatar 2004).', 'Using available UAS-RNAi lines, 50 of the 193 IPC-enriched genes were tested for their influence on growth. Transgenes that encode RNAi were engineered to be active specifically in IPCs (Figure S1A). RNAi lines for individual genes from either the Vienna Drosophila RNAi Center (VDRC) or Transgenic RNAi Project (TRiP) collections were crossed with Ilp2-Gal4, and the adult progeny were examined for their weight (Dietzl et al. 2007; Ni et al. 2008) (Figure S1A). The Ilp2 promoter drives gene expression in IPCs starting during late embryogenesis and continuing into adulthood (Rulifson et al. 2002; Slaidina et al. 2009) (<xref ref-type="fig" rid="175fig1">Figure 1</xref>). RNAi inhibition in IPCs of ). RNAi inhibition in IPCs of amon, foxo, Rab26, unc-104, hth, ald, Pkc98E, Vap-33-1, Vha26, and CG13506 had strong effects on adult fly size (>10% reduction) while inhibition of other genes had little or no effect (Figure S1A).'], '175fig2': ['In the third instar, IPCs are clustered in two symmetrically organized groups of seven neurons (<xref ref-type="fig" rid="175fig2">Figure 2A</xref>). The neurons’ structural characteristics and arrangement at this stage make them convenient for identification and laser capture. We chose early third instar larvae as the IPC source for two reasons: (1) the rapid growth during second and third instars is indicative of the need for IPC-secreted ILPs. (2) Neurite structures of IPCs do not change much during the third instar (). The neurons’ structural characteristics and arrangement at this stage make them convenient for identification and laser capture. We chose early third instar larvae as the IPC source for two reasons: (1) the rapid growth during second and third instars is indicative of the need for IPC-secreted ILPs. (2) Neurite structures of IPCs do not change much during the third instar (<xref ref-type="fig" rid="175fig1">Figure 1</xref>), making it more likely that active genes contribute to IPC-regulated ILP production rather than IPC neurite development.), making it more likely that active genes contribute to IPC-regulated ILP production rather than IPC neurite development.', 'Frozen brain sections containing IPC cell bodies were identified using Ilp2-Gal4 > mCD8-GFP and Ilp2-Gal4 > nuclearRFP. During larval stages, the Ilp2-Gal4 driver is expressed at low levels in imaginal discs and at high levels in salivary glands and the 14 brain IPCs. In the brain, expression of the Ilp2-Gal4 drive is not detectable outside IPCs (Brogiolo et al. 2001; Rulifson et al. 2002). The successful capture of IPCs was demonstrated by the absence of GFP-labeled tissue from the residual sections (<xref ref-type="fig" rid="175fig2">Figure 2, B and C</xref>). In total, 46 IPC cell bodies from 10 brains were captured and equally divided into two groups (IPC1 and IPC2). Two samples (control 1 and control 2) of ∼100 non-IPC, non-GFP cells each were collected from the same sections from which IPCs were captured. These adjacent regions of the brain are enriched in neurons that form the superior lateral, superior medial, and ventrolateral protocerebrum, dorsolateral neurons, mushroom body, optic tubercle, and lateral horn.). In total, 46 IPC cell bodies from 10 brains were captured and equally divided into two groups (IPC1 and IPC2). Two samples (control 1 and control 2) of ∼100 non-IPC, non-GFP cells each were collected from the same sections from which IPCs were captured. These adjacent regions of the brain are enriched in neurons that form the superior lateral, superior medial, and ventrolateral protocerebrum, dorsolateral neurons, mushroom body, optic tubercle, and lateral horn.'], '175fig3': ['The small amount of RNA isolated from laser-captured cells necessitated amplification of the RNA sequences while trying to minimally skew relative mRNA abundances. We performed two rounds of RNA amplification from each of the four initial samples. In each round of RNA amplification, poly(A)RNA was reversed-transcribed to cDNA, which was then used as template for T7-based in vitro transcription to produce amplified RNA (Wang 2005). One limitation of this RNA amplification method is the lower 5′ representation of amplified RNA due to inefficient reverse transcription at each round of the amplification step (Baugh et al. 2001; Wang 2005) (<xref ref-type="fig" rid="175fig3">Figure 3A</xref>). Two rounds of amplification may be insufficient for amplifying the low-copy-number mRNA species. Despite these potential problems, this amplification strategy produces a high correlation of gene expression profiles between unamplified RNA and RNA amplified from low-input RNA (500 pg of total RNA) (). Two rounds of amplification may be insufficient for amplifying the low-copy-number mRNA species. Despite these potential problems, this amplification strategy produces a high correlation of gene expression profiles between unamplified RNA and RNA amplified from low-input RNA (500 pg of total RNA) (Van Gelder et al. 1990; Wang et al. 2000; Baugh et al. 2001; Lang et al. 2009). To evaluate the fidelity of the LCM and RNA amplification, we used RT-PCR to verify that the major larval Ilp mRNAs were enriched in amplified samples from captured IPCs, not in laser-dissected control samples (<xref ref-type="fig" rid="175fig3">Figure 3B</xref>).).', 'After aligning the sequencing reads to the reference D. melanogaster genome and transcriptome (dm3/BDGP Release 5 from UCSC genome browser, Table S1; Materials and Methods) (Adams et al. 2000; Fujita et al. 2011), we deduced the presence of transcripts representing 1851 genes that were common to both IPC samples and 2941 genes that were common to both control samples (RPKM ≥ 5.0 for moderate-to-high gene expression level) (<xref ref-type="fig" rid="175fig3">Figure 3, C and D</xref>; ; Table S2). The number of expressed genes (total of 3373 from IPC and control samples) is 56% fewer than the number of genes detectably represented in third instar larval whole-brain mRNA-seq data (7704 genes, or 55% of the total fly genes). The lower transcriptome representation of our laser-dissected samples may be due to (1) the mRNAs from the 46 captured IPC cells and the ∼200 captured cells presumably from a subset of the genes that are expressed in the whole brain and (2) RNA degradation during LCM and/or insufficient mRNA amplification during IPC and control sample preparation.', 'Comparing IPCs to the control samples from adjacent regions, 1419 genes are expressed in both. A total of 432 genes are expressed only in IPC samples (IPC1 and IPC2), and 1522 genes are expressed only in control samples (control 1 and control 2). Among the 1419 shared genes, the majority showed little variance in gene expression level between IPCs and controls; most of them are clustered around the diagonal (<xref ref-type="fig" rid="175fig3">Figure 3C</xref>; see also ; see also Table S2).'], '175fig4': ['Immunofluorescence labeling of ILP2, one of the major ILPs, together with mCD8-labeled IPC neuronal processes showed that ILP2 is localized mainly in 14 cell bodies and some of their neurites (<xref ref-type="fig" rid="175fig4">Figure 4A</xref>). To determine whether these ILP2-positive neurites have axon and/or dendrite features, we expressed axonal (Tau) and dendritic (Khc::nod) markers (). To determine whether these ILP2-positive neurites have axon and/or dendrite features, we expressed axonal (Tau) and dendritic (Khc::nod) markers (<xref ref-type="fig" rid="175fig4">Figure 4, B–D</xref>) () (Rolls 2011). Tau-labeled IPC axonal bundles project contralaterally and posteriorly (<xref ref-type="fig" rid="175fig4">Figure 4B</xref>, arrowhead), make a U-turn, and extend anteriorly (arrow) to terminals outside the brain. ILP2 resides mainly in these axonal projections (, arrowhead), make a U-turn, and extend anteriorly (arrow) to terminals outside the brain. ILP2 resides mainly in these axonal projections (<xref ref-type="fig" rid="175fig4">Figure 4A</xref>, white arrows; , white arrows; <xref ref-type="fig" rid="175fig4">Figure 4B</xref>). The two main populations of Khc::nod-labeled dendritic arborizations extend laterally and posteriorly from the cell bodies (). The two main populations of Khc::nod-labeled dendritic arborizations extend laterally and posteriorly from the cell bodies (<xref ref-type="fig" rid="175fig4">Figure 4C</xref>, yellow arrows). ILP2 was invisible, or at very low levels, in these neurite structures (, yellow arrows). ILP2 was invisible, or at very low levels, in these neurite structures (<xref ref-type="fig" rid="175fig4">Figure 4A</xref>, yellow arrows). The pattern of GFP-tagged DCV marker atrial natriuretic factor (ANF) in IPCs showed that DCV-carried neuropeptides are tightly associated with ILP2 granules in IPC cell bodies. This suggests that ILP2, like mammalian insulin, is packaged in DCVs for transport (, yellow arrows). The pattern of GFP-tagged DCV marker atrial natriuretic factor (ANF) in IPCs showed that DCV-carried neuropeptides are tightly associated with ILP2 granules in IPC cell bodies. This suggests that ILP2, like mammalian insulin, is packaged in DCVs for transport (<xref ref-type="fig" rid="175fig4">Figure 4E</xref>) () (Dean 1973; Rao et al. 2001; Takahashi et al. 2004). Since Ilp2 mRNA is made in the cell bodies (Brogiolo et al. 2001; Rulifson et al. 2002), ILP2 protein carried in DCVs is transported out of the cell bodies and along the axonal projections of IPCs.'], '175fig5': ['Depletion of Unc-104 from IPCs during early larval developmental stages reduced the number of IPCs, disrupted IPC morphology, and imposed ∼1 day of developmental delay in adult eclosion (<xref ref-type="fig" rid="175fig5">Figure 5A</xref>), indicating that ), indicating that unc-104 is required during IPC development. Unc-104 is required for fly embryonic motor neuron synapse formation, larval synaptic terminal outgrowth, and dendrite morphogenesis of larval multidendritic neurons (Pack-Chung et al. 2007; Kern et al. 2013). In these past studies, a possible role of Unc-104 in insulin production would have been obscured by early developmental defects. To look specifically at Unc-104 function in ILP production, we employed tub-Gal80ts as a temporal Gal4 switch. Gal80ts inhibits Gal4 until increased temperature inactivates Gal80 and allows Gal4 to trigger gene transcription, in this case transcription of a gene encoding interfering unc-104 mRNA. We allowed Unc-104 to function through late larval development at 18°, by which time IPC neurite structure has fully developed (<xref ref-type="fig" rid="175fig1">Figure 1</xref>), and then changed the temperature to 29° for 24 hr to induce production of ), and then changed the temperature to 29° for 24 hr to induce production of unc-104 RNAi. This strategy successfully prevented occurrence of any visible defects in IPC development (<xref ref-type="fig" rid="175fig5">Figure 5B</xref>). In control IPCs with functional Unc-104, low-level ILP2 was detected in the cell bodies. In contrast, 24-hr depletion of ). In control IPCs with functional Unc-104, low-level ILP2 was detected in the cell bodies. In contrast, 24-hr depletion of unc-104 mRNA during the second–third larval instar transition caused striking accumulation of ILP2 in IPC cell bodies, as well as enrichment of ILP2 in neurites extending from the IPC cell bodies (<xref ref-type="fig" rid="175fig5">Figure 5B</xref>). Quantitation showed that reducing ). Quantitation showed that reducing unc-104 function in IPCs caused a twofold increase in ILP2 in the cell bodies of IPCs (<xref ref-type="fig" rid="175fig5">Figure 5C</xref>). qPCR analysis indicated no obvious increase in brain ). qPCR analysis indicated no obvious increase in brain Ilp2 mRNA level after knockdown of unc-104 for 24 hr (Figure S2). Thus insulin secretion was likely inhibited after depletion of unc-104 mRNA.', 'Unc-104 is predominantly expressed in the nervous system in larval and adult flies (Chintapalli et al. 2007; Pack-Chung et al. 2007). To study the role of Unc-104 in IPCs, we examined the localization of Unc-104 protein in IPCs using tagged Unc-104 produced in IPCs (Ilp2 > Unc-104-mCherry, <xref ref-type="fig" rid="175fig5">Figure 5D</xref>). Expression of this tagged Unc-104 in the nervous system is sufficient to rescue ). Expression of this tagged Unc-104 in the nervous system is sufficient to rescue unc-104 mutant phenotypes (Pack-Chung et al. 2007; Kern et al. 2013). In IPCs, Unc-104-mCherry strongly colocalized with axons (<xref ref-type="fig" rid="175fig5">Figure 5D</xref>). Unc-104-mCherry was also in IPC dendrites, but at a much lower level compared to axons. A similar pattern of Unc-104 localization was observed when GFP-tagged Unc-104 was expressed in IPCs (). Unc-104-mCherry was also in IPC dendrites, but at a much lower level compared to axons. A similar pattern of Unc-104 localization was observed when GFP-tagged Unc-104 was expressed in IPCs (<xref ref-type="fig" rid="175fig5">Figure 5E</xref>) () (Barkus et al. 2008).', 'When unc-104 was depleted, ILP2 accumulated in IPC axons. The accumulation was limited to regions proximal to the cell bodies (<xref ref-type="fig" rid="175fig5">Figure 5B</xref>, bottom). In view of the known role of Unc-104 in transporting DCVs in other neurons (, bottom). In view of the known role of Unc-104 in transporting DCVs in other neurons (Pack-Chung et al. 2007; Barkus et al. 2008), the localization of Unc-104 in IPCs and their axonal processes is consistent with a role for the motor protein in transporting ILP2 in DCVs along axons, especially in regions proximal to the cell bodies.'], '175fig6': ['To identify additional proteins involved in fly ILP production, 29 dominant-negative fly Rab constructs (UAS-Rab DN) were screened by crossing at least one line for each Rab gene to the Ilp2-Gal4 driver (Rulifson et al. 2002; Zhang et al. 2007). Among 43 lines tested, only IPC-specific expression of Rab1-DN resulted in a dramatic (>40%) reduction in adult fly weight (<xref ref-type="fig" rid="175fig6">Figure 6A</xref> and and Figure S1B). The other Rab-DN lines, including Rab27-DN, had little effect on fly weights when expressed in IPCs (Figure S1B). Rab27 is the fly ortholog of mammalian Rab27a, which is involved in insulin granule exocytosis (Yi et al. 2002). In the larval brain lobes, Rab27 is expressed in the mushroom bodies and developing antennal lobes, but not in IPCs (Chan et al. 2011), which explains why expression of Rab27-DN in IPCs did not cause a growth phenotype. Expressing Rab26-DN in IPCs had a weaker effect on growth inhibition (7% reduction in size), compared with a 20% reduction in size achieved with Rab26 RNAi, suggesting that the DN construct is less effective than the RNAi construct (Figure S1). Rab1 is expressed ubiquitously in the brain including in all 14 IPCs (<xref ref-type="fig" rid="175fig6">Figure 6C</xref>) () (Chan et al. 2011). Producing Rab1-DN in IPCs dramatically reduced pupal size to only 61% of the controls, in keeping with the RNAi data (<xref ref-type="fig" rid="175fig6">Figure 6B</xref> and and Figure S3A).', 'Reducing ILP production or secretion inhibits fly growth and developmental progression (Geminard et al. 2009; Zhang et al. 2009; Gronke et al. 2010). We tested whether inhibiting Rab1 in IPCs affects developmental timing. Flies producing Rab1-DN in IPCs eclosed and pupated, on average, ∼2 days later than control flies (<xref ref-type="fig" rid="175fig6">Figure 6, D and E</xref>). One direct consequence of reduced ILP production or secretion is elevated levels of circulating sugars in hemolymph (). One direct consequence of reduced ILP production or secretion is elevated levels of circulating sugars in hemolymph (Rulifson et al. 2002). With larvae producing Rab1-DN in IPCs, hemolymph levels of trehalose and glucose were elevated compared to control larvae (<xref ref-type="fig" rid="175fig6">Figure 6F</xref>). The average combined trehalose and glucose levels for control larvae and ). The average combined trehalose and glucose levels for control larvae and Ilp2 > Rab1-DN larvae were 2372 and 2904 mg/dl, respectively. The values of carbohydrate concentration for the Rab1-DN larvae resembled the levels of IPC-ablated larvae (Rulifson et al. 2002). This elevated sugar level, together with developmental delay and growth inhibition caused by Rab1-DN produced in IPCs, suggests that Rab1 promotes ILP production by IPCs.'], '175fig7': ['To explore the mechanism by which Rab1-DN inhibits ILP production, we first looked for any IPC developmental defects caused by constitutive inhibition of Rab1 in IPCs. IPC morphology and cell number were examined using mCD8-GFP and a RFP-conjugated nuclear marker, combined with Rab1-DN produced in the IPCs. Under these conditions, there were fewer IPCs in these larvae (<xref ref-type="fig" rid="175fig7">Figure 7, A and B</xref>). The average IPC count was reduced from 14 ± 0.0 in wild-type larvae to 8.7 ± 0.4 in ). The average IPC count was reduced from 14 ± 0.0 in wild-type larvae to 8.7 ± 0.4 in Ilp2 > Rab1-DN larvae. IPC neuronal morphology was dramatically disrupted. In Ilp2 > Rab1-DN larvae, dendritic arborizations were missing (<xref ref-type="fig" rid="175fig7">Figure 7A</xref>, white arrows), while the axonal bundles were mostly intact. To specifically examine whether Rab1 controls ILP production, we employed the Gal80, white arrows), while the axonal bundles were mostly intact. To specifically examine whether Rab1 controls ILP production, we employed the Gal80ts stretegy described earlier to inhibit Rab1 after IPC neurite structures had fully developed. As with unc-104 inhibition, late inhibition of Rab1 for 24 hr with the tub-Gal80ts system avoided IPC developmental defects but caused a 1.6-fold increase in ILP2 level in IPC cell bodies (<xref ref-type="fig" rid="175fig7">Figure 7, C and D</xref>). The level of ILP2 was reduced in the axonal projections that connect the IPC cell bodies, suggesting that ILP2 was trapped in cell bodies rather than transported along axons. qPCR assays indicated no obvious changes in brain ). The level of ILP2 was reduced in the axonal projections that connect the IPC cell bodies, suggesting that ILP2 was trapped in cell bodies rather than transported along axons. qPCR assays indicated no obvious changes in brain Ilp2 mRNA level after expression of Rab1-DN for 24 hr (Figure S2). As an alternative approach to inhibit Rab1 function, we expressed Rab1 RNAi using the Ilp2-Gal4 driver. At 29°, the RNAi caused strong accumulation of ILP2 in IPC cell bodies but left IPC morphology intact (Figure S3, B–D). Brains expressing Rab1 RNAi had the normal 14 IPCs, yet the RNAi reduced fly weight by 20%. The RNAi effect was milder than the effect of Rab1-DN (Figure S3A and <xref ref-type="fig" rid="175fig6">Figure 6A</xref>). ). Rab1 RNAi in IPCs caused only a slight developmental delay (about one-half day delayed for pupation and adult eclosion) and allowed axons and dendrites of IPCs to be formed and maintained properly. There was some loss of left–right symmetry (Figure S3B), perhaps as a consequence of perturbing global growth. Despite the mild effects on development and IPC morphology, Rab1 RNAi caused a doubling of ILP2 accumulation in IPC cell bodies (Figure S3, C and D). Both types of depletion of Rab1 function suggest a role for Rab1 in controlling intracellular trafficking of ILP2.', 'When Rab1 is inhibited in IPCs, ILP secretion is inhibited during transit from cell bodies to the axonal tracts. To investigate whether Rab1 directly influences ILP transport in IPCs, ILP2 was labeled with antibodies in wild-type IPCs expressing Rab1-YFP. Rab1-YFP was localized within IPC cell bodies with very low fluorescence in IPC axons or dendrites (<xref ref-type="fig" rid="175fig7">Figure 7E</xref>). Rab1-YFP and ILP2 immunofluorescent labeling displayed punctate patterns in IPC cell bodies. Strikingly, the majority of ILP2 punctae overlapped with Rab1 punctae, suggesting that Rab1-containing vesicles directly transport ILP granules along the route of ILP secretion.). Rab1-YFP and ILP2 immunofluorescent labeling displayed punctate patterns in IPC cell bodies. Strikingly, the majority of ILP2 punctae overlapped with Rab1 punctae, suggesting that Rab1-containing vesicles directly transport ILP granules along the route of ILP secretion.', 'Rab1 controls ER-to-Golgi transport (Stenmark 2009), so we investigated whether ILP2 granules reside in the Golgi. Producing the GFP-labeled Golgi marker Grasp65 in IPCs revealed punctate Golgi patterns that colocalized with ILP2 granules (<xref ref-type="fig" rid="175fig7">Figure 7E</xref>). As a negative control, the endosome marker FYVE-GFP was produced in IPCs. The FYVE-labeled endosomes were in a punctate pattern, like Grasp65-GFP and Rab1-YFP, but had few overlaps with ILP2 granules. These data, together with the elevated ILP levels in IPCs after Rab1 inhibition, suggested that ILP is delivered by Rab1 to the Golgi in IPC cell bodies. The failed delivery of ILPs outside the cell bodies, due to Rab1 inhibition, results in failed secretion.). As a negative control, the endosome marker FYVE-GFP was produced in IPCs. The FYVE-labeled endosomes were in a punctate pattern, like Grasp65-GFP and Rab1-YFP, but had few overlaps with ILP2 granules. These data, together with the elevated ILP levels in IPCs after Rab1 inhibition, suggested that ILP is delivered by Rab1 to the Golgi in IPC cell bodies. The failed delivery of ILPs outside the cell bodies, due to Rab1 inhibition, results in failed secretion.']}
|
Drosophila Insight into Insulin Secretion from Transcriptome and Genetic Analysis of Insulin-Producing Cells of
|
[
"insulin",
"pancreas",
"{'italic': 'Drosophila'}",
"Unc-104",
"kinesin",
"Rab1",
"Golgi",
"ER",
"RNA-seq",
"laser microdissection",
"transport"
] |
Genetics
|
1400569200
|
In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4(+) T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4(+) T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.
|
[
"Animals",
"Basic Helix-Loop-Helix Transcription Factors",
"Cell Differentiation",
"Cell Movement",
"DNA-Binding Proteins",
"Down-Regulation",
"Germinal Center",
"Humans",
"Inhibitor of Differentiation Proteins",
"Mice",
"Mutation",
"Proto-Oncogene Proteins c-bcl-6",
"Receptors, CCR7",
"Receptors, CXCR5",
"T-Lymphocytes, Helper-Inducer",
"Th17 Cells",
"Transcription, Genetic",
"Up-Regulation"
] |
other
|
PMC4012477
| null |
30
|
[
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"{'Citation': 'Xu H, et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature. 2013;496:523–527.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '23619696'}}}",
"{'Citation': 'Liu X, et al. Bcl6 expression specifies the T follicular helper cell program in vivo. The Journal of experimental medicine. 2012;209:1841–1852. S1841–1824.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3457730'}, {'@IdType': 'pubmed', '#text': '22987803'}]}}",
"{'Citation': 'van der Flier LG, et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell. 2009;136:903–912.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19269367'}}}",
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] |
Genetics. 2014 May 20; 197(1):175-192
|
NO-CC CODE
|
|
Applications of trendsceek on spatial and single-cell gene expression data.(A) Spatial transcriptomics data from mouse olfactory bulb (replicate 3, n=269 array-spots). Left: hematoxylin and eosin stained tissue-sections (from Ståhl et al4), followed by examples of genes with significant expression trends. Expression was scaled to the range zero to one by unity-based normalization. (B) Density plots of gene expression with cells in regions of significantly elevated expression coloured red. (C) Spatial transcriptomics data from mouse olfactory bulb (replicate 12, n=280 array-spots), as in (A). (D) As in (C) for mouse olfactory bulb, replicate 12. (E) Spatial transcriptomics data from breast cancer biopsy (histological section “Layer 2”, n=251 array-spots), with examples of genes with significant expression trends. The distance between array-spots is 200μm, each spot covering multiple cells. (F) Examples of distinct spatial expression patterns identified by trendsceek within E6.5 mouse epiblast cells (cluster 3 in Scialdone et al.5, n=481 cells). (G) Identification of spatial patterns related to the positions of male and female cells within the cluster, with mutually exclusive expression of Xist (expressed in female cells) and Eif2s3y (located on the Y-chromosome, only expressed in male cells). (H) Examples of spatial expression patterns identified in mouse hippocampus seqFISH data (cells imaged; H=93, O=89, T=208). Left: Cartoon of hippocampus with the 21 imaged regions labelled according to previous publication3. P-values represent (A-E) mark-correlation (F-G) mark-variogram and (H) Emark (two-sided, Benjamini-Hochberg adjusted).
|
emss-76329-f002
|
2
|
5662fa1ca7a77466b63adc0da40e778ba87736dc4bf632dc6af88b7121ff84a8
|
emss-76329-f002.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
905
] |
[{'image_id': 'emss-76329-f001', 'image_file_name': 'emss-76329-f001.jpg', 'image_path': '../data/media_files/PMC6314435/emss-76329-f001.jpg', 'caption': 'Illustrating trendsceek on simulated data.(A) Simulated mark distributions with local hotspot, step gradients, non-radial streaks and linear gradient patters. Expression values were sampled from empirical seqFISH data3 and cells in certain regions were spiked by sampling from the upper quantile of the expression distribution (cells n=500, spiked cells n=˜50, mean expression spiked cells / mean expression background = ˜10). (B) Marked point pattern statistics (Methods) for the simulated hotspot shown in (A). The mark correlation and mark variogram clearly indicate a significant spatial pattern as the band, which indicates the 5% significance level of a null distribution based on resampling of the mark distribution, is exceeded at certain radii. (C) 3D-representations of the spatial expression trend, where the green surfaces show weighted kernel density estimation (wKDE) of the simulated datasets. The blue surfaces indicate the upper 5% quantile of a null distribution generated by wKDE of the resampled mark distribution for each dataset. (D) Density plot of the simulated datasets (A) with cells colored red if they exceeded a 5% significance level based on wKDE, indicated by the blue surface in (C). Scale bars in (A) and (D) apply to all items of the figure, including the radius shown in (B).', 'hash': '27ce86cb571e81d9addb688473385fa2505825d1212d23efe0a82b07b75c9a3f'}, {'image_id': 'emss-76329-f002', 'image_file_name': 'emss-76329-f002.jpg', 'image_path': '../data/media_files/PMC6314435/emss-76329-f002.jpg', 'caption': 'Applications of trendsceek on spatial and single-cell gene expression data.(A) Spatial transcriptomics data from mouse olfactory bulb (replicate 3, n=269 array-spots). Left: hematoxylin and eosin stained tissue-sections (from Ståhl et al4), followed by examples of genes with significant expression trends. Expression was scaled to the range zero to one by unity-based normalization. (B) Density plots of gene expression with cells in regions of significantly elevated expression coloured red. (C) Spatial transcriptomics data from mouse olfactory bulb (replicate 12, n=280 array-spots), as in (A). (D) As in (C) for mouse olfactory bulb, replicate 12. (E) Spatial transcriptomics data from breast cancer biopsy (histological section “Layer 2”, n=251 array-spots), with examples of genes with significant expression trends. The distance between array-spots is 200μm, each spot covering multiple cells. (F) Examples of distinct spatial expression patterns identified by trendsceek within E6.5 mouse epiblast cells (cluster 3 in Scialdone et al.5, n=481 cells). (G) Identification of spatial patterns related to the positions of male and female cells within the cluster, with mutually exclusive expression of Xist (expressed in female cells) and Eif2s3y (located on the Y-chromosome, only expressed in male cells). (H) Examples of spatial expression patterns identified in mouse hippocampus seqFISH data (cells imaged; H=93, O=89, T=208). Left: Cartoon of hippocampus with the 21 imaged regions labelled according to previous publication3. P-values represent (A-E) mark-correlation (F-G) mark-variogram and (H) Emark (two-sided, Benjamini-Hochberg adjusted).', 'hash': '5662fa1ca7a77466b63adc0da40e778ba87736dc4bf632dc6af88b7121ff84a8'}]
|
{'emss-76329-f001': ['We first investigated the method on simulated spatial expression data (sampled from empirical seqFISH data; Methods) where cells with higher expression were located in local hotspots, step-gradients or non-radial streaks, or where the expression followed a linear gradient (<xref ref-type="fig" rid="emss-76329-f001">Figure 1A</xref>). Exemplifying the analyses of the hotspot pattern, the mark-correlation and mark-variogram were significant at low ). Exemplifying the analyses of the hotspot pattern, the mark-correlation and mark-variogram were significant at low r values (<xref ref-type="fig" rid="emss-76329-f001">Figure 1B</xref>, , P < 0.05) as determined via 1,000 randomly permuted expression distributions of the same marks (the 5% critical rejection band of these randomizations are shown as grey areas in <xref ref-type="fig" rid="emss-76329-f001">Figure 1B</xref>). Power analysis of the four metrices as a function of the number of cells, expression level, expression level difference and the size of the region with elevated expression are presented in ). Power analysis of the four metrices as a function of the number of cells, expression level, expression level difference and the size of the region with elevated expression are presented in Supplementary Figures 1, 2. The analysis revealed that spatial structures are reliably identified if at least 5% of sampled cells have differing expression levels, in particular when the total number of analysed cells exceed 500. For these patterns, the mark-variogram and mark-correlation based tests had the highest detection power, but E-mark and V-mark can have higher power in other cases (see results from real data below). We conclude that the method has sufficient power to reveal a variety of spatial patterns involving a small number of cells.', 'Next, we developed an approach to identify the cells belonging to regions with elevated expression patterns. Using wKDE, we identified cells exceeding a 5% significance level (green surface in <xref ref-type="fig" rid="emss-76329-f001">Figure 1C</xref>) by comparison to the upper 5% quantile of a two-dimensional null distribution generated by resampling the mark distribution (blue surface in ) by comparison to the upper 5% quantile of a two-dimensional null distribution generated by resampling the mark distribution (blue surface in <xref ref-type="fig" rid="emss-76329-f001">Figure 1C</xref>). Significant cells are shown on top of the density estimate of the expression in ). Significant cells are shown on top of the density estimate of the expression in <xref ref-type="fig" rid="emss-76329-f001">Figure 1D</xref>. Having developed a method for the identification of genes with spatial expression trends and an approach to pinpoint the cells belonging to regions of interest within the pattern, we next explored recently published gene expression data.. Having developed a method for the identification of genes with spatial expression trends and an approach to pinpoint the cells belonging to regions of interest within the pattern, we next explored recently published gene expression data.'], 'emss-76329-f002': ['We first analysed spatial transcriptomics data from olfactory bulb tissue4, and trendsceek identified 35 significant genes (<xref ref-type="fig" rid="emss-76329-f002">Figures 2A-B</xref>, , Supplementary Figures 3A, 4A, 5A, 6A-B and Supplementary Tables 1, 2, 3; P < 0.05, Benjamini-Hochberg adjusted) with expression primarily in non-granular cells. These genes included Ptn, Nr2f2 and Fabp7 which has been detected as tissue-domain restricted using principal component analyses (PCA)4 and with clear spatial RNA in situ signatures in the Allen Brain Institute atlas (see Supplementary Figure 9 in Ståhl et al. 20164). We also identified 45 genes with significant expression primarily in granular regions of the bulb (<xref ref-type="fig" rid="emss-76329-f002">Figures 2C-D</xref>, , Supplementary Figures 3B, 4B, 5B, 6C-D and Supplementary Tables 1, 4, 5) including known genes such as Nrgn, Camk4 and Pcp4 but also novel genes such as Gpsm1 (<xref ref-type="fig" rid="emss-76329-f002">Figure 2C</xref>). A strength of spatial transcriptomics is the ability to profile tumour tissues. Applying ). A strength of spatial transcriptomics is the ability to profile tumour tissues. Applying trendsceek to spatial profiling of human breast cancer tissue (layer 2)4 identified 14 genes with significant spatial expression (<xref ref-type="fig" rid="emss-76329-f002">Figure 2E</xref>, , Supplementary Figures 3C, 4C, 7, 8 and Supplementary Tables 1, 6, 7). Several genes implicated in breast cancer had significant spatial patterns, including the transcription factor KLF68, the transmembrane protein TMEPA19, and twelve genes related to the extracellular matrix (ECM). We conclude that trendsceek can be broadly applied to spatial transcriptomics data to find genes with significant spatial trends.', 'The vast majority of single-cell RNA-sequencing has been performed on dissociated cells that lack spatial information. The analysis of single-cell gene expression data often include clustering and visualization of cells in low-dimensional spaces (using e.g. PCA or t-SNE). We determined whether trendsceek could find spatial patterns within two-dimensional representations of dissociated single-cell data. t-SNE analysis of scRNA-seq data from a mouse gastrulation dataset identified a larger cluster of 481 epiblast cells from E6.5 mice5, still trendsceek identified 107 genes with significant spatial expression patterns (P < 0.05, Benjamini-Hochberg adjusted) within that cluster of cells (Supplementary Figure 9). The vast majority among the significant genes were characterized by an expression gradient with higher expression at the narrow part of the cell cluster (Supplementary Figure 10, exemplified by T and Fgf8 in <xref ref-type="fig" rid="emss-76329-f002">Figure 2F</xref>). Moreover, we identified hotspots of male cells that separated from female cells with significant expression of ). Moreover, we identified hotspots of male cells that separated from female cells with significant expression of Eif2s3y and Xist, respectively (<xref ref-type="fig" rid="emss-76329-f002">Figure 2G</xref> and and Supplementary Tables 1, 8, 9; P < 0.05, Benjamini-Hochberg adjusted). Different sets of genes were identified by the mark-correlation and mark-variogram based tests, indicating that the inclusion of multiple summary statistics improves sensitivity (Supplementary Figure 4D,H). We conclude that trendsceek can reveal diverse spatial patterns manifesting as gradients or hotspots within low-dimensional projections of dissociated single-cell data, in a fully unbiased manner without incorporating a priori knowledge about candidate genes.', 'Finally, we applied trendsceek to sequential FISH data from 21 mouse hippocampus regions3 that in total included approximately 3,500 cells and 249 genes. We identified genes with significant spatial patterns in 15 out of 21 regions (median of 54 genes per region) (Supplementary Figures 11, 12 and Supplementary Tables 10, 11), representative spatial patterns for five genes are shown in <xref ref-type="fig" rid="emss-76329-f002">Figure 2H</xref>. Regions A, C-F and Q contained no significant genes which may indicate greater homogeneity in these regions. This analysis demonstrated that . Regions A, C-F and Q contained no significant genes which may indicate greater homogeneity in these regions. This analysis demonstrated that trendsceek can identify a variety of spatial gene expression patterns in multiplexed FISH data.']}
|
Identification of spatial expression trends in single-cell gene expression data
| null |
Nat Methods
|
1526713200
|
Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George-the iconic last member of Chelonoidis abingdonii-and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations.
|
[
"Aging",
"Animals",
"DNA Repair",
"Evolution, Molecular",
"Genome",
"HEK293 Cells",
"Humans",
"Inflammation Mediators",
"Male",
"Neoplasms",
"Phylogeny",
"Population Density",
"Turtles"
] |
other
|
PMC6314435
| null |
67
|
[
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] |
Nat Methods. 2018 May 19; 15(5):339-342
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NO-CC CODE
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Both Rab29 and Rac1 rescued neurite shortening induced by LRRK2G2019S in differentiated SH-SY5Y cells.
A: Overexpressed 3HA-LRRK2G2019S in differentiated SH-SY5Y cells reduced neurite length compared with that for wild type protein. Na+-K+ ATPase was used to track plasma membrane of the cells. B: Co-overexpression LRRK2G2019S with Rab29WT or Rab29CA but not Rab29DN could significantly rescue neurite retraction induced by LRRK2G2019S. C: Co-overexpression LRRK2G2019S with Rac1WT or Rac1CA but not Rac1DN could significantly rescue neurite retraction induced by LRRK2G2019S. These results suggested that Rab29 and Rac1 can act as downstream of LRRK2 mediated neurite outgrowth. Quantification analysis of the relative neurite length (normalized by the WT) was presented in A, B, and C. Data presented were mean±SEM from two independent experiments. *P < 0.01 and ***P < 0.001, unpaired t-test.
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1674-8301-32-2-145-fig5
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2
|
53ef520bd5ad59daf15194c9f38f22ac8d936efd5b3c2b30e87d88fa1d619db2
|
1674-8301-32-2-145-fig5.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
403,
600
] |
[{'image_id': '1674-8301-32-2-145-fig1', 'image_file_name': '1674-8301-32-2-145-fig1.jpg', 'image_path': '../data/media_files/PMC5895569/1674-8301-32-2-145-fig1.jpg', 'caption': "LRRK2 preferentially interacted with Rac1and Rab29.A: Co-Immunoprecipitation of EGFP-Rab29 or EGFP-Rac1 with 3HALRRK2WT overexpressed in COS7 cells. The data indicated that Rac1 and Rab29 specifically interacted with LRRK2. B: Schematic representation of LRRK2 constructs used. C&D: Co-Immunoprecipitation of GFP-Rac1 (C) or 3Flag-Rab29 (D) with different fragments of 3HA-LRRK2 (full length, Roc, COR or Kinase domains) overexpressed in COS7 cells. The data indicated that Rac1 specifically bound to COR and kinase domain while Rab29 mainly interacted with COR domain of LRRK2. E&F: Quantification analysis of the relative Rab29 levels (normalized by the WT) was presented in C and D, Data presented were mean±SEM from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey's post hoc test.", 'hash': '811a6e5113626613c478d838403ef7651a495218843c9086040fcc8dbbe221f2'}, {'image_id': '1674-8301-32-2-145-fig3', 'image_file_name': '1674-8301-32-2-145-fig3.jpg', 'image_path': '../data/media_files/PMC5895569/1674-8301-32-2-145-fig3.jpg', 'caption': "Overexpressed Rab29 mutants but not Rac1 accelerated CI-M6PR degradation. A-C: CI-M6PR was co-transfected in HeLa Swiss cells with indicated plasmids and the cells were treated with 100 mg/mL CHX and collected samples at the indicate time points (0, 6 and 12 hours) for detecting the levels of the overexpressed proteins. The data suggested that LRRK2G2019S reduced the half-life of CI-M6PR compared with that in wild type or LRRK2K1906M group (A). Furthermore, overexpressed mutant Rab29s but not Rac1 reduced the half-life of CI-M6PR compared with that in wild type group (B&C). The arbitrary densitometry value in A, B and C was measured using imaging analysis software Image J. Data (mean±SEM) were from the indicated number of independent experiments and comparisons were analyzed usingone-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001.", 'hash': 'f8721fbc33fa6e6bdd8bc7b0172c8babe5abc418ac5b07e652a33fdf2cac15c8'}, {'image_id': '1674-8301-32-2-145-fig4', 'image_file_name': '1674-8301-32-2-145-fig4.jpg', 'image_path': '../data/media_files/PMC5895569/1674-8301-32-2-145-fig4.jpg', 'caption': "Rab29 but not Rac1 is a downstream effector of LRRK2 involved in retrograde trafficking of CI-M6PR. A: HeLa Swiss cells overexpressing Myc-CI-M6PR, 3HA-LRRK2G2019S and 3Flag-Rab29WT or EGFP-Rac1WT were immunostained, which indicated that Rab29 but not Rac1 could rescue the altered subcellular distribution of CI-M6PR induced by 3HA-LRRK2G2019S. B: Co-overexpression of LRRK2G2019S and Rab29WT or Rac1WT HeLa in Swiss cells was used to determine the half-life of CI-M6PR. The data showed that Rab29WT could rescue the reduced half-life of CI-M6PR induced by LRRK2G2019S. C: Quantitative analysis of the relative CI-M6PR levels (normalized by β-actin, time point 0 hour) in (B). Data (mean±SEM) were from the indicated number of independent experiments and comparisons were made using one-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001.", 'hash': 'c61704921c043182f0573890b65e3d1323ce7029310d2a73aa3e00b85d479430'}, {'image_id': '1674-8301-32-2-145-fig5', 'image_file_name': '1674-8301-32-2-145-fig5.jpg', 'image_path': '../data/media_files/PMC5895569/1674-8301-32-2-145-fig5.jpg', 'caption': 'Both Rab29 and Rac1 rescued neurite shortening induced by LRRK2G2019S in differentiated SH-SY5Y cells. \nA: Overexpressed 3HA-LRRK2G2019S in differentiated SH-SY5Y cells reduced neurite length compared with that for wild type protein. Na+-K+ ATPase was used to track plasma membrane of the cells. B: Co-overexpression LRRK2G2019S with Rab29WT or Rab29CA but not Rab29DN could significantly rescue neurite retraction induced by LRRK2G2019S. C: Co-overexpression LRRK2G2019S with Rac1WT or Rac1CA but not Rac1DN could significantly rescue neurite retraction induced by LRRK2G2019S. These results suggested that Rab29 and Rac1 can act as downstream of LRRK2 mediated neurite outgrowth. Quantification analysis of the relative neurite length (normalized by the WT) was presented in A, B, and C. Data presented were mean±SEM from two independent experiments. *P < 0.01 and ***P < 0.001, unpaired t-test.', 'hash': '53ef520bd5ad59daf15194c9f38f22ac8d936efd5b3c2b30e87d88fa1d619db2'}, {'image_id': '1674-8301-32-2-145-fig2', 'image_file_name': '1674-8301-32-2-145-fig2.jpg', 'image_path': '../data/media_files/PMC5895569/1674-8301-32-2-145-fig2.jpg', 'caption': 'Rab29 but not Rac1 acts as downstream of LRRK2 signaling in regulating retrograde trafficking of CI-M6PR. A: Double immunostaining of HeLa Swiss cells transiently expressing Myc-CI-M6PR with wild type, G2019S or K1906M of 3HA-LRRK2, respectively. The data showed that LRRK2G2019S altered the perinuclear localization of CI-M6PR with diffused staining pattern. B: Double immunostaining of HeLa Swiss cells transiently expressing Myc-CI-M6PR and wild type or mutant 3Flag-Rab29, respectively. The data showed that Rab29 mutations (CA and DN) altered the perinuclear localization of CI-M6PR. C: Double immunostaining of HeLa Swiss cells overexpressing Myc-CI-M6PR and wild type or mutant EGFP-Rac1, respectively. The data showed that Rac1 did not alter the perinuclear localization of CI-M6PR. Fluorescence intensity profiles of stained proteins were shown in the green and red channels of the regions indicated by the white lines. The CIM6PR fluorescence intensity near the nucleus represents the quantitative perinuclear distribution. Scale bar = 10 μm.', 'hash': 'f13964e2e8e266adf4eb83f291ac825dce11e5237ac005508de996155368f09d'}]
|
{}
|
Distinctive roles of Rac1 and Rab29 in LRRK2 mediated membrane trafficking and neurite outgrowth
|
[
"Parkinson’s disease",
"LRRK2",
"Rac1",
"Rab29",
"retrograde trafficking"
] |
J Biomed Res
|
1522047600
|
Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment. : X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. 2018;7:173-178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.
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[] |
other
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PMC5895569
| null |
56
|
[
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] |
J Biomed Res. 2018 Mar 26; 32(2):145-156
|
NO-CC CODE
|
|
Site-specific
incorporation of 1 and 2 into proteins in
mammalian cells and their rapid and specific labeling
with tetrazine fluorophores. (a) Western blots demonstrate that the
expression of full-length mCherry(TAG)eGFP-HA is dependent on the
presence of 1 or 2 and tRNACUA. BCNRS and TCORS were FLAG-tagged. (b) Specific and ultrarapid labeling
of a cell-surface protein in live mammalian cells. Left: EGFR-GFP
bearing 1, 2, or 5 at position
128 is visible as green fluorescence at the membrane of transfected
cells. Middle: treatment of cells with 11 (400 nM) selectively
labels EGFR containing 1 or 2. Right: merged
green and red fluorescence images with differential interference contrast
(DIC). Cells were imaged 2 min after the addition of 11. (c) Specific and rapid labeling of a nuclear protein in live mammalian
cells. Left: jun-1-mCherry and jun-5-mCherry
are visible as red fluorescence in the nuclei of transfected cells.
Middle: Selective labeling of jun-1-mCherry with 17 (200 nM). Right: merged red and green fluorescence with
DIC. No labeling was observed for cells bearing jun-5-mCherry.
|
ja-2012-02832g_0007
|
2
|
e2eb9cbf7b08bd72e794a1bb11c7be68142d45b3f15d17adfeb6c93164e51627
|
ja-2012-02832g_0007.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
664,
541
] |
[{'image_id': 'ja-2012-02832g_0003', 'image_file_name': 'ja-2012-02832g_0003.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0003.jpg', 'caption': 'Structures of unnatural amino acids 1–5 and tetrazine derivatives 6–17 used in this study. For the structures of\nTAMRA-X, Bodipy TMR-X,\nBodipy-FL, and CFDA, see Figure S4.', 'hash': '4e1d63be77db86a4df5c2c29f65385ff9dcbda452cdf36b84be0d9be3e258c57'}, {'image_id': 'ja-2012-02832g_0004', 'image_file_name': 'ja-2012-02832g_0004.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0004.jpg', 'caption': 'Characterization of the reaction of BCN with 7. (a)\nStopped-flow kinetics of the reaction. The inset shows the conjugation\nof 7 to 5-norbornene-2-ol (Nor); the different time scales\nshould be noted. Conditions: c7 = 0.05 mM and cBCN = cNor = 5 mM in 55:45 MeOH/H2O at 25 °C.\n(b) Determination of the second-order rate constant k for the reaction of 7 and BCN. (c) Fluorogenic reaction\nof 11 with BCN.', 'hash': '4c6d9854e73203842a707564221f1c25e1d75cc3755bf16b797d9aeab88db4c9'}, {'image_id': 'ja-2012-02832g_0005', 'image_file_name': 'ja-2012-02832g_0005.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0005.jpg', 'caption': 'Efficient genetically encoded incorporation of unnatural\namino\nacids in E. coli. (a) Amino acid-dependent\noverexpression of sfGFP-His6 bearing an amber codon at\nposition 150. The expressed protein was detected in lysates using\nan anti-His6 antibody and Coomassie staining. (b) Coomassie-stained\ngel showing purified proteins. (c–e) ESI-MS data for amino\nacid incorporation. For sfGFP-1-His6: found,\n28017.54 Da; calcd, 28017.62 Da. For sfGFP-2-His6: found, 27993.36 Da; calcd, 27992.82 Da. For sfGFP-His6 produced with 3 as described: found, 28019.34\nDa; calcd, 28019.63 Da. The minor peaks in the mass spectra correspond\nto loss of the N-terminal methionine.', 'hash': '749886f8475408ef829e40ee42300fdc03407bc3a300e36ebd6b5541e3c52848'}, {'image_id': 'ja-2012-02832g_0008', 'image_file_name': 'ja-2012-02832g_0008.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0008.jpg', 'caption': 'Genetic Encoding\nand Fluorogenic Labeling of Unnatural Amino Acids 1 and 2', 'hash': 'c4dddda441b45517be1d7336e5d2ca3a063570cdca739fc1bb1a1ebd246b4e17'}, {'image_id': 'ja-2012-02832g_0001', 'image_file_name': 'ja-2012-02832g_0001.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0001.jpg', 'caption': 'No caption found', 'hash': 'c2d6776e0cd1419468f4b70fda5feaf7d3e0b91514f94809c091e201b64591ce'}, {'image_id': 'ja-2012-02832g_0006', 'image_file_name': 'ja-2012-02832g_0006.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0006.jpg', 'caption': 'Rapid and specific labeling of recombinant proteins with\ntetrazine–fluorophore\nconjugates. (a) Specific labeling of sfGFP bearing 1, 2, or 4 with 11 (10 equiv) demonstrated\nby SDS-PAGE and in-gel fluorescence. (b) Quantitative labeling of\nsfGFP-1 with 11 demonstrated by ESI-MS.\nBefore bioconjugation (blue): found, 28018.1 Da; calcd, 28017.6 Da.\nAfter bioconjugation (red): found, 28824.2 Da; calcd, 28823.2 Da.\n(c) Quantitative labeling of sfGFP-2 with 11 demonstrated by ESI-MS. Before bioconjugation (blue): found, 27993.2\nDa; calcd, 27992.8 Da. After bioconjugation (red): found, 28799.4\nDa; calcd, 28799.1 Da. (d) No labeling with 11 of sfGFP-His6 expressed in the presence of 3 could be detected\nby MS. (e) Very rapid labeling of proteins containing 1 or 2.', 'hash': '25695c885d98f497e426f14f4664dcfb75537d88f04d9dfd15d60ce7405147bf'}, {'image_id': 'ja-2012-02832g_0007', 'image_file_name': 'ja-2012-02832g_0007.jpg', 'image_path': '../data/media_files/PMC3687367/ja-2012-02832g_0007.jpg', 'caption': 'Site-specific\nincorporation of 1 and 2 into proteins in\nmammalian cells and their rapid and specific labeling\nwith tetrazine fluorophores. (a) Western blots demonstrate that the\nexpression of full-length mCherry(TAG)eGFP-HA is dependent on the\npresence of 1 or 2 and tRNACUA. BCNRS and TCORS were FLAG-tagged. (b) Specific and ultrarapid labeling\nof a cell-surface protein in live mammalian cells. Left: EGFR-GFP\nbearing 1, 2, or 5 at position\n128 is visible as green fluorescence at the membrane of transfected\ncells. Middle: treatment of cells with 11 (400 nM) selectively\nlabels EGFR containing 1 or 2. Right: merged\ngreen and red fluorescence images with differential interference contrast\n(DIC). Cells were imaged 2 min after the addition of 11. (c) Specific and rapid labeling of a nuclear protein in live mammalian\ncells. Left: jun-1-mCherry and jun-5-mCherry\nare visible as red fluorescence in the nuclei of transfected cells.\nMiddle: Selective labeling of jun-1-mCherry with 17 (200 nM). Right: merged red and green fluorescence with\nDIC. No labeling was observed for cells bearing jun-5-mCherry.', 'hash': 'e2eb9cbf7b08bd72e794a1bb11c7be68142d45b3f15d17adfeb6c93164e51627'}]
|
{'ja-2012-02832g_0003': ['The rate constants for the reactions of various\ndienophiles [BCN, trans-cyclooctene-4-ol (TCO), and\nbicyclo[6.1.0]non-4-ene-9-ylmethanol\n(sTCO)] with tetrazines have been determined.3−5,9,11 However, in many cases,\ndifferent tetrazines, solvent systems, or measurement methods were\nused, making it challenging to compare quantitatively the reactivities\nof the dienophiles with tetrazines of interest. Our initial experiments\nconfirmed that the reactions of the dienophiles 1–3 with tetrazine 6 (Figure <xref rid="ja-2012-02832g_0003" ref-type="fig">1</xref>) were too fast to study by manual mixing under pseudo-first-order\nconditions. We therefore turned to stopped-flow techniques to determine\nthe pseudo-first-order rate constants for these reactions. By following\nthe exponential decay of the tetrazine absorbance at 320 nm upon reaction\nwith a 10–100-fold excess of BCN in 55:45 methanol/water, we\ndetermined the rate constants for the reactions of BCN with ) were too fast to study by manual mixing under pseudo-first-order\nconditions. We therefore turned to stopped-flow techniques to determine\nthe pseudo-first-order rate constants for these reactions. By following\nthe exponential decay of the tetrazine absorbance at 320 nm upon reaction\nwith a 10–100-fold excess of BCN in 55:45 methanol/water, we\ndetermined the rate constants for the reactions of BCN with 6 and 7 as 437 ± 13 and 1245 ± 45 M–1 s–1, respectively [Figure <xref rid="ja-2012-02832g_0004" ref-type="fig">2</xref>a,b and Figure S2a,b in the a,b and Figure S2a,b in the Supporting Information (SI)]. LC–MS and NMR analysis\nconfirmed the products (see the SI and Figure S1). Under the same conditions, the rate\nconstants for the reactions of TCO with 6 and 7 were 5235 ± 258 and 17248 ± 3132 M–1 s–1, respectively (Figure\nS3). The reaction between BCN and 6 is ∼1000\ntimes faster than the reaction between 5-norbornene-2-ol and 6,7 while the TCO reaction is 10–15\ntimes faster than that with BCN. The sTCO reaction was too fast for\naccurate measurements by stopped-flow techniques, and we estimate\nthat it is at least 50 times faster than the TCO reaction. Similar\nrate accelerations were observed for the reactions of BCN with 7, 8, and 9 (SI Table 1 and Figure S2).', 'Several tetrazine–fluorophore conjugates,\nincluding 11, 13, 14, and 16 (Figure <xref rid="ja-2012-02832g_0003" ref-type="fig">1</xref> and and Figure S4) are substantially\nquenched with respect to the free fluorophores.7,18 We\nfound that the reaction of BCN with 11, 13, 14, and 16 leads to a 5–10-fold\nincrease in fluorescence, suggesting that the formation of the pyridazine\nproduct efficiently relieves the fluorophore quenching (Figure <xref rid="ja-2012-02832g_0004" ref-type="fig">2</xref>c and c and Figure S5). These\nfluorogenic reactions with BCN, like those between strained alkenes\nand tetrazines,7,18 are advantageous for imaging\nexperiments since they maximize the labeling signal while minimizing\nthe fluorescence arising from the free tetrazine fluorophore.', 'Next, we aimed to design, synthesize, and genetically encode amino\nacids bearing BCN, TCO, and sTCO for site-specific protein labeling\nwith a diverse range of probes both in vitro and in cells. The pyrrolysyl-tRNA\nsynthetase (PylRS)/tRNACUA pairs from Methanosarcina species, including M. barkeri (Mb) and M. mazei (Mm), and their evolved derivatives have been used to direct\nthe site-specific incorporation of a growing list of structurally\ndiverse unnatural amino acids in response to the amber codon19−26 in a range of hosts, allowing synthetases evolved in E. coli to be used for genetic code expansion in\na growing list of cells and organisms, including E.\ncoli, Salmonella typhimurium, yeast, human cells, and Caenorhabditis elegans.7,27−31 We designed the unnatural amino acids 1, 2, and 3 (Figure <xref rid="ja-2012-02832g_0003" ref-type="fig">1</xref>) with the goal\nof incorporating them into proteins using the PylRS/tRNA) with the goal\nof incorporating them into proteins using the PylRS/tRNACUA pair or an evolved derivative. The amino acids were synthesized\nas described in the SI.'], 'ja-2012-02832g_0005': ['We screened\nthe MbPylRS/tRNACUA pair\nalong with a panel of MbPylRS mutants for their ability\nto direct the incorporation of 1–3 in response to an amber codon introduced at position 150 in a C-terminally\nHis6-tagged superfolder green fluorescent protein (sfGFP).\nCells containing a mutant of MbPylRS with the three\namino acid substitutions Y271M, L274G, and C313A32 in the enzyme active site [which we named BCN-tRNA synthetase\n(BCNRS)] and a plasmid that encodes MbtRNACUA and sfGFP-His6 with an amber codon at position 150 (psfGFP150TAGPylT-His6) led to the amino acid-dependent\nsynthesis of full-length sfGFP-His6 (Figure <xref rid="ja-2012-02832g_0005" ref-type="fig">3</xref>a and a and Figure S6). We found an additional\nsynthetase mutant bearing the mutations Y271A, L274 M, and C313A,32 which we named TCO-tRNA synthetase (TCORS).\nThe TCORS/tRNACUA pair led to the amino acid-dependent\nsynthesis of sfGFP from psfGFP150TAGPylT-His6 in the presence of 2. Finally, we found that\nboth the BCNRS/tRNACUA pair and the TCORS/tRNACUA pair lead to the amino acid-dependent synthesis of sfGFP from psfGFP150TAGPylT-His6 in the presence of 3. For each amino acid, sfGFP was isolated in good yield after\nHis-tag and gel-filtration purification (6–12 mg/L of culture;\nFigure <xref rid="ja-2012-02832g_0005" ref-type="fig">3</xref>b). This is comparable to the yields\nobtained for other well-incorporated unnatural amino acids, including b). This is comparable to the yields\nobtained for other well-incorporated unnatural amino acids, including 5. Electrospray ionization mass spectrometry (ESI-MS) data\nfor sfGFP produced from psfGFP150TAGPylT-His6 in the presence of each unnatural amino acid were consistent\nwith their site-specific incorporation (Figure <xref rid="ja-2012-02832g_0005" ref-type="fig">3</xref>c–e).c–e).'], 'ja-2012-02832g_0006': ['To demonstrate that the tetrazine–dye probes\nreact efficiently\nand specifically with recombinant proteins bearing site-specifically\nincorporated 1, we labeled purified sfGFP-1-His6 with 10 equiv of tetrazine–fluorophore conjugate 11 for 1 h at room temperature. SDS-PAGE and ESI-MS analyses\nconfirmed the quantitative labeling of sfGFP-1 (Figure <xref rid="ja-2012-02832g_0006" ref-type="fig">4</xref>a,b). Control experiments demonstrated that sfGFP-a,b). Control experiments demonstrated that sfGFP-4 was labeled under the same conditions and that no nonspecific\nlabeling occurred with sfGFP-5. ESI-MS showed that sfGFP-1 could be efficiently and specifically derivatized with 6–9 (Figure S7) and with 12–14 and 16 (Figure S8). We also demonstrated that\npurified sfGFP-2 could be quantitatively labeled with 11 (Figure <xref rid="ja-2012-02832g_0006" ref-type="fig">4</xref>a,c). Interestingly, we\nobserved only very weak labeling of sfGFP-Hisa,c). Interestingly, we\nobserved only very weak labeling of sfGFP-His6 purified\nfrom cells expressing TCORS/tRNACUA and psfGFP150TAGPylT-His6 and grown in the presence of 3 (Figure <xref rid="ja-2012-02832g_0006" ref-type="fig">4</xref>a,d) and substoichiometric labeling of this protein\nprior to purification (a,d) and substoichiometric labeling of this protein\nprior to purification (Figure S9). Since\nthe sfGFP expressed in the presence of 3 has a mass corresponding\nto the incorporation of 3, these observations are consistent\nwith the in vivo conversion of a fraction of the trans-alkene in 3 to its unreactive cis isomer.\nThis isomerization is known to occur in the presence of thiols.4', 'To investigate whether the rates\nof the BCN– and TCO–tetrazine\ncycloadditions observed for small molecules translate into exceptionally\nrapid protein labeling, we compared the labeling of purified sfGFP\nbearing 1, 2, or 4 with 10\nequiv of 11. In-gel fluorescence imaging of the labeling\nreaction as a function of time (Figure <xref rid="ja-2012-02832g_0006" ref-type="fig">4</xref>e)\nindicated that the reaction of sfGFP-e)\nindicated that the reaction of sfGFP-4 reached completion\nin ∼1 h. In contrast, the labeling of sfGFP-1 and\nsfGFP-2 was complete within the few seconds it took to\nmeasure the first time point, demonstrating that the rate acceleration\nof the BCN– and TCO–tetrazine reactions translates into\nmuch more rapid protein labeling.'], 'ja-2012-02832g_0007': ['To demonstrate the incorporation\nof amino acids 1 and 2 into mammalian cells,\nwe transplanted the mutations allowing\nthe incorporation of 1 or 2 into a mammalian-optimized MbPylRS. Western blots showed that both 1 and 2 can be genetically encoded with high efficiency into proteins\nin mammalian cells using the BCNRS/tRNACUA and TCORS/tRNACUA pairs, respectively (Figure <xref rid="ja-2012-02832g_0007" ref-type="fig">5</xref>a).a).', 'To investigate whether the rapid BCN–tetrazine\nligation\nwould provide advantages for site-specific labeling of proteins on\nmammalian cells, we expressed an epidermal growth factor receptor\n(EGFR)–GFP fusion bearing an amber codon at position 128 (EGFR(128TAG)GFP) in HEK-293 cells containing the BCNRS/tRNACUA pair cultured in the presence of 1 (0.5 mM).\nFull-length EGFR-1-GFP was produced in the presence of 1, resulting in bright green fluorescence at the cell membrane.\nTo label 1 with tetrazine–fluorophore conjugates,\nwe incubated cells with 11 (400 nM), changed the medium,\nand imaged the red fluorescence arising from TAMRA labeling. The TAMRA\nfluorescence colocalized nicely with the cell-surface EGFR-GFP fluorescence.\nClear labeling of cells bearing EGFR-1-GFP was observed\nwithin 2 min, the first time point we could measure; additional time\npoints demonstrated that the labeling was saturated within 2 min (Figure <xref rid="ja-2012-02832g_0007" ref-type="fig">5</xref>b and b and Figures S11–S14); similar results were obtained with 12. Incorporation\nof 2 into the EGFR–GFP fusion led to similarly\nrapid and efficient labeling with 11 (Figure <xref rid="ja-2012-02832g_0007" ref-type="fig">5</xref>b and b and Figures S15 and S16). In contrast, it took 2 h before we observed any specific labeling\nof cells bearing EGFR-4-GFP under identical conditions\n(Figure S14).7 In control experiments we observed neither labeling of cells bearing\nEGFR-5-GFP nor nonspecific labeling of cells that did\nnot express EGFR-GFP. We observed weak but measurable labeling of\nEGFR-GFP expressed in HEK-293 cells from EGFR(128TAG)GFP in the presence of the BCNRS/tRNACUA pair and 3 (Figure S17). These observations are\nconsistent with the isomerization of a fraction of 3 in\nmammalian cells and with our observations in E. coli.', 'To demonstrate the rapid labeling of an intracellular protein\nin\nmammalian cells, we expressed a transcription factor, jun, with a\nC-terminal mCherry fusion from a gene bearing an amber codon in the\nlinker between JunB (jun) and mCherry. In the presence of amino acid 1 and the BCNRS/tRNACUA pair, the jun-1-mCherry protein was produced in HEK cells and, as expected, localized\nin the nuclei of cells (Figure <xref rid="ja-2012-02832g_0007" ref-type="fig">5</xref>c and c and Figure S18). Labeling with cell-permeable conjugate 17 (200 nM) resulted in green fluorescence that colocalized\nnicely with the mCherry signal at the first time point analyzed (after\n15 min of labeling and 90 min of washing). No specific labeling was\nobserved in nontransfected cells in the same sample or in control\ncells expressing jun-5-mCherry, further confirming the\nspecificity of intracellular labeling.']}
|
trans Genetic Encoding of Bicyclononynes
and -Cyclooctenes for Site-Specific Protein
Labeling in Vitro and in
Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions
| null |
J Am Chem Soc
|
1340780400
|
[{'@Label': 'INTRODUCTION', '@NlmCategory': 'BACKGROUND', '#text': 'A significant number of mania patients fail to respond to current pharmacotherapy, thereby there is need for novel augmentation strategies. The results of some early studies showed the effectiveness of cholinomimetics in the treatment of mania. One open case series suggested the efficacy of donepezil in the treatment of bipolar disorder. Our aim was to explore whether an oral cholinesterase inhibitor, donepezil, administered during a 4-week treatment period, would benefit patients with acute mania.'}, {'@Label': 'METHODS', '@NlmCategory': 'METHODS', '#text': 'We conducted a 4-week double-blind, placebo-controlled trial of donepezil as an adjunctive treatment to lithium in patients with acute mania. Eligible subjects were randomly assigned to receive donepezil or placebo in addition to lithium. Donepezil was started at 5 mg/day, and increased to 10 mg/day in the first week. Patients were rated with the Young Mania Rating Scale (YMRS) and Brief Psychiatric Rating Scale (BPRS) at baseline, day 1, week 1, week 2, and week 4.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': "Out of the 30 patients who were enrolled, 15 were on donepezil and 15 were on placebo. All patients completed the 4-week trial. On the first day, there was a difference of 1.97 units on the psychomotor symptoms scale of the YMRS in the donepezil group as compared to the placebo group (t = 2.39, P = 0.02). There was a difference of 0.57 units (t = 2.09, P = 0.04) in the speech item and a difference of 0.29 units in the sexual interest item (t = 2.11, P = 0.04) in the donepezil group as compared to the placebo group. The total YMRS difference on the first day approached the conventional significance level (1.97 units, t = 1.84, P = 0.07). Over the course of 4 weeks, we failed to find that donepezil produced any significant difference in the YMRS (6.71 units difference, t = -1.44, P = 0.16) or the BPRS scale (1.29 units difference, t = -0.33, P = 0.75) as compared to placebo. Ten subjects (66.67%) in both groups met the criteria for clinical response (Fisher's exact P = 1.00). Five subjects (33.33%) in the donepezil group met the criteria for clinical remission while nine subjects (60.00%) in the placebo group met the remission criteria (Fisher's exact P = 0.27)."}, {'@Label': 'CONCLUSION', '@NlmCategory': 'CONCLUSIONS', '#text': 'Use of the oral anticholinergic donepezil had some benefit in the augmentation of lithium treatment on the first day, but did not provide any significant benefits in the long-term.'}]
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other
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PMC3687367
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39
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[
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"{'Citation': 'Young AH, Hammond JM. Lithium in mood disorders: increasing evidence base, declining use? Br J Psychiatry. 2007;191(6):474–476.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18055949'}}}",
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"{'Citation': 'Scherk H, Pajonk FG, Leucht S. Second-generation antipsychotic agents in the treatment of acute mania: a systematic review and meta-analysis of randomized controlled trials. Arch Gen Psychiatry. 2007;64(4):442–455.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17404121'}}}",
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] |
J Am Chem Soc. 2012 Jun 27; 134(25):10317-10320
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NO-CC CODE
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Interaction of YWHAB with PCV2 ORF5 protein.(A) Exogenous co-IP analysis the binding of ORF5 and YWHAB. HEK293 cells were co-transfected with CMV-Flag-YWHAB and pEGFP-ORF5 plasmids for 48 h. Cells co-transfected with pEGFP-C1 and CMV-Flag-YWHAB were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a mouse anti-Flag pAb.(B) GST pull-down assay analysis the interaction of ORF5 and YWHAB. The GST and GST-YWHAB proteins were produced from Escherichia coli Rosetta (DE3) cells and were immobilized on a glutathione agarose beads for 2 h at 4 °C, followed by incubation of the resin with the cell lysates containing GFP-ORF5 protein (HEK293 cells were transfected with pEGFP-ORF5 plasmid for 48 h).(C) HEK293 cells were co-transfected with pDsRed-YWHAB and pEGFP-ORF5 plasmids for 48 h. Cells co-transfected with pEGFP-C1 and pDsRed-N1 were used as negative control. Cells were fixed and stained with DAPI (blue) for 10 min at room temperature. Scale bar, 10 μm. Data in (A, B and C) are one representative of those from three independent experiments.
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gr1_lrg
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030cbc4cc4cf9e96803e43bd301918f9cdfb2777628b7878c6db0f1b1b9e2b4d
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gr1_lrg.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
716,
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[{'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr1_lrg.jpg', 'caption': 'Interaction of YWHAB with PCV2 ORF5 protein.(A) Exogenous co-IP analysis the binding of ORF5 and YWHAB. HEK293 cells were co-transfected with CMV-Flag-YWHAB and pEGFP-ORF5 plasmids for 48 h. Cells co-transfected with pEGFP-C1 and CMV-Flag-YWHAB were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a mouse anti-Flag pAb.(B) GST pull-down assay analysis the interaction of ORF5 and YWHAB. The GST and GST-YWHAB proteins were produced from Escherichia coli Rosetta (DE3) cells and were immobilized on a glutathione agarose beads for 2 h at 4 °C, followed by incubation of the resin with the cell lysates containing GFP-ORF5 protein (HEK293 cells were transfected with pEGFP-ORF5 plasmid for 48 h).(C) HEK293 cells were co-transfected with pDsRed-YWHAB and pEGFP-ORF5 plasmids for 48 h. Cells co-transfected with pEGFP-C1 and pDsRed-N1 were used as negative control. Cells were fixed and stained with DAPI (blue) for 10 min at room temperature. Scale bar, 10 μm. Data in (A, B and C) are one representative of those from three independent experiments.', 'hash': '030cbc4cc4cf9e96803e43bd301918f9cdfb2777628b7878c6db0f1b1b9e2b4d'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr5_lrg.jpg', 'caption': 'YWHAB knockdown potentiates the PCV2-induced endoplasmic reticulum stress.Real-time qRT-PCR analysis of GRP78 (A) and GRP94 (B) gene expression in YWHAB knockdown (shYWHAB), lentivirus control (shN) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(C) Immunoblot analysis of GRP78 and GRP94 protein expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.The intensity represents GRP78 (D) and GRP94 (E) protein level normalized to that of β-actin across three independent experiments. Data in (A, B, D and E) are shown as the mean ± SD of three independent experiments. Data in (C) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.', 'hash': 'fe159ca8096fef75ab50e518dda548ff929cbd07b153055e030ec413b22b0f33'}, {'image_id': 'gr8_lrg', 'image_file_name': 'gr8_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr8_lrg.jpg', 'caption': 'YWHAB inhibits PCV2-induced apoptosis.Flow cytometry analysis of cell apoptosis in YWHAB overexpressed (A, CTR, untransduced control; LV, lentivirus control; LV-YWHAB, YWHAB overexpressed cells) or knockdown (B, CTR, untransduced control; shN, lentivirus control; shYWHAB, YWHAB knockdown cells) PK-15 cells infected with PCV2 at an MOI of 1. At 24 h post-infection, the cells were resuspended and stained with 5 μL Annexin V-FITC and 5 μL 7-aminoactinomycin D (7-AAD) for 10 min in the dark at room temperature. Fluorescent signals were detected by flow cytometry analysis. Data is one representative of those from three independent experiments.', 'hash': '73cb325d88ff9b5667f8a212c21e938e32585b6d1380624101ad00092c394289'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr4_lrg.jpg', 'caption': 'YWHAB alleviates the PCV2-induced endoplasmic reticulum stress.Real-time qRT-PCR analysis of GRP78 (A) and GRP94 (B) gene expression in YWHAB overexpressed, lentivirus control (LV) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(C) Immunoblot analysis of GRP78 and GRP94 protein expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.The intensity represents GRP78 (D) and GRP94 (E) protein level normalized to that of β-actin across three independent experiments.Data in (A, B, D and E) are shown as the mean ± SD of three independent experiments. Data in (C) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.', 'hash': 'e0a07f8abf15a4bda7a51a2c6d50a4d2ec6dd07bb1cef2115496ffccd85b0618'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr2_lrg.jpg', 'caption': 'PCV2 and ORF5 activated YWHAB expression.(A) Real-time qRT-PCR analysis of YWHAB mRNA expression level in PK-15 cells infected with PCV2 at an MOI of 1 or Mock infected at 24 h or 48 h post-infection.(B) Immunoblot analysis of YWHAB protein expression in PK-15 cells infected with PCV2 at an MOI of 1 or Mock infected at 24 h or 48 h post-infection.(C) The intensity represents YWHAB protein level normalized to that of β-actin across three independent experiments.(D) Real-time qRT-PCR analysis of YWHAB mRNA expression level in PK-15 cells infected with PCV2 at an indicated MOI (0.5, 1, 1.5, 2) at 48 h post-infection.(E) Immunoblot analysis of YWHAB and PCV2 Cap protein expression in PK-15 cells infected with PCV2 at an indicated MOI (0.5, 1, 1.5, 2) at 48 h post-infection.(F) The intensity represents YWHAB protein level normalized to that of β-actin across three independent experiments.(G) Real-time qRT-PCR analysis of YWHAB mRNA expression in PK-15 cells transfected with pEGFP-ORF5 plasmid at 24 h or 48 h post-transfection.(H) Immunoblot analysis of YWHAB protein expression in PK-15 cells transfected with pEGFP-ORF5 plasmid at 24 h or 48 h post-transfection.(I) The intensity represents YWHAB protein level normalized to that of β-actin across three independent experiments. Data in (A, C, D, F, G and I) are shown as the mean ± SD of three independent experiments. Data in (B, E and H) are one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.', 'hash': '53f70459ee56df61d78b249327146367bb6aaddd75d4948936e220a3fa614b9f'}, {'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr6_lrg.jpg', 'caption': 'YWHAB inhibits the PCV2-induced autophagy.(A) Real-time qRT-PCR analysis of Beclin1 gene expression in YWHAB overexpressed, lentivirus control (LV) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection. (B) Immunoblot analysis of LC3II protein expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(C) The intensity represents LC3II protein level normalized to that of β-actin across three independent experiments.(D) Real-time qRT-PCR analysis of Beclin1 gene expression in YWHAB knockdown (shYWHAB), lentivirus control (shN) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(E) Immunoblot analysis of LC3II protein expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(F) The intensity represents LC3II protein level normalized to that of β-actin across three independent experiments.Data in (A, C, D and F) are shown as the mean ± SD of three independent experiments. Data in (B and E) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.', 'hash': 'a6a3cda8a038e15dfd7f3dfb9db9b83315b20935e98414157af7967e8352c7ad'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr3_lrg.jpg', 'caption': 'YWHAB inhibits PCV2 replication.(A) Immunoblot analysis of YWHAB protein expression in PK-15 cells transduced with lentivirus that overexpress YWHAB (LV-YWHAB), control lentivirus (LV) or untransduced (CTR).(B) Real-time qRT-PCR analysis of PCV2 viral RNA expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at indicated time points (12, 24, 36 and 48 h post-infection).(C) Immunoblot analysis of PCV2 Cap protein expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(D) The intensity represents PCV2 Cap protein level normalized to that of β-actin across three independent experiments.(E) Real-time qRT-PCR analysis of PCV2 ORF5 RNA expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(F) Immunoblot analysis of YWHAB protein expression in PK-15 cells transduced with lentivirus that knockdown YWHAB (shYWHAB-1, 2 or 3) or control lentivirus (shN) or untransduced (CTR).(G) Real-time qRT-PCR analysis of PCV2 viral RNA expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at indicated time points (12, 24, 36 and 48 h post-infection).(H) Immunoblot analysis of PCV2 Cap protein expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(I) The intensity represents PCV2 Cap protein level normalized to that of β-actin across three independent experiments.(J) Real-time qRT-PCR analysis of PCV2 ORF5 RNA expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.Data in (B, D, E, G, I and J) are shown as the mean ± SD of three independent experiments. Data in (A, C, F and H) are one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.', 'hash': '29ac14c5727691e709c9c7bd7a4481b2412c06c2f7346efc62f2d6e13f040bad'}, {'image_id': 'gr7_lrg', 'image_file_name': 'gr7_lrg.jpg', 'image_path': '../data/media_files/PMC7568206/gr7_lrg.jpg', 'caption': 'YWHAB inhibits PCV2-induced intracellular ROS.Flow cytometry analysis of ROS level in YWHAB overexpressed or knockdown PK-15 cells infected with PCV2 at an MOI of 1. At 24 h post-infection, the cells were incubated with 2′,7′-dichlorofluorescein diacetate (DHE, 5 μM) at 37 °C for 30 min and the ROS related fluorescent signal was detected by flow cytometry analysis.Data are shown as the mean ± SD of three independent experiments.**p < 0.01; ***p < 0.001.', 'hash': '4fa6172ad59b26873a3e9b5e02a101e305f4ff67d6c4ac32286ed40493a36e04'}]
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{'gr1_lrg': ['In our previous study, by using the yeast two-hybrid assay, several proteins (YWHAB, GPNMB, CYP1A1, ZNF511 and SRSF3) were identified as ORF5-interacting host factors. To further validate the interaction between YWHAB (also known as 14-3-3β/α) and ORF5 protein, the CMV-Flag-YWHAB and pEGFP-ORF5 plasmids were co-transfected into HEK293 cells. The result showed a clear binding between YWHAB and PCV2 ORF5 (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>\nA), suggesting the potential interaction between ORF5 and YWHAB. Next, the GST pull-down assay also confirmed a specific interaction between YWHAB and PCV2 ORF5 protein (\nA), suggesting the potential interaction between ORF5 and YWHAB. Next, the GST pull-down assay also confirmed a specific interaction between YWHAB and PCV2 ORF5 protein (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B), which purified GST-YWHAB protein and cell lysis from ORF5-overexpressed cells were employed. Furthermore, colocalization of YWHAB and PCV2 ORF5 protein was observed in the cytoplasm of pEGFP-ORF5 and pDsRed-YWHAB vectors co-transfected porcine alveolar macrophages 3D4/2 (PAMs) cells (B), which purified GST-YWHAB protein and cell lysis from ORF5-overexpressed cells were employed. Furthermore, colocalization of YWHAB and PCV2 ORF5 protein was observed in the cytoplasm of pEGFP-ORF5 and pDsRed-YWHAB vectors co-transfected porcine alveolar macrophages 3D4/2 (PAMs) cells (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>C), which further fostered an interaction between ORF5 and YWHAB. Together, these results confirmed that PCV2 ORF5 interacted with the host factor YWHAB.C), which further fostered an interaction between ORF5 and YWHAB. Together, these results confirmed that PCV2 ORF5 interacted with the host factor YWHAB.Fig. 1Interaction of YWHAB with PCV2 ORF5 protein.(A) Exogenous co-IP analysis the binding of ORF5 and YWHAB. HEK293 cells were co-transfected with CMV-Flag-YWHAB and pEGFP-ORF5 plasmids for 48 h. Cells co-transfected with pEGFP-C1 and CMV-Flag-YWHAB were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a mouse anti-Flag pAb.(B) GST pull-down assay analysis the interaction of ORF5 and YWHAB. The GST and GST-YWHAB proteins were produced from Escherichia coli Rosetta (DE3) cells and were immobilized on a glutathione agarose beads for 2 h at 4 °C, followed by incubation of the resin with the cell lysates containing GFP-ORF5 protein (HEK293 cells were transfected with pEGFP-ORF5 plasmid for 48 h).(C) HEK293 cells were co-transfected with pDsRed-YWHAB and pEGFP-ORF5 plasmids for 48 h. Cells co-transfected with pEGFP-C1 and pDsRed-N1 were used as negative control. Cells were fixed and stained with DAPI (blue) for 10 min at room temperature. Scale bar, 10 μm. Data in (A, B and C) are one representative of those from three independent experiments.Fig. 1', 'The identification of host factors that interact with viral proteins is essential to understand the pathogenesis of virus. In our previous study, five host proteins (YWHAB, GPNMB, CYP1A1, ZNF511 and SRSF3) were identified as PCV2 ORF5-interacting factors by used yeast two-hybrid assay (Lv et al., 2015). The interaction between GPNMB and PCV2 ORF5 was confirmed through different approaches and we surprisingly found that GPNMB inhibits PCV2 replication and ORF5 expression by regulating the Cyclin A expression in host cells (Guo et al., 2018). This study validated our previous screening approach and prompt us to investigate the role of other ORF5-interacting proteins in PCV2 infection. Intrigued by its role in regulating diverse cellular processes and affecting virus replication, we focused on the interaction between YWHAB and PCV2 in present study. A specifical interaction between PCV2-ORF5 and YWHAB was validated via diffident approaches (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>). Then, we surprisingly found that PCV2 infection and ORF5 transfection strongly activated YWHAB expression. This is consistent with a previous study that showed pancreatic necrosis virus (IPNV) induced the expression of a large number of genes including YWHAB (14-3-3β), but they did not illustrate the role of YWHAB against IPNV infection (). Then, we surprisingly found that PCV2 infection and ORF5 transfection strongly activated YWHAB expression. This is consistent with a previous study that showed pancreatic necrosis virus (IPNV) induced the expression of a large number of genes including YWHAB (14-3-3β), but they did not illustrate the role of YWHAB against IPNV infection (Villalba et al., 2017). In this study, we further showed that YWHAB inhibits PCV2 replication. Several isoforms of 14-3-3 proteins have been reported to affect viral infection (Aoki et al., 2000; Diao et al., 2001; Kino et al., 2005). In particular, a study shows that the M protein of parainfluenza virus 5 (PIV5) interacts with host protein 14-3-3 β and the production of PIV5 particles was negatively affected (Schmitt and Lamb, 2004). Together, we believe that YWHAB is an anti-PCV2 host factor, in which the expression is induced upon viral infection. However, the molecular mechanism of how the YWHAB is induced needs further investigation.'], 'gr2_lrg': ['We have already shown that PCV2 ORF5 interacts with YWHAB. However, whether PCV2 infection or ORF5 transfection could affect the expression of YWHAB remains unknown. To this aim, the endogenous YWHAB levels were measured upon PCV2 infection and ORF5 transfection. We found that PCV2 infection substantially induces YWHAB expression at mRNA and protein levels from 24 h post-infection (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\nA, B and C). Correspondingly, the expression of YWHAB was induced to a higher level with the increase of infection MOI (\nA, B and C). Correspondingly, the expression of YWHAB was induced to a higher level with the increase of infection MOI (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>D, E and F). The interaction between PCV2 ORF5 and host factor YWHAB prompts us to test whether the PCV2 ORF5 protein could affect YWHAB expression. As expected, the mRNA expression of YWHAB was significantly induced by ORF5 transfection (D, E and F). The interaction between PCV2 ORF5 and host factor YWHAB prompts us to test whether the PCV2 ORF5 protein could affect YWHAB expression. As expected, the mRNA expression of YWHAB was significantly induced by ORF5 transfection (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>G). Consistently, the protein expression of YWHAB was upregulated by PCV2 ORF5 (G). Consistently, the protein expression of YWHAB was upregulated by PCV2 ORF5 (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>H and I). Together, these results demonstrated that PCV2 infection and ORF5 transfection induced YWHAB expression at both transcriptional and translational levels.H and I). Together, these results demonstrated that PCV2 infection and ORF5 transfection induced YWHAB expression at both transcriptional and translational levels.Fig. 2PCV2 and ORF5 activated YWHAB expression.(A) Real-time qRT-PCR analysis of YWHAB mRNA expression level in PK-15 cells infected with PCV2 at an MOI of 1 or Mock infected at 24 h or 48 h post-infection.(B) Immunoblot analysis of YWHAB protein expression in PK-15 cells infected with PCV2 at an MOI of 1 or Mock infected at 24 h or 48 h post-infection.(C) The intensity represents YWHAB protein level normalized to that of β-actin across three independent experiments.(D) Real-time qRT-PCR analysis of YWHAB mRNA expression level in PK-15 cells infected with PCV2 at an indicated MOI (0.5, 1, 1.5, 2) at 48 h post-infection.(E) Immunoblot analysis of YWHAB and PCV2 Cap protein expression in PK-15 cells infected with PCV2 at an indicated MOI (0.5, 1, 1.5, 2) at 48 h post-infection.(F) The intensity represents YWHAB protein level normalized to that of β-actin across three independent experiments.(G) Real-time qRT-PCR analysis of YWHAB mRNA expression in PK-15 cells transfected with pEGFP-ORF5 plasmid at 24 h or 48 h post-transfection.(H) Immunoblot analysis of YWHAB protein expression in PK-15 cells transfected with pEGFP-ORF5 plasmid at 24 h or 48 h post-transfection.(I) The intensity represents YWHAB protein level normalized to that of β-actin across three independent experiments. Data in (A, C, D, F, G and I) are shown as the mean ± SD of three independent experiments. Data in (B, E and H) are one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.Fig. 2'], 'gr3_lrg': ['Although has been shown that YWHAB expression was greatly induced by PCV2 infection, the role of YWHAB during PCV2 infection is still enigmatic. To investigate the potential function of YWHAB in affecting PCV2 infection, the lentiviral-based overexpression and knockdown approaches were employed. As shown in <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\nA, a successful overexpression of YWHAB was observed in LV-YWHAB cell line. Remarkably, the overexpression YWHAB significantly suppressed the replication of PCV2 (\nA, a successful overexpression of YWHAB was observed in LV-YWHAB cell line. Remarkably, the overexpression YWHAB significantly suppressed the replication of PCV2 (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>B). Consistently, the protein expression of PCV2 Cap protein and mRNA expression of ORF5 was strongly decreased in YWHAB-overexpressed cells (B). Consistently, the protein expression of PCV2 Cap protein and mRNA expression of ORF5 was strongly decreased in YWHAB-overexpressed cells (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C, D and E).C, D and E).Fig. 3YWHAB inhibits PCV2 replication.(A) Immunoblot analysis of YWHAB protein expression in PK-15 cells transduced with lentivirus that overexpress YWHAB (LV-YWHAB), control lentivirus (LV) or untransduced (CTR).(B) Real-time qRT-PCR analysis of PCV2 viral RNA expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at indicated time points (12, 24, 36 and 48 h post-infection).(C) Immunoblot analysis of PCV2 Cap protein expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(D) The intensity represents PCV2 Cap protein level normalized to that of β-actin across three independent experiments.(E) Real-time qRT-PCR analysis of PCV2 ORF5 RNA expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(F) Immunoblot analysis of YWHAB protein expression in PK-15 cells transduced with lentivirus that knockdown YWHAB (shYWHAB-1, 2 or 3) or control lentivirus (shN) or untransduced (CTR).(G) Real-time qRT-PCR analysis of PCV2 viral RNA expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at indicated time points (12, 24, 36 and 48 h post-infection).(H) Immunoblot analysis of PCV2 Cap protein expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(I) The intensity represents PCV2 Cap protein level normalized to that of β-actin across three independent experiments.(J) Real-time qRT-PCR analysis of PCV2 ORF5 RNA expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.Data in (B, D, E, G, I and J) are shown as the mean ± SD of three independent experiments. Data in (A, C, F and H) are one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.Fig. 3', 'As shown in <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>F, a successful knockdown of YWHAB was obtained and following studies were performed on the shYWHAB-3 cell lines (F, a successful knockdown of YWHAB was obtained and following studies were performed on the shYWHAB-3 cell lines (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>F). Conversely, the PCV2 replication level was significantly increased in YWHAB-knockdown cells (F). Conversely, the PCV2 replication level was significantly increased in YWHAB-knockdown cells (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>G). The Cap protein expression and ORF5 gene expressions were also up-regulated in YWHAB silenced cells (G). The Cap protein expression and ORF5 gene expressions were also up-regulated in YWHAB silenced cells (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>H, I and J). Together, these results convincingly demonstrated that YWHAB inhibits PCV2 replication.H, I and J). Together, these results convincingly demonstrated that YWHAB inhibits PCV2 replication.', 'The PCV2 ORF5 protein has been evidenced to induce the Endoplasmic Reticulum Stress (ERS) and unfolded protein response (UPR) (Ouyang et al., 2019). In the present study, we found that a host factor YWHAB alleviates PCV2-induced ERS. The hallmark of ERS is the upregulation of glucose-regulated protein 78 (GRP78) and 94 (GRP94) (Bailey and O’Hare, 2007). During ERS, BiP/GRP78 or GRP94 binds to misfolded proteins and releases PERK, ATF6, and IRE1, resulting in their activation and initiation of the UPR (Tu and Weissman, 2004). In this study, we found that YWHAB significantly inhibits GRP78 and GRP94 expression upon PCV2 infection (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>, , <xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>). PCV2 infection triggers autophagy to facilitate its replication (). PCV2 infection triggers autophagy to facilitate its replication (Zhai et al., 2019). Our previous work showed that ORF5 protein induces autophagy through PERK-eIF2a-ATF4 and AMPK-ERK1/2-mTOR pathways to promotes viral replication (Lv et al., 2020). Given the importance of autophagy in regulating virus replication, it is not surprising that both PCV2 infection and host factors YWHAB can alter autophagy. Although no study has been reported that PCV2 ORF5 protein induces ROS production, PCV2 infection increases ROS production to facilitate PCV2 replication (Chen et al., 2012). PCV2 infection induced ROS production elicits dynamic relative protein1 (Drp1) phosphorylation and activation of the PINK1/Parkin pathway, which eventually activates mitophagy and mitochondrial apoptosis (Zhang et al., 2020). In this study, PCV2 infection induced ROS production is also observed (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>) and YWHAB potently decreases this effect. However, whether YWHAB inhibits PCV2 infection by suppressing GRP78/GRP94, autophagy or ROS production needs further investigation. Together, these results showed that the ORF5-interacting protein YWHAB potently alleviates the ORF5-elicited ERS, autophagy and ROS production. Nevertheless, further study is required to elucidate the underlying molecular mechanism of YWHAB in mitigating these cellular responses.) and YWHAB potently decreases this effect. However, whether YWHAB inhibits PCV2 infection by suppressing GRP78/GRP94, autophagy or ROS production needs further investigation. Together, these results showed that the ORF5-interacting protein YWHAB potently alleviates the ORF5-elicited ERS, autophagy and ROS production. Nevertheless, further study is required to elucidate the underlying molecular mechanism of YWHAB in mitigating these cellular responses.'], 'gr4_lrg': ['PCV2 infection and ORF5 protein induce the Endoplasmic Reticulum Stress (ERS) (Lv et al., 2015). To investigate whether the YWHAB protein could affect the PCV2-induced ERS, we measured the expression of GRP78 and GRP94, which are the hall markers of ERS, in YWHAB overexpressed or silenced cells upon PCV2 infection. Both mRNA expressions of GRP78 and GRP94 were significantly decreased in YWHAB overexpressed cells at 24 and 48 h post PCV2 infection (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\nA and B). Consistently, the protein expression levels of GRP78 and GRP94 was also induced to a lower level by PCV2 in YWHAB overexpressed cells (\nA and B). Consistently, the protein expression levels of GRP78 and GRP94 was also induced to a lower level by PCV2 in YWHAB overexpressed cells (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>C, D and E). In contrast, the gene expression of GRP78 and GRP94 was elevated in YWHAB silenced cells at 24 h and 48 h post PCV2 infection (C, D and E). In contrast, the gene expression of GRP78 and GRP94 was elevated in YWHAB silenced cells at 24 h and 48 h post PCV2 infection (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>\nA and B). Importantly, similar results were obtained for the protein expression level of GRP78 and GRP94 in YWHAB silenced cells (\nA and B). Importantly, similar results were obtained for the protein expression level of GRP78 and GRP94 in YWHAB silenced cells (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>C, D and E). Together, we revealed that YWHAB alleviated the PCV2-induced endoplasmic reticulum stress.C, D and E). Together, we revealed that YWHAB alleviated the PCV2-induced endoplasmic reticulum stress.Fig. 4YWHAB alleviates the PCV2-induced endoplasmic reticulum stress.Real-time qRT-PCR analysis of GRP78 (A) and GRP94 (B) gene expression in YWHAB overexpressed, lentivirus control (LV) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(C) Immunoblot analysis of GRP78 and GRP94 protein expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.The intensity represents GRP78 (D) and GRP94 (E) protein level normalized to that of β-actin across three independent experiments.Data in (A, B, D and E) are shown as the mean ± SD of three independent experiments. Data in (C) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.Fig. 4Fig. 5YWHAB knockdown potentiates the PCV2-induced endoplasmic reticulum stress.Real-time qRT-PCR analysis of GRP78 (A) and GRP94 (B) gene expression in YWHAB knockdown (shYWHAB), lentivirus control (shN) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(C) Immunoblot analysis of GRP78 and GRP94 protein expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.The intensity represents GRP78 (D) and GRP94 (E) protein level normalized to that of β-actin across three independent experiments. Data in (A, B, D and E) are shown as the mean ± SD of three independent experiments. Data in (C) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.Fig. 5'], 'gr6_lrg': ['Our previous work reported that ORF5 protein induces autophagy through PERK-eIF2a-ATF4 and AMPK-ERK1/2-mTOR pathways to promotes viral replication (Lv et al., 2020). To determine whether YWHAB affects the PCV2-induced autophagy, the autophagy markers Beclin1 and LC3-II were measured in YWHAB overexpressed or silenced cells. Importantly, PCV2 infection induced lower level of Beclin-1 gene expression in YWHAB overexpressed cells compared to that in control cells (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>\nA). In addition, the protein expression of LC3-II, the autophagy marker, was also less activated in YWHAB overexpressed cells (\nA). In addition, the protein expression of LC3-II, the autophagy marker, was also less activated in YWHAB overexpressed cells (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>B and C). In contrast, PCV2 infection induced stronger gene expression of Beclin-1 in YWHAB knockdown cells (B and C). In contrast, PCV2 infection induced stronger gene expression of Beclin-1 in YWHAB knockdown cells (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>D) and similar results were obtained for the protein expression of LC3-II (D) and similar results were obtained for the protein expression of LC3-II (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>E and F). Together, these results demonstrated that YWHAB inhibits the PCV2-induced autophagy.E and F). Together, these results demonstrated that YWHAB inhibits the PCV2-induced autophagy.Fig. 6YWHAB inhibits the PCV2-induced autophagy.(A) Real-time qRT-PCR analysis of Beclin1 gene expression in YWHAB overexpressed, lentivirus control (LV) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection. (B) Immunoblot analysis of LC3II protein expression in YWHAB overexpressed PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(C) The intensity represents LC3II protein level normalized to that of β-actin across three independent experiments.(D) Real-time qRT-PCR analysis of Beclin1 gene expression in YWHAB knockdown (shYWHAB), lentivirus control (shN) or untransduced (CTR) PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(E) Immunoblot analysis of LC3II protein expression in YWHAB knockdown PK-15 cells infected with PCV2 at an MOI of 1 at 24 h or 48 h post-infection.(F) The intensity represents LC3II protein level normalized to that of β-actin across three independent experiments.Data in (A, C, D and F) are shown as the mean ± SD of three independent experiments. Data in (B and E) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.Fig. 6'], 'gr7_lrg': ['Intracellular ROS production is an important indicator of cellular stresses. Previous studies showed that PCV2 infection induces the production of intracellular ROS (Sun et al., 2020; Zhang et al., 2019). To investigate the role of YWHAB in regulating PCV2-induced ROS production, we measured the intracellular ROS in YWHAB overexpressed and knockdown cells by using flow cytometry. In line with a previous study (Zhang et al., 2019), we confirmed that PCV2 infection strongly induced intracellular ROS level. Importantly, the intracellular ROS was significantly decreased in YWHAB upregulated cells (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>\n). Conversely, the intracellular ROS level was significantly elevated in the cells that have lower YWHAB expression (\n). Conversely, the intracellular ROS level was significantly elevated in the cells that have lower YWHAB expression (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>). This result indicated that YWHAB inhibits PCV2-induced ROS.). This result indicated that YWHAB inhibits PCV2-induced ROS.Fig. 7YWHAB inhibits PCV2-induced intracellular ROS.Flow cytometry analysis of ROS level in YWHAB overexpressed or knockdown PK-15 cells infected with PCV2 at an MOI of 1. At 24 h post-infection, the cells were incubated with 2′,7′-dichlorofluorescein diacetate (DHE, 5 μM) at 37 °C for 30 min and the ROS related fluorescent signal was detected by flow cytometry analysis.Data are shown as the mean ± SD of three independent experiments.**p < 0.01; ***p < 0.001.Fig. 7'], 'gr8_lrg': ['It has been demonstrated that PCV2 infection induces apoptosis in both cell culture model and animal model (Chang et al., 2007; Resendes et al., 2011). To explore whether the YWHAB affects the PCV2-induced apoptosis, the apoptosis in PCV2-infected YWHAB overexpressed or knockdown cells was measured at 24 h post-infection. As shown in <xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>\nA, the PCV2-induced apoptosis was strongly attenuated in YWHAB overexpressed cells compared to that in Lv control cells. As expected, the PCV2 infection activated apoptosis was potentiated in YWHAB silenced cells (\nA, the PCV2-induced apoptosis was strongly attenuated in YWHAB overexpressed cells compared to that in Lv control cells. As expected, the PCV2 infection activated apoptosis was potentiated in YWHAB silenced cells (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>B). Together, this result suggested that YWHAB inhibits cells from PCV2-induced apoptosis.B). Together, this result suggested that YWHAB inhibits cells from PCV2-induced apoptosis.Fig. 8YWHAB inhibits PCV2-induced apoptosis.Flow cytometry analysis of cell apoptosis in YWHAB overexpressed (A, CTR, untransduced control; LV, lentivirus control; LV-YWHAB, YWHAB overexpressed cells) or knockdown (B, CTR, untransduced control; shN, lentivirus control; shYWHAB, YWHAB knockdown cells) PK-15 cells infected with PCV2 at an MOI of 1. At 24 h post-infection, the cells were resuspended and stained with 5 μL Annexin V-FITC and 5 μL 7-aminoactinomycin D (7-AAD) for 10 min in the dark at room temperature. Fluorescent signals were detected by flow cytometry analysis. Data is one representative of those from three independent experiments.Fig. 8']}
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A novel PCV2 ORF5-interacting host factor YWHAB inhibits virus replication and alleviates PCV2-induced cellular response
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[
"Porcine circovirus type 2",
"Open reading frame 5 (ORF5)",
"YWHAB (14-3-3β/α)",
"Endoplasmic reticulum stress (ERS)",
"Autophagy",
"Apoptosis",
"Reactive oxygen species (ROS)"
] |
Vet Microbiol
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1608192000
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None
| null |
other
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PMC7568206
| null | null |
[
""
] |
Vet Microbiol. 2020 Dec 17; 251:108893
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NO-CC CODE
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Rac-independent functions of TIPE2 in chemotaxing cells(a-f) Confocal microscopy for subcellular distribution of TIPE2 (a-c), Rac-GTP (a,c,e) and F-actin (d,f) in wild-type (WT) and Tipe2−/− bone marrow neutrophils, which were rested or stimulated with CXCL8 (point source), with or without pretreatments with Rac inhibitor (NSC24766) (a,b) or PI(3)K inhibitor (LY294002) (a,c-f). Panels a, e and f show the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of the indicated molecules, whereas panels b-d show representative images (scale bars are 5 µm). ns, not significant. The experiments were repeated at least two times; a, e-f, n ≥ 30. *, P < 0.05; ***, P < 0.001; ns, not significant.
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nihms910780f4
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2
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225ff1527a323939f3f9451a5ea6cf81c74fde102c19fc4d6b8052a748588fef
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nihms910780f4.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
748,
1020
] |
[{'image_id': 'nihms910780f3', 'image_file_name': 'nihms910780f3.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f3.jpg', 'caption': 'Rac-dependent functions of TIPE2 in chemotaxing cells(a) Relative spreading areas of wild-type (WT) and Tipe2−/− bone marrow neutrophils, which were either rested or stimulated with CXCL8 from a point source (designated as stim), with or without pretreatments with Rac inhibitor (NSC24766) or PI(3)K inhibitor (LY294002). Values represent means ± SEM. The experiments were repeated at least two times, n ≥ 30. **, P < 0.01; ns, not significant; RU, relative units. (b-d) Co-immunoprecipitation (co-IP) analysis of TIPE2 interaction with Rac. The experiments were performed at least three times. The results of a representative experiments are shown. (b) Cytoplasmic (Cyt) and membrane (M) protein fractions of 293T cells expressing recombinant Flag-TIPE2 and HA-Rac proteins were subjected to co-IP with anti-Flag or control IgG. (c) Lysates of WT and Tipe2−/− BMDMs cultured with or without L929 cell supernatant (designated as L929 sup) or CCL2 (stimulated for 5 min) were subjected to co-IP with anti-Rac or control IgG. (d) Lysates of dHL-60T cells were subjected to co-IP with anti-TIPE2 or control IgG. The precipitates were analyzed by immunoblot for the indicated proteins. (e-h) The subcellular distributions of F-actin (e,g) and pAKT(308) (f,h) in WT and Tipe2−/− bone marrow neutrophils pretreated with Rac inhibitor and stimulated with CXCL8 (point source) were determined by confocal microscopy. Panels e and f show representative images of each cell types (scale bars are 5 µm), whereas panels g and h show the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of the indicated molecules in each cell type. e-h, The experiments were repeated three times; g and h, n ≥ 30. ***, P < 0.0001; ns, not significant.', 'hash': 'b766c0f033c8505572dacf8cb7e78c7c676b743b265326ae73ec2a89144261a8'}, {'image_id': 'nihms910780f4', 'image_file_name': 'nihms910780f4.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f4.jpg', 'caption': 'Rac-independent functions of TIPE2 in chemotaxing cells(a-f) Confocal microscopy for subcellular distribution of TIPE2 (a-c), Rac-GTP (a,c,e) and F-actin (d,f) in wild-type (WT) and Tipe2−/− bone marrow neutrophils, which were rested or stimulated with CXCL8 (point source), with or without pretreatments with Rac inhibitor (NSC24766) (a,b) or PI(3)K inhibitor (LY294002) (a,c-f). Panels a, e and f show the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of the indicated molecules, whereas panels b-d show representative images (scale bars are 5 µm). ns, not significant. The experiments were repeated at least two times; a, e-f, n ≥ 30. *, P < 0.05; ***, P < 0.001; ns, not significant.', 'hash': '225ff1527a323939f3f9451a5ea6cf81c74fde102c19fc4d6b8052a748588fef'}, {'image_id': 'nihms910780f5', 'image_file_name': 'nihms910780f5.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f5.jpg', 'caption': 'TIPE2 functions as a PtdIns(4,5)P2 transfer protein in PtdIns(3,4,5)P3-enriched lipid bilayers(a,b) The percentages of TIPE2, 15/16Q, and control protein (a), or α0-eGFP, α0 15/16Q-eGFP, and α0 4Q-eGFP (wild-type or lysine-mutated TIPE2 α0 helixes fused with eGFP) (b) bound to small unilamellar vesicles (SUV) containing the indicated lipids, as determined in the phosphoinositide binding assay. (c) The percentages of TopFluor (TF)-PtdIns(4,5)P2 extracted from SUV containing the indicated lipids by TIPE2 or control protein, as determined in the phosphoinositide extraction assay. FIU, fluorescence intensity units. (d) The percentages of TIPE2 or control protein remained unbound to SUV containing the indicated lipids, as determined in the phosphoinositide extraction assay. Values represent means ± SD. *, P < 0.05; **, P < 0.01; the experiments were repeated at least three times (n ≥ 3).', 'hash': '241252b847f587996c32cc41dbd7fed6c7b97b0c505197f0e30914cea3defc21'}, {'image_id': 'nihms910780f2', 'image_file_name': 'nihms910780f2.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f2.jpg', 'caption': 'TIPE2 is required for chemoattractant-induced leukocyte polarization(a) Time-lapse confocal microscopy for PtdIns(3,4,5)P3 distribution in control dHL-60C and TIPE2-deficient dHL-60T neutrophils subjected to point-source stimulation with CXCL8 over the indicated times. The PtdIns(3,4,5)P3 distribution was probed with eGFP-GRP1-PH domain; results are presented as degree of PtdIns(3,4,5)P3 polarization. Values represent means ± SD. The experiments were repeated two times, n ≥ 45. (b-g) Confocal microscopy for the indicated molecules in wild-type (WT) and Tipe2−/− bone marrow neutrophils subjected to point-source stimulations with CXCL8. Panels b, d and f show representative images of each cell types (scale bars are 5 µm), whereas panels c, e, and g show the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of the indicated molecules in each cell type. The experiments were repeated three times, n ≥ 30. ***, P < 0.0001.', 'hash': 'dac56fadc2ddf03ea687372ff445616c944ebe87d8506c022f6812b09ccfc0d2'}, {'image_id': 'nihms910780f1', 'image_file_name': 'nihms910780f1.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f1.jpg', 'caption': 'LY2940021. TIPE2 promotes leukocyte chemotaxis both in vivo and in vitro(a) Wild-type (WT) mice with acute peritonitis were intravenously injected with CFSE-labeled Tipe2−/− CD45.2+ and WT CD45.1+ bone marrow cells mixed at 1:1 ratio. The percentages of injected WT and Tipe2−/− Ly6G+ cells in the blood and peritoneal cavity were determined 16 h later by flow cytometry. Values represent means ± SEM. The experiments were repeated two times with n = 5. (b-d) Chemotaxis indexes of WT and Tipe2−/− bone marrow-derived macrophages (b), WT and Tipe2−/− bone marrow neutrophils (c), or control dHL-60C, TIPE2-deficient dHL-60T, and TIPE2-expressing dHL-60T neutrophils (d) migrating through transwell filters toward CCL2, CXCL2 or CXCL8 as indicated. Values represent means ± SD. The experiments were performed in duplicates and repeated three times, n=6. (e,f) Directionality (e) and velocity (f) of WT and Tipe2−/− blood neutrophils migrating toward CXCL1 (200 ng/ml) on µ-slides were measured as described in Methods. The experiments were performed at least three times. The results of a representative experiment are shown, n ≥ 145. DMI, directional migration index. Values represent means ± SEM; *, P < 0.05; **, P < 0.01.', 'hash': '4db45aec781b4d2bbceea68863b713a597dd1ef87b38c5fd442fe09b33015597'}, {'image_id': 'nihms910780f6', 'image_file_name': 'nihms910780f6.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f6.jpg', 'caption': 'TIPE2 controls phosphoinositide signaling through PtdIns(3,4,5)P3-dependent mechanisms(a) Time course of PI(3)K-catalyzed generation of PtdIns(3,4,5)P3 in the absence or presence of TIPE2 or 15/16Q at the indicated concentrations. RU, relative units. (b) Percentages of cofilin bound to small unilamellar vesicles (SUV) containing the indicated lipids in the absence or presence of TIPE2, 15/16Q, or control protein, as determined in the phosphoinositide binding assay. (c) The percentages of TIPE2, 15/16Q, or control protein bound SUV, as determined in the phosphoinositide binding assay of cofilin. (d-f) The degree of cofilin-dependent F-actin depolymerization in the presence of control protein, control protein plus SUV, or TIPE2 plus SUV was analyzed as described in Methods. FIU, fluorescence intensity units. For panels a, b and c, values represent means ± SD. For panels d-f, values represent means ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant; the experiments were repeated at least three times (n ≥ 3).', 'hash': 'f5d3e0293719d693cf37a577a52d54ae7d2787134257c81802745971226f05e9'}, {'image_id': 'nihms910780f7', 'image_file_name': 'nihms910780f7.jpg', 'image_path': '../data/media_files/PMC5690821/nihms910780f7.jpg', 'caption': 'Reduced encephalomyelitis and leukocyte infiltration in the nervous tissue of Tipe2−/− mice(a) Tipe2−/− and wild-type (WT) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce experimental autoimmune encephalomyelitis (EAE), and the clinical scores of the disease were recorded daily. The experiments were repeated three times, n = 8; P < 0.0001 for differences after day 10. (b) Twenty-five days after immunization, Tipe2−/− and WT mice were sacrificed, and their spinal cords collected, sectioned, and stained with hematoxylin and eosin. The experiments were repeated three times, n = 8; representative images of the spinal cord sections are shown; scale bars are 500 µm. (c) WT mice were sub-lethally irradiated, and injected intravenously with either Tipe2−/− or WT bone marrow cells. Seven weeks later, mice were immunized with MOG to induce EAE, and the clinical scores of the disease were recorded daily. The experiments were repeated three times, n = 10. P = 0.0005 for differences after day 16. (d,e) WT mice were sub-lethally irradiated, and injected with mixed WT and Tipe2−/− bone marrow cells (at a ratio of 1:1). Seven weeks later, mice were immunized with MOG peptide to induce EAE, and sacrificed on the day of the disease onset. The percentages of WT and Tipe2−/− leukocytes (Total cells) and CD11b+Ly6G+ cells in the blood and spinal cord leukocyte preparations were determined by flow cytometry. The experiments were repeated two times, n = 3. For panels a, c, d and e, values represent means ± SEM; *, P < 0.05; **, P < 0.01.', 'hash': 'cd452be2df9306dda1c7a74900a2bf6e6be6b6f6fb4ea4cb122f6f05104c98a4'}]
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{'nihms910780f1': ['To understand the role of TIPE2 in chemotaxis, we studied the migration of circulating Tipe2−/− and wild-type leukocytes into peritoneal cavity in a murine model of acute peritonitis. Significantly fewer TIPE2-deficient Ly6G+ myeloid cells migrated into the peritoneal cavity, leaving markedly more of them in the blood as compared to WT myeloid cells in the same mice (<xref ref-type="fig" rid="nihms910780f1">Fig. 1a</xref>). To study TIPE2-dependent chemotaxis ). To study TIPE2-dependent chemotaxis in vitro, we used transwell chambers (for transmigration) and µ-slides (for two-dimensional chemotaxis). Murine Tipe2−/− bone marrow-derived macrophages (BMDMs) and bone marrow neutrophils (BMNs), and human TIPE2-deficient HL-60 neutrophils (dHL-60T) all showed significant defects in migration through transwell filters following chemoattractant gradients, but did not exhibit any defect in random migration as compared to WT controls (<xref ref-type="fig" rid="nihms910780f1">Fig. 1b - d</xref> and and Supplementary Fig. 1a). The chemotaxis defect of dHL-60T cells could be fully rescued by expressing a wild-type TIPE2 transgene (<xref ref-type="fig" rid="nihms910780f1">Fig. 1d</xref> and and Supplementary Fig. 1a). Importantly, in the µ-slide chemotaxis assay, Tipe2−/− neutrophils showed significant reductions in both directionality (by ~62% as compared to WT cells) and velocity (by ~25%) (<xref ref-type="fig" rid="nihms910780f1">Fig. 1e,f</xref> and and Supplementary Fig. 1b,c). Taking together, these results indicate that TIPE2 controls leukocyte chemotaxis in vivo and in vitro.'], 'nihms910780f2': ['To explore how TIPE2 controls chemotaxis, we studied polarization of neutrophils in response to point-source chemoattractants. To visualize polarization, we expressed in cells an enhanced green fluorescent protein (eGFP)-tagged PtdIns(3,4,5)P3-specific probe (the GRP1-PH domain), or stained cells for F-actin, Rac-GTP (the active form of Rac), or p-AKT(T308), the active form of AKT phosphorylated at threonine 308 that serves as an indicator of PI(3)K activation2,3. By time-lapse microscopy, we compared polarization of WT and TIPE2-deficient dHL-60 neutrophils in response to point-source CXCL8 stimulation over a period of 400 seconds (<xref ref-type="fig" rid="nihms910780f2">Fig. 2a</xref> and and Supplementary Fig. 1d). CXCL8-induced polarization of WT dHL-60 control (dHL-60C) cells occurred almost immediately after chemokine exposure, with more than 60% of cells polarized 180 seconds later. Chemokine-induced polarization was markedly reduced in dHL-60T neutrophils, with only <14% of cells polarized at the end of the observation period.', 'Similarly, 150 seconds after the exposure to chemoattractants, the vast majority of wild-type BMNs were polarized, with F-actin, Rac-GTP, and p-AKT(T308) primarily localized at the leading edge of cells; by contrast, most Tipe2−/− BMNs were not polarized (<xref ref-type="fig" rid="nihms910780f2">Fig. 2b - g</xref> and and Supplementary Fig. 2a - e). Importantly, the polarization defect of dHL-60T neutrophils could be rescued by expressing a wild-type TIPE2 transgene (Supplementary Fig. 2f - h). TIPE2-deficient cells were sensitive to chemoattractant stimulation, as evidenced by elevated global abundance of p-AKT(T308) and F-actin (<xref ref-type="fig" rid="nihms910780f2">Fig. 2b,d,f</xref>, , Supplementary Notes and Supplementary Figs. 2-4). In addition, in response to chemoattractant stimulation, Tipe2−/− BMNs increased their spreading areas more significantly, but failed to acquire an elongated shape as compared to wild-type BMNs (<xref ref-type="fig" rid="nihms910780f3">Fig. 3a</xref>). Taken together, these results indicate that TIPE2 controls the stable polarization of the signaling and actin regulatory molecules that are essential for the formation of the leading and trailing edges in chemotaxing neutrophils.). Taken together, these results indicate that TIPE2 controls the stable polarization of the signaling and actin regulatory molecules that are essential for the formation of the leading and trailing edges in chemotaxing neutrophils.'], 'nihms910780f3': ['Inhibitory proteins of the chemoattractant-induced signaling and cytoskeletal activities are predicted to be essential for cell polarization and chemotaxis1,4–6. Consistent with our previous report, TIPE2 interacted with Rac in several cell types (<xref ref-type="fig" rid="nihms910780f3">Fig. 3b - d</xref>, and , and Supplementary Notes)18. We hypothesized that TIPE2-dependent inhibition of Rac was required for effective cell polarization20. Pretreatment of Tipe2−/− BMNs with a specific Rac inhibitor NSC24766 prior to chemokine stimulation significantly reduced cell spreading and abolished the difference in spreading areas between wild-type and Tipe2−/− BMNs (<xref ref-type="fig" rid="nihms910780f3">Fig. 3a</xref>). By contrast, PI(3)K inhibitor LY294002 did not have the same effect on cell spreading, suggesting that TIPE2 controls cell spreading through the interaction with Rac (). By contrast, PI(3)K inhibitor LY294002 did not have the same effect on cell spreading, suggesting that TIPE2 controls cell spreading through the interaction with Rac (<xref ref-type="fig" rid="nihms910780f3">Fig. 3a</xref>). Importantly, Rac inhibition partially rescued the polarization defect of ). Importantly, Rac inhibition partially rescued the polarization defect of Tipe2−/− BMNs and dHL-60T. F-actin and p-AKT(T308) were excluded from the edges of the cells opposite to the source of chemoattractants (<xref ref-type="fig" rid="nihms910780f3">Fig. 3e - h</xref> and and Supplementary Fig. 5a,b). Thus, TIPE2 served as a cellular Rac inhibitor, promoting trailing edge formation and suppressing the generation of secondary leading edges. However, Tipe2−/− BMNs and dHL-60T neutrophils treated with Rac inhibitor failed to form well-defined leading edges, strongly suggesting a Rac-GTP-independent role of TIPE2 in leading edge formation (<xref ref-type="fig" rid="nihms910780f3">Fig. 3e,f</xref> and and Supplementary Fig. 5b). Importantly, TIPE2 exhibited a polarized localization in migrating BMNs, dHL-60C and TIPE2-expressing dHL-60T. It is highly enriched at the leading edge, although also present throughout the cells (<xref ref-type="fig" rid="nihms910780f4">Fig. 4a</xref>, and , and Supplementary Fig. 5c). Since the interaction of TIPE2 with Rac took place in both the cytoplasm and plasma membrane, we asked if Rac-GTP, localized exclusively at the leading edge, was responsible for the polarized localization of TIPE2. Rac inhibition did not significantly change the localization of TIPE2 (<xref ref-type="fig" rid="nihms910780f4">Fig. 4a,b</xref> and and Supplementary Fig. 5b,c). However, pretreatment of wild-type BMNs, dHL-60C and TIPE2-expressing dHL-60T with a PI(3)K inhibitor abolished the polarized distribution of TIPE2 and F-actin (<xref ref-type="fig" rid="nihms910780f4">Fig. 4a,c-f</xref> and and Supplementary Fig. 5c,d). By contrast, the PI(3)K inhibitor only slightly altered Rac-GTP distribution. Rac-GTP was enriched in the leading edge, but was also detected at the trailing edge at low amounts (<xref ref-type="fig" rid="nihms910780f4">Fig. 4a,c,e</xref>). These data suggest that Rac-independent, but PI(3)K-dependent mechanisms are likely responsible for the polarized TIPE2 localization in migrating cells. Taken together, our results indicate that TIPE2 may play dual roles in the polarization of migrating cells. TIPE2 controls the trailing edge formation through inhibition of Rac, and employs its Rac-independent, PI(3)K-dependent activities for the formation of the leading edge.). These data suggest that Rac-independent, but PI(3)K-dependent mechanisms are likely responsible for the polarized TIPE2 localization in migrating cells. Taken together, our results indicate that TIPE2 may play dual roles in the polarization of migrating cells. TIPE2 controls the trailing edge formation through inhibition of Rac, and employs its Rac-independent, PI(3)K-dependent activities for the formation of the leading edge.'], 'nihms910780f5': ['To explore the Rac-independent actions of TIPE2 at leading edges, we investigated the functional significance of TIPE2 binding to phosphoinositides. We hypothesized that TIPE2 acted differently at membranes containing PtdIns(4,5)P2 and PtdIns(4,5)P2 plus PtdIns(3,4,5)P3, which are characteristics of trailing and leading edges, respectively. Using small unilamellar vesicles (SUV) made of phospholipid bilayers, we found that TIPE2 protein could effectively bind to SUV containing 10% PtdIns(4,5)P2 (with ~65% TIPE2 bound to the vesicle) and to a lesser extent to SUV containing 10% PtdIns(3,4,5)P3 (with ~14% TIPE2 bound), but it only very weakly bound to SUV containing both 10% PtdIns(4,5)P2 and 10% PtdIns(3,4,5)P3 (with ~4% TIPE2 bound) (<xref ref-type="fig" rid="nihms910780f5">Fig. 5a</xref> and and Supplementary Fig. 6a). Consistent with this finding, TIPE2 showed reduced binding to SUV containing 5% PtdIns(4,5)P2 and 5% PtdIns(3,4,5)P3 as compared to SUV containing 10% PtdIns(4,5)P2 (<xref ref-type="fig" rid="nihms910780f5">Fig. 5a</xref>). TIPE2 did not bind SUV containing PtdIns(4)P or only phosphatidylcholine (PC) (). TIPE2 did not bind SUV containing PtdIns(4)P or only phosphatidylcholine (PC) (<xref ref-type="fig" rid="nihms910780f5">Fig. 5a</xref>). Therefore, TIPE2 exhibited differential binding to SUV containing PtdIns(4,5)P). Therefore, TIPE2 exhibited differential binding to SUV containing PtdIns(4,5)P2, PtdIns(3,4,5)P3, or both of the phosphoinositides. Previously, we demonstrated that positively charged residues of α0 helix of TIPE3 mediates the formation of electrostatic interactions with the negatively charged phosphate groups of phosphoinositides10. To evaluate whether positively charged residues of α0 helix of TIPE2 contributed to this protein’s interaction with phosphoinositides, we generated TIPE2 K15/16Q mutant (15/16Q), in which lysines 15 and 16 in the α0 helix were replaced with glutamines (Supplementary Fig. 6b). Similar to TIPE2, 15/16Q could strongly bind SUV containing 10% PtdIns(4,5)P2 (<xref ref-type="fig" rid="nihms910780f5">Fig. 5a</xref> and and Supplementary Fig. 6a). However, this mutant almost completely lost the binding to SUV containing 10% PtdIns(3,4,5)P3 (<xref ref-type="fig" rid="nihms910780f5">Fig. 5a</xref>). In addition, 15/16Q could interact with PtdIns(4)P-containing SUV (). In addition, 15/16Q could interact with PtdIns(4)P-containing SUV (<xref ref-type="fig" rid="nihms910780f5">Fig. 5a</xref>). To determine to what degree the α0 helix contributed to TIPE2 binding to phosphoinositides, we fused eGFP with wild-type or lysine-mutated TIPE2 α0 helix. We found that the TIPE2 α0 helix could interact with phosphoinositide-containing SUV with the following order of preference: PtdIns(3,4,5)P). To determine to what degree the α0 helix contributed to TIPE2 binding to phosphoinositides, we fused eGFP with wild-type or lysine-mutated TIPE2 α0 helix. We found that the TIPE2 α0 helix could interact with phosphoinositide-containing SUV with the following order of preference: PtdIns(3,4,5)P3 > PtdIns(4,5)P2 > PtdIns(4)P, and that lysine mutations within the α0 helix negated this interaction (<xref ref-type="fig" rid="nihms910780f5">Fig. 5b</xref> and and Supplementary Fig. 6a). Importantly, unlike PtdIns(4,5)P2 that showed markedly reduced binding to the α0 helix as compared to its binding to the full-length TIPE2, PtdIns(3,4,5)P3 binding to the helix was similar to its binding to the full-length TIPE2 (Supplementary Fig. 6c). These results strongly suggest that TIPE2 interacts with PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, mostly through the electrostatic interactions formed between positively charged amino acids of its α0 helix and negatively charged phosphate groups of the phosphoinositide.', 'We asked whether the marked reduction in TIPE2 binding to SUV containing both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 resulted from PtdIns(3,4,5)P3-dependant extraction of PtdIns(4,5)P2 by TIPE2 from the vesicles. We hypothesized that the α0 helix of TIPE2 functioned as a flexible lid of its hydrophobic cavity, and that the conformational change induced by PtdIns(3,4,5)P3 binding displaced the lid and allowed TIPE2 to extract PtdIns(4,5)P2 from the lipid bilayer and transfer it to the solution10,21. Indeed, TIPE2 extracted and transferred PtdIns(4,5)P2 from the SUV containing both PtdIns(4,5)P2 and PtdIns(3,4,5)P3, but not from SUV containing no PtdIns(3,4,5)P3 (<xref ref-type="fig" rid="nihms910780f5">Fig. 5c</xref>). As expected, more than 81% of TIPE2 was not bound by SUV containing both PtdIns(4,5)P). As expected, more than 81% of TIPE2 was not bound by SUV containing both PtdIns(4,5)P2 and PtdIns(3,4,5)P3, which was significantly reduced in the absence of PtdIns(3,4,5)P3 in the SUV (<xref ref-type="fig" rid="nihms910780f5">Fig. 5d</xref>). Thus, TIPE2 may function as a PtdIns(4,5)P). Thus, TIPE2 may function as a PtdIns(4,5)P2 transfer protein only on membranes that contain PtdIns(3,4,5)P3, a characteristic of the leading edge of chemotaxing cells (Supplementary 6d).'], 'nihms910780f6': ['Since TIPE2 is capable of both binding and extracting PtdIns(4,5)P2, we examined whether TIPE2 could regulate PtdIns(4,5)P2-dependent signaling. First, we analyzed the ability of TIPE2 to promote the phosphorylation of PtdIns(4,5)P2 by active PI(3)Ks, a key process occurring at the leading edge of migrating cells. We found that TIPE2 indeed increased PI(3)K-catalyzed conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 for up to 5 fold, in a dose-dependent manner (<xref ref-type="fig" rid="nihms910780f6">Fig. 6a</xref>). 15/16Q that strongly bound PtdIns(4,5)P). 15/16Q that strongly bound PtdIns(4,5)P2 but almost completely lost its ability to bind PtdIns(3,4,5)P3 had a weak or no effect on PI(3)K-catalyzed PtdIns(3,4,5)P3 generation (<xref ref-type="fig" rid="nihms910780f6">Fig. 6a</xref>). Second, we tested if, through its interaction with PtdIns(4,5)P). Second, we tested if, through its interaction with PtdIns(4,5)P2 and PtdIns(3,4,5)P3, TIPE2 could affect actin remodeling, which is required for leading edge formation. For this study we selected cofilin, an actin-severing protein, whose activity to drive actin remodeling is inhibited by its binding to phosphoinositides22,23. We found that TIPE2 binding to SUV containing either PtdIns(4,5)P2 or PtdIns(3,4,5)P3 led to a small decrease in the phosphoinositide interaction with cofilin (<xref ref-type="fig" rid="nihms910780f6">Fig. 6b,c</xref>). By contrast, the interaction of cofilin with SUV containing both PtdIns(4,5)P). By contrast, the interaction of cofilin with SUV containing both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 was reduced for more than 7 fold by TIPE2 (<xref ref-type="fig" rid="nihms910780f6">Fig. 6b,c</xref>). The effect of 15/16Q on the cofilin binding to the vesicles was similar to that of wild-type TIPE2, but to a lesser degree, due presumably to its weakened binding to phosphoinositides (). The effect of 15/16Q on the cofilin binding to the vesicles was similar to that of wild-type TIPE2, but to a lesser degree, due presumably to its weakened binding to phosphoinositides (<xref ref-type="fig" rid="nihms910780f6">Fig. 6b,c</xref>). The expression of 15/16Q in dHL-60T neutrophils only partially rescued the polarization defect (). The expression of 15/16Q in dHL-60T neutrophils only partially rescued the polarization defect (Supplementary Fig. 6e).', 'To understand the impact of TIPE2 binding to phosphoinositides on cofilin activity, we examined cofilin-induced F-actin depolymerization in the presence or absence of purified TIPE2 protein and phosphoinositides. SUV containing either PtdIns(4,5)P2 or PtdIns(4,5)P2 plus PtdIns(3,4,5)P3 significantly decreased cofilin-induced F-actin depolymerization (<xref ref-type="fig" rid="nihms910780f6">Fig. 6d,e</xref> and and Supplementary Fig. 7a,b). However, TIPE2 could only rescue the decrease in F-actin depolymerization caused by SUV containing both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 (<xref ref-type="fig" rid="nihms910780f6">Fig. 6d,e</xref> and and Supplementary Fig. 7a,b). PtdIns(3,4,5)P3, TIPE2, or control protein alone had no effect on F-actin depolymerization (<xref ref-type="fig" rid="nihms910780f6">Fig. 6f</xref>, , Supplementary Fig. 7c, and data not shown). These results indicate that the ability of TIPE2 to extract PtdIns(4,5)P2 from PtdIns(3,4,5)P3-rich membranes impacts cofilin-induced F-actin remodeling.'], 'nihms910780f7': ['Directional migration of leukocytes into the central nervous system is crucial for the development of multiple sclerosis in humans, and experimental autoimmune encephalomyelitis (EAE) in mice24,25. Importantly, Tipe2−/− mice exhibited significantly delayed EAE onset and reduced clinical score relative to wild-type mice (<xref ref-type="fig" rid="nihms910780f7">Fig. 7a</xref>). Histological examination of spinal cord sections of mice with EAE revealed more severe leukocyte infiltration in the wild-type group than in the ). Histological examination of spinal cord sections of mice with EAE revealed more severe leukocyte infiltration in the wild-type group than in the Tipe2−/− group (<xref ref-type="fig" rid="nihms910780f7">Fig. 7b</xref>). Bone marrow chimeric experiments established that TIPE2 expressed by bone marrow-derived cells contributed to the difference in EAE between wild-type and ). Bone marrow chimeric experiments established that TIPE2 expressed by bone marrow-derived cells contributed to the difference in EAE between wild-type and Tipe2−/− mice. Wild-type mice that received Tipe2−/− bone marrow cells developed significantly delayed and diminished EAE as compared to wild-type mice that received wild-type bone marrow cells (<xref ref-type="fig" rid="nihms910780f7">Fig. 7c</xref>). Importantly, the resistance of ). Importantly, the resistance of Tipe2−/− mice to EAE was not caused by a reduction in T cell responses to myelin oligodendrocyte glycoprotein (MOG). Tipe2−/− splenocytes isolated from mice 9 days after the EAE induction produced normal or slightly increased concentrations of interleukin 2 (IL-2), interferon-γ and IL-17 after MOG-stimulation in vitro, as compared to wild-type splenocytes (Supplementary Fig. 8). To test whether reduced EAE in Tipe2−/− mice could be related to decreased leukocyte migration, we generated mixed bone marrow chimeric mice containing both Tipe2−/− and wild-type bone marrow-derived cells. At EAE onset, we observed significantly less Tipe2−/− total and CD11b+Ly6G+ leukocytes present in the spinal cord as compared to wild-type cells in the same mice (<xref ref-type="fig" rid="nihms910780f7">Fig. 7d,e</xref>). Thus, TIPE2 plays a crucial role in controlling leukocyte infiltration during neural inflammation.). Thus, TIPE2 plays a crucial role in controlling leukocyte infiltration during neural inflammation.']}
|
Directing leukocyte polarization and migration by the phosphoinositide transfer protein TIPE2
| null |
Nat Immunol
|
1514016000
|
Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients' outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients.
|
[
"Animals",
"Antineoplastic Agents",
"Antineoplastic Combined Chemotherapy Protocols",
"Cell Cycle Proteins",
"Cell Line, Tumor",
"Cell Survival",
"Cisplatin",
"Gene Expression Regulation, Neoplastic",
"Humans",
"Lung Neoplasms",
"M Phase Cell Cycle Checkpoints",
"Mesothelioma",
"Mesothelioma, Malignant",
"Mice, Inbred BALB C",
"Neoplasms, Experimental",
"Pemetrexed",
"Protein Kinase Inhibitors",
"Protein Serine-Threonine Kinases",
"Protein-Tyrosine Kinases",
"Pyrazoles",
"Pyrimidines",
"Survival Analysis"
] |
other
|
PMC5690821
| null |
58
|
[
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] |
Nat Immunol. 2017 Dec 23; 18(12):1353-1360
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Mst3b mediates the response of DRG neurons to NGF. (a–f) Representative photos of DRG neurons transfected with plasmids expressing shRNA constructs and/or variant forms of Mst3b as shown on the left. Cells were exposed to NGF (50 ng/ml) or media alone. Three days later, cells were fixed and immunostained to detect EGFP, a marker for transfected cells, and βIII-tubulin to identify neurons (antibody TuJ1). (g) Quantitation of axon growth. (a,b,g) Cells expressing the control shRNA extend neurites when treated with NGF (b,g). (c,g) Cells expressing mst3b shRNA (arrowheads) fail to respond to NGF; an nontransfected cell in the same field shows outgrowth (arrow). (d,g) NGF-induced outgrowth is restored when cells expressing mst3b shRNA (green) are co-transfected with a plasmid expressing human Mst3b. (e,g) Cells expressing kinase-dead (k/d) Mst3b (arrowheads) fail to extend neurites when treated with NGF; a non-transfected cell (arrows) in the same field shows outgrowth. (f,g) expression of constitutively active (c/a) Mst3b (arrowhead) is sufficient to induce outgrowth in the absence of NGF; arrow points to an untransfected cell in the same field that is not growing. Scale bar in a–f: 100 μm. Each experiment included 4 blinded, independent observations of about 10 cells per well, and each experiment was performed three times. **,***increase relative to baseline (control shRNA-transfected cells without NGF) significant at P < 0.01 or P < 0.001, respectively. †, ††† decrease relative to control shRNA-transfected cells treated with NGF significant at P<0.05 or P<0.001, respectively. Error bars represent s.e.m.
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
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[{'image_id': 'nihms144945f5', 'image_file_name': 'nihms144945f5.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f5.jpg', 'caption': 'Mst3b knockdown in DRG neurons. Sections through the rat DRG four weeks after injecting AAV2 expressing EGFP and either control or mst3b shRNA. (a) Infection of DRG neurons is demonstrated by co-immunostaining (arrows) with antibodies to the neuronal marker, βIII tubulin (TuJ1, red), and to virally expressed EGFP (green). Overall transfection rates were 21% for the control shRNA group (N = 10) and 35% for the mst3b shRNA group (N = 13). (b) AAV2 expressing EGFP and mst3b shRNA infects small IB4-positive (red) non-peptidergic neurons (arrows) as well as small IB-4-negative neurons (arrowheads). (c) AAV2 also infects large neurons (arrow) that stain positively with antibodies to the neurofilament protein NF200 (red). Scale bar: 40 μm. (d) Knockdown of Mst3b in DRG neurons. Ganglia were immunostained with antibodies to Mst3b (red) and EGFP (green) 4 weeks after infecting DRGs with AAV2 expressing EGFP and either control (top) or mst3b shRNA (bottom). Mst3b can be visualized in cells expressing control shRNA (arrows) (top), but not in cells expressing mst3b shRNA (arrowheads) (bottom). Scale bar: 40 μm.', 'hash': '5d74043e762e5947f41a4eeb6a50b7a81781b2a12c2dfce4d8a306ce02280ebc'}, {'image_id': 'nihms144945f2', 'image_file_name': 'nihms144945f2.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f2.jpg', 'caption': 'Infection of RGCs with AAV2 expressing shRNAs in vivo. (a) Animals received intraocular injections of AAV2 expressing EGFP and either a control shRNA or mst3b shRNA 4 weeks prior to dissection. Cross-sections through the retina show double-labeled cells (arrows) expressing TuJ1 (to identify RGCs: red) and EGFP (a marker for transfected cells: green). Transfection efficiencies averaged 58% for the virus expressing control shRNA and 68% for the virus expressing mst3b shRNA. (b) Suppression of Mst3b expression. Cross-sections through the retinas of animals injected 4 weeks earlier with AAV2 expressing EGFP and either control shRNA (top) or mst3b shRNA (bottom). Double-immunostaining with antibodies to Mst3b (red) and EGFP (green) shows co-expression of the two markers in cells expressing control shRNA (arrows, top), but a loss of Mst3b in RGCs expressing mst3b shRNA (arrowheads, bottom). Scale bar: 20 μm. (c) Quantitation of Mst3b expression. EGFP-positive cells expressing either control or mst3b shRNA were analyzed for intensity of Mst3b immunostaining in cross-sections through all cases using Image J. (d) Lack of macrophage infection: Four weeks after intravitreal injections of AAV2 expressing mst3b shRNA and EGFP, the lens was injured to induce an inflammatory response. Double immunostaining shows ED-1-positive macrophages to be uninfected, as evidenced by an absence of EGFP. Scale bar: 40 μm. Error bars represent s.e.m.', 'hash': '38849fd93e4a3bd9749af86e02fdbcd908b6b3ff9c95c84f2c08e94a4abc5ee9'}, {'image_id': 'nihms144945f3', 'image_file_name': 'nihms144945f3.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f3.jpg', 'caption': 'Mst3b is essential for optic nerve regeneration. RGCs were infected with AAV2 expressing EGFP plus either control or mst3b shRNA. Four weeks later, the optic nerve was crushed and the lens was injured to induce inflammation and transform RGCs into an active growth state. Regeneration was quantified 2 weeks later. (a) Longitudinal sections through the optic nerve stained for GAP-43 (red, a marker for regenerating axons) and EGFP (green, a marker for transfected neurons). Asterisk denotes the site of nerve injury; box shows the region magnified in b. Scale bar: 100 μm. (b) Area distal to the injury site from a case expressing mst3b shRNA. Merged image shows that GAP-43-positive axons that arise from uninfected RGCs (red), but not double-labeled axons (yellow) that arise from transfected neurons, extend distal to the injury site. Arrows indicate regenerating axons from uninfected RGCs. Scale bar: 100 μm. (c) Mean number of axons that regenerate ≥ 500 μm beyond the injury site. The set of bars on the left shows axons that arise from transfected RGCs. The number of regenerating axons was normalized by the percentage of transfected cells to account for differences in infection efficiency. The set of bars on the right shows axons of noninfected cells, normalized by the percentage of noninfected cells. Results are based on 7 animals per group, 4-6 sections per animal, and 0-24 axons per section. †††decrease relative to cases transfected with control shRNA significant at P < 0.001. Error bars represent s.e.m.', 'hash': '99845da04ddf7153bae0c508c2d41a5177daf55b3f561ddb83da06c6c7d71004'}, {'image_id': 'nihms144945f4', 'image_file_name': 'nihms144945f4.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f4.jpg', 'caption': 'Mst3b mediates the response of DRG neurons to NGF. (a–f) Representative photos of DRG neurons transfected with plasmids expressing shRNA constructs and/or variant forms of Mst3b as shown on the left. Cells were exposed to NGF (50 ng/ml) or media alone. Three days later, cells were fixed and immunostained to detect EGFP, a marker for transfected cells, and βIII-tubulin to identify neurons (antibody TuJ1). (g) Quantitation of axon growth. (a,b,g) Cells expressing the control shRNA extend neurites when treated with NGF (b,g). (c,g) Cells expressing mst3b shRNA (arrowheads) fail to respond to NGF; an nontransfected cell in the same field shows outgrowth (arrow). (d,g) NGF-induced outgrowth is restored when cells expressing mst3b shRNA (green) are co-transfected with a plasmid expressing human Mst3b. (e,g) Cells expressing kinase-dead (k/d) Mst3b (arrowheads) fail to extend neurites when treated with NGF; a non-transfected cell (arrows) in the same field shows outgrowth. (f,g) expression of constitutively active (c/a) Mst3b (arrowhead) is sufficient to induce outgrowth in the absence of NGF; arrow points to an untransfected cell in the same field that is not growing. Scale bar in a–f: 100 μm. Each experiment included 4 blinded, independent observations of about 10 cells per well, and each experiment was performed three times. **,***increase relative to baseline (control shRNA-transfected cells without NGF) significant at P < 0.01 or P < 0.001, respectively. †, ††† decrease relative to control shRNA-transfected cells treated with NGF significant at P<0.05 or P<0.001, respectively. Error bars represent s.e.m.', 'hash': '9e1e874d8f091ac5e4671b91596c6cfcc4169d26efd895607676bdb23b735163'}, {'image_id': 'nihms144945f7', 'image_file_name': 'nihms144945f7.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f7.jpg', 'caption': 'Mst3b activation and regulation of p42/44 MAPK phosphorylation in vivo. (a) Mst3b was immunoprecipitated from lysates of DRG neurons 3 days after peripheral nerve crush or sham surgery. Kinase assays were performed using HF1 as a pseudo-substrate and γ-[32P]-ATP, with or without 6-thioguanine present. Reaction mixes were separated by SDS-PAGE and kinase activity was visualized by autoradiography (top). Protein loading was visualized by Coomassie Blue staining (bottom). Position of molecular weight markers are shown on the right. (b) Quantitation of Mst3b kinase activity based on densitometry of autoradiograms using Image J (NIH) averaged from 3 separate experiments. (c,d) Cross-sections through the DRG of animals injected 4 weeks earlier with AAV2 expressing EGFP and either control shRNA (top) or mst3b shRNA (bottom). Arrows point to infected, EGFP-positive neurons. (c) Double-immunostaining with antibodies to phospho-p42/44 MAPK (red) and EGFP (green) reveal a selective reduction of phospho-MAPK in DRG neurons expressing the mst3b shRNA. (d) Double-immunostaining with antibodies to p42/p44 MAPK (red) and EGFP (green) reveal that Mst3b knock-down does not alter overall p42/44 MAPK levels. (e) Quantitation of changes in the overall expression and phosphorylation of p42/p44 MAPK based on 4-6 sections from 3 animals per condition. Scale bar: 40 μm. **Increase relative to controls significant at P < 0.01; †, ††Decrease relative to induced state significant at P < 0.05 or P < 0.01, respectively. Error bars represent s.e.m.', 'hash': '65c23af72ff98b0258eae8de4c57785c17b2c61e45c87d4c5e81efefd3e89d02'}, {'image_id': 'nihms144945f1', 'image_file_name': 'nihms144945f1.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f1.jpg', 'caption': 'Mst3b mediates the response of retinal ganglion cells (RGCs) to growth factors. (a–g) Representative photos of RGCs transfected with plasmids expressing the shRNA constructs and/or Mst3b variants shown on the left. Cells were exposed to media alone or to a combination of oncomodulin, mannose, and forskolin (Ocm/m/f) as indicated. Cells were fixed 3 days later and immunostained for EGFP, a marker for transfected cells, and either βIII-tubulin, a marker for RGCs (antibody TuJ1), or the myc-his-tag present on recombinant human Mst3b. (h) Quantitation of axon outgrowth. RGCs transfected with control shRNA extended axons when exposed to Ocm/m/f (a,b,h), whereas cells expressing mst3b shRNA did not (c,h). Outgrowth was restored when RGCs expressing mst3b shRNA were co-transfected with a plasmid expressing his-tagged human Mst3b (d,h). Expression of human Mst3b was verified by immunostaining for the his tag (e). Expression of kinase-dead (k/d) Mst3b blocked the effects of growth factors (f,h), whereas expression of constitutively active (c/a) Mst3b resulted in outgrowth even with growth factors absent (g,h). Scale bar for a–g: 40 μm. Each experiment included 4 blinded, independent observations (10–100 cells per well), and each experiment was repeated 3 times. *, **, ***increase relative to untreated controls significant at P < 0.05, P < 0.01, or P < 0.001, respectively. †††decrease relative to control cells treated with growth factors significant at P < 0.001. Error bars represent s.e.m.', 'hash': '872436f53081cf6903f104b1207fc0f24ee396bc83e4fa6946df798fcbe85dee'}, {'image_id': 'nihms144945f6', 'image_file_name': 'nihms144945f6.jpg', 'image_path': '../data/media_files/PMC2770175/nihms144945f6.jpg', 'caption': 'Mst3b knockdown attenuates peripheral nerve regeneration. (a–d) The radial nerve was crushed 4 weeks after infecting cervical DRG neurons with AAV2 expressing EGFP and either mst3b- or control shRNA. Axons extending beyond the injury site (arrowheads) were visualized 3 days later in sections stained with antibodies to detect GAP-43 (red) and EGFP (green). The box in b is magnified in panel e. Only profiles that are labeled with both markers are considered as regenerating axons that arise from infected neurons (arrows). Scale bar for a–d: 200 μm. (e) High-power images show regenerating axons that arise from neurons expressing control shRNA: many axons are positive for both GAP-43 (red) and EGFP (green) (arrows). Scale bar: 100μm. (f) Quantitation of GAP-43+/EGFP+ axons. The number of regenerating axons was normalized by the percent of infected cells to account for differences in infection efficiency. Note the marked attenuation of axon growth in cells expressing mst3b shRNA. (g) Quantitation of GAP-43+/EGFP- axons from non-infected neurons shows similar levels of regeneration irrespective of the virus infecting neighboring neurons. Data show mean number of axons at the indicated distances from the injury site (8-9 animals per group, 4-6 sections per animal, 0-10 axons per section). **,***Differences between number of axons arising from cells expressing control vs. mst3b shRNA are significant at P < 0.01, P < 0.001, respectively. Error bars represent s.e.m.', 'hash': '77bd6178108eaecb0513f0f9449af61ec072b58000e5b72afd775a29b7798a27'}]
|
{'nihms144945f1': ['We transfected RGCs with plasmids expressing mst3b shRNA to investigate whether these cells require Mst3b to regenerate axons in response to appropriate growth factors in culture. We dissociated retinas from normal, intact animals, placed these cells into culture, and transfected them with plasmids expressing enhanced green fluorescent protein (EGFP) and either a control shRNA or an mst3b-specific shRNA that was previously shown to knock down Mst3b expression16. The control shRNA differs from mst3b shRNA in 2 nucleotide positions and does not affect axon outgrowth16. After 3 days of treatment with the combination of oncomodulin (Ocm), mannose, and forskolin, RGCs expressing the control shRNA showed a 3-fold increase in axon outgrowth relative to untreated controls (difference significant at P < 0.001: <xref rid="nihms144945f1" ref-type="fig">Fig. 1a,b,h</xref>). In contrast, RGCs expressing ). In contrast, RGCs expressing mst3b shRNA showed little response to these factors (<xref rid="nihms144945f1" ref-type="fig">Fig. 1,c,h</xref>).).', 'To investigate whether the reduced outgrowth seen in this study is specifically related to a reduction in Mst3b levels, we co-transfected cells with 2 plasmids, one expressing mst3b shRNA and the other expressing his-tagged human Mst3b. The mRNA sequence of human mst3b differs significantly from that of the rat and its expression is not blocked by the shRNA construct used here16. We verified the presence of exogenous human Mst3b by staining cells with an anti-his antibody that recognizes his-tagged Mst3b (<xref rid="nihms144945f1" ref-type="fig">Fig. 1e</xref>). Co-transfecting RGCs with the two plasmids restored RGCs\' ability to extend axons in response to growth factors (). Co-transfecting RGCs with the two plasmids restored RGCs\' ability to extend axons in response to growth factors (P < 0.01; <xref rid="nihms144945f1" ref-type="fig">Fig. 1d,e,h</xref>). Thus, the effect of ). Thus, the effect of mst3b shRNA in blocking outgrowth depends specifically on its ability to knock down Mst3b expression and not on off-target effects.', 'To determine whether Mst3b exerts its effects through its kinase activity, we transfected RGCs with a plasmid expressing a mutant form of the protein with an amino acid change in the ATP-binding site16. Expression of this kinase-dead (k/d) Mst3b mutant eliminated the response of RGCs to growth factors (<xref rid="nihms144945f1" ref-type="fig">Fig. 1f,h</xref>).).', 'To investigate whether the kinase activity of Mst3b plays an active role in axon growth, we next transfected RGCs with a plasmid expressing a constitutively active (c/a) mutant of Mst3b. To create c/a Mst3b, we mutated threonine190 to aspartate, a change that has been shown to activate Mst3, an isotype of Mst3b23. Expression of c/a Mst3b enabled RGCs to grow axons even with growth factors absent (<xref rid="nihms144945f1" ref-type="fig">Fig 1g,h</xref>). Thus, the kinase activity of Mst3b is necessary and sufficient for axon outgrowth in RGCs. Low magnification photographs illustrating the transfection efficiency and outgrowth in these cultures are shown in ). Thus, the kinase activity of Mst3b is necessary and sufficient for axon outgrowth in RGCs. Low magnification photographs illustrating the transfection efficiency and outgrowth in these cultures are shown in Supplementary Fig.1 online.', 'To verify that virally mediated Mst3b reduction still suppressed axon growth after the time delay used in our in vivo studies, we waited 4 weeks after injecting AAVs, euthanized animals, dissected and dissociated retinas, and cultured cells6 in the presence or absence of Ocm, mannose, and forskolin6,10,27. In RGCs expressing the control shRNA, the addition of Ocm, forskolin and mannose caused a two-fold increase in average axon length over baseline (difference significant at P < 0.001: Supplementary Fig. 2 on line). RGCs expressing mst3b shRNA extended much shorter axons with or without growth factors present (difference from control-infected RGCs significant at P < 0.001: Supplementary Fig. 2 online). Thus, the effect of Mst3b knockdown remains strong several weeks after viral infection and results in a reduction of both baseline- and growth factor-stimulated levels of axon outgrowth. The lower overall outgrowth seen in <xref rid="nihms144945f1" ref-type="fig">Fig. 1h</xref> compared to that in compared to that in Supplementary Fig. 2e may reflect a general decrease in RGCs\' ability to extend axons after being exposed to the reagents used for the transient transfections.'], 'nihms144945f2': ['To investigate the role of Mst3b in vivo, we injected adult rats intravitreally with AAV2 expressing EGFP from a CMV promoter and, from a U6 promoter, either the shRNA that blocks Mst3b expression or the control shRNA. Animals survived for 4 weeks after viral infections prior to undergoing surgery to injure the optic nerve and induce intravitreal inflammation. The 4-week delay allowed for high levels of shRNA expression and degradation of preexisting Mst3b. In conformity with earlier reports on the high infection efficiency of AAV224-26, we found that AAV2 expressing EGFP and either of the two shRNAs enabled us to visualize EGFP in up to 68% of RGCs 4 weeks after infection (<xref rid="nihms144945f2" ref-type="fig">Fig 2a</xref>). Infecting RGCs with AAV2 expressing ). Infecting RGCs with AAV2 expressing mst3b shRNA strongly suppressed Mst3b expression. Using an antibody that recognizes the N-terminal region of Mst3b16, we detected the protein in essentially every EGFP-positive RGC expressing control shRNA, but observed an ∼85% reduction in EGFP-positive RGCs expressing mst3b shRNA (<xref rid="nihms144945f2" ref-type="fig">Fig. 2b,c</xref>). As expected from the known tropism of AAV2 and the 4 week separation between viral injections and macrophage induction, intraocular injections of AAV2 expressing EGFP and ). As expected from the known tropism of AAV2 and the 4 week separation between viral injections and macrophage induction, intraocular injections of AAV2 expressing EGFP and mst3b shRNA did not infect macrophages nor block their activation (<xref rid="nihms144945f2" ref-type="fig">Fig. 2d</xref>).).'], 'nihms144945f3': ['In vivo, regenerating axons that arise from infected RGCs were distinguished by virtue of staining positively for both EGFP and GAP-43. The membrane phosphoprotein GAP-43 is normally undetectable in the mature optic nerve, but is strongly induced in axons undergoing regeneration2,3,28. Profiles that are GAP-43+/EGFP-, on the other hand, are expected to reflect regenerating axons that arise from uninfected neurons. Remnants of degenerating axons that already contained EGFP before injury, but which are not regenerating, would be GAP-43-/EGFP+. Animals infected with control shRNA (N = 7) showed many GAP-43+/EGFP+ axons regenerating ≥ 0.5 mm past the lesion site (<xref rid="nihms144945f3" ref-type="fig">Fig. 3a</xref>, , top). In contrast, animals with Mst3b knocked down (N = 7) showed a 97% reduction in GAP-43+/GFP+ regenerating axons (<xref rid="nihms144945f3" ref-type="fig">Fig. 3b</xref>) (difference significant at ) (difference significant at P < 0.001: <xref rid="nihms144945f3" ref-type="fig">Fig. 3a, bottom and 3c</xref>). In all cases, the number of axons was normalized by the percentage of RGCs found to be EGFP-positive to account for individual variations in infection rates. As an internal control, we also quantified the number of regenerating axons arising from ). In all cases, the number of axons was normalized by the percentage of RGCs found to be EGFP-positive to account for individual variations in infection rates. As an internal control, we also quantified the number of regenerating axons arising from non-infected RGCs (GAP-43+/EGFP-) and found this number to be similar between animals infected with AAV2 expressing control vs. mst3b shRNA (<xref rid="nihms144945f3" ref-type="fig">Fig. 3c</xref>). Thus, Mst3b suppression essentially eliminated axon regeneration in the mature optic nerve, but did not alter the growth capacity of neighboring non-infected neurons.). Thus, Mst3b suppression essentially eliminated axon regeneration in the mature optic nerve, but did not alter the growth capacity of neighboring non-infected neurons.'], 'nihms144945f4': ['In contrast to the optic nerve, axon regeneration occurs readily in peripheral nerves after injury. Axons that arise from neurons in cervical DRGs 7 and 8 begin to regenerate past a lesion site in the radial nerve within a day or two, with full recovery of function taking place over a few weeks22,31. As with RGCs, we first used transient transfection assays to evaluate whether Mst3b mediates the response of adult DRG neurons to trophic factors, in this case nerve growth factor (NGF). We dissociated DRG neurons from non-infected, non-lesioned animals and transfected these with plasmids expressing EGFP and either control or mst3b-specific shRNA. After being exposed to NGF for 3 days, DRG neurons expressing the control shRNA showed a 2-fold increase in average axon length relative to untreated controls (difference significant at P < 0.001: <xref rid="nihms144945f4" ref-type="fig">Fig. 4a,b,g</xref>), whereas DRG neurons expressing ), whereas DRG neurons expressing mst3b shRNA showed no response (<xref rid="nihms144945f4" ref-type="fig">Fig. 4c,g</xref>). To investigate whether this failure to respond to NGF was due specifically to reduced Mst3b expression, we co-transfected DRG neurons with plasmids expressing ). To investigate whether this failure to respond to NGF was due specifically to reduced Mst3b expression, we co-transfected DRG neurons with plasmids expressing mst3b shRNA and human Mst3b, as above. Co-expression of the two plasmids restored DRG neurons\' ability to respond to NGF (P < 0.01; <xref rid="nihms144945f4" ref-type="fig">Fig. 4d,g</xref>). Thus, the effect of ). Thus, the effect of mst3b shRNA in blocking outgrowth is due specifically to knockdown of Mst3b expression. These studies show that Mst3b is essential for DRG neurons\' ability to extend axons in response to NGF.', 'As in RGCs, we next investigated whether Mst3b participates in axon outgrowth through changes in its kinase activity. To do this, we transfected DRG neurons with either the kinase-dead or constitutively active form of Mst3b described above. Expression of k/d Mst3b eliminated the ability of DRG neurons to extend axons in response to NGF (<xref rid="nihms144945f4" ref-type="fig">Fig. 4e,g</xref>). In contrast, transfection with c/a Mst3b caused these cells to extend long axons even in the absence of NGF (). In contrast, transfection with c/a Mst3b caused these cells to extend long axons even in the absence of NGF (<xref rid="nihms144945f4" ref-type="fig">Fig. 4f,g</xref>). Thus, the axon-promoting effect of NGF on DRG neurons appears to be mediated via changes in the kinase activity of Mst3b.). Thus, the axon-promoting effect of NGF on DRG neurons appears to be mediated via changes in the kinase activity of Mst3b.'], 'nihms144945f5': ['Four weeks after injecting AAV2 expressing EGFP and either control shRNA or mst3b shRNA into cervical ganglia, the percentage of TuJ1+ neurons found to be transfected was 21.4 ± 2.9% and 34.9 ± 2.1%, respectively. Transduction was neuron-specific (<xref rid="nihms144945f5" ref-type="fig">Fig. 5a</xref>) and included small peptidergic (IB4) and included small peptidergic (IB4-) and non-peptidergic (IB4+) neurons, as well as large NF200+ neurons (<xref rid="nihms144945f5" ref-type="fig">Fig. 5b,c</xref>). Compared to cells expressing control shRNA, Mst3b levels were reduced by ∼ 85% in DRG neurons transfected with AAV2 expressing ). Compared to cells expressing control shRNA, Mst3b levels were reduced by ∼ 85% in DRG neurons transfected with AAV2 expressing mst3b shRNA (<xref rid="nihms144945f5" ref-type="fig">Fig. 5d</xref>).).'], 'nihms144945f6': ['To investigate the role of Mst3b in peripheral nerve regeneration in vivo, we prepared a separate group of animals in which cervical DRG neurons were injected with AAV2 expressing EGFP and either the control or mst3b-specific shRNA. We then crushed the radial nerve 4 weeks later and evaluated axon regeneration after three days, a time point at which injured axons have normally grown several millimeters beyond the injury site (<xref rid="nihms144945f6" ref-type="fig">Fig. 6</xref>). As in the optic nerve, we assessed regeneration arising from infected neurons as the number of axons beyond the injury site that were positive for both GAP-43 and EGFP, and then normalized this number to the percentage of cells that were transfected to account for individual variations in transfection efficiency. In cases injected with AAV2 expressing the control shRNA, many EGFP). As in the optic nerve, we assessed regeneration arising from infected neurons as the number of axons beyond the injury site that were positive for both GAP-43 and EGFP, and then normalized this number to the percentage of cells that were transfected to account for individual variations in transfection efficiency. In cases injected with AAV2 expressing the control shRNA, many EGFP+/GAP-43+ axons extended 1-2 mm distal to the injury site, and some extended as far as 5 mm (<xref rid="nihms144945f6" ref-type="fig">Fig. 6a,b,e,f</xref>: N = 8). Animals expressing : N = 8). Animals expressing mst3b shRNA showed an 80 – 85% reduction in axon growth (N = 9; difference from controls significant at P < 0.001 at 0.5 mm, P < 0.01 at 1, and 2 mm; N.S. at 3, 4, and 5 mm: <xref rid="nihms144945f6" ref-type="fig">Fig. 6c,d,f</xref>). We also evaluated regeneration in non-infected DRG neurons in these same cases, that is, the number of axons that were GAP-43-positive and EGFP-negative, normalized by the percentage of cells that were uninfected. Axons arising from non-infected DRG neurons regenerated to a similar extent regardless of whether their neighbors expressed the control or ). We also evaluated regeneration in non-infected DRG neurons in these same cases, that is, the number of axons that were GAP-43-positive and EGFP-negative, normalized by the percentage of cells that were uninfected. Axons arising from non-infected DRG neurons regenerated to a similar extent regardless of whether their neighbors expressed the control or mst3b shRNA (<xref rid="nihms144945f6" ref-type="fig">Fig. 6g</xref>). In sum, knocking down Mst3b expression in DRG neurons strongly attenuated, but did not eliminate, the regeneration of injured axons ). In sum, knocking down Mst3b expression in DRG neurons strongly attenuated, but did not eliminate, the regeneration of injured axons in vivo.'], 'nihms144945f7': ['To determine whether the activity of Mst3b in DRG neurons increases during regeneration, we isolated ganglia 3 days after peripheral nerve injury and compared their Mst3b kinase activity with that of control DRG neurons using immunoprecipitation-kinase (IP-kinase) assays as described16. We precipitated Mst3b from DRG lysates using the antibody to the N-terminal region of the protein, and evaluated kinase activity using histone protein HF1 as an experimental substrate. DRG neurons undergoing axon regeneration showed a 2.5-fold increase in Mst3b kinase activity compared with control neurons (P < 0.01). 6-thioguanine, an inhibitor of Mst3b, attenuated this increase (P < 0.05: <xref rid="nihms144945f7" ref-type="fig">Fig. 7a,b</xref>).).', 'The MAP kinase pathway contributes to the development and regeneration of peripheral axons32,33 and to the effect of NGF in inducing outgrowth in PC12 cells34,35. The observation that knocking down Mst3b inhibits outgrowth in both sensory neurons and PC12 cells16 is consistent with the possibility that it acts via a MAPK pathway. To examine how Mst3b fits into the signal transduction cascade that leads to axon outgrowth, we compared p42/44 MAPK phosphorylation in neurons expressing control vs. mst3b shRNA. In sections through DRGs expressing mst3b shRNA, immunostaining for phospho-p42/44 MAPK was detected in 11% of neurons, compared to 87% of neurons expressing the control shRNA (<xref rid="nihms144945f7" ref-type="fig">Fig. 7c–e</xref>). The level of total p42/44 MAPK did not differ between the two groups.). The level of total p42/44 MAPK did not differ between the two groups.']}
|
Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways
| null |
Nat Neurosci
|
1259136000
|
Mammalian sterile 20-like kinase-3b (Mst3b, encoded by Stk24), regulates axon outgrowth in embryonic cortical neurons in culture, but its role in vivo and in neural repair is unknown. Here we show that Mst3b mediates the axon-promoting effects of trophic factors in mature rat retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons, and is essential for axon regeneration in vivo. Reducing Mst3b levels using short hairpin RNA prevented RGCs and DRG neurons from regenerating axons in response to growth factors in culture, as did expression of a kinase-dead Mst3b mutant. Conversely, expression of constitutively active Mst3b enabled both types of neurons to extend axons without growth factors. In vivo, RGCs lacking Mst3b failed to regenerate injured axons when stimulated by intraocular inflammation. DRG neurons regenerating axons in vivo showed elevated Mst3b activity, and reducing Mst3b expression attenuated regeneration and p42/44 MAPK activation. Thus, Mst3b regulates axon regeneration in both CNS and PNS neurons.
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[
"Animals",
"Calcium-Binding Proteins",
"Central Nervous System",
"Disease Models, Animal",
"Ganglia, Spinal",
"Gene Expression Regulation",
"Green Fluorescent Proteins",
"Intercellular Signaling Peptides and Proteins",
"Male",
"Nerve Regeneration",
"Neurons",
"Optic Nerve Injuries",
"Peripheral Nerves",
"Protein Serine-Threonine Kinases",
"RNA, Antisense",
"Rats",
"Rats, Inbred F344",
"Rats, Sprague-Dawley",
"Retina",
"Spinal Cord Injuries",
"Tubulin"
] |
other
|
PMC2770175
| null |
50
|
[
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] |
Nat Neurosci. 2009 Nov 25; 12(11):1407-1414
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NO-CC CODE
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Therapeutic effects of miR-34aa. Tail vein-injected miR-34a inhibited orthotopic PC3 tumor growth (n = 9 each). b–d. Tail vein-injected miR-34a oligos inhibited metastasis (GFP+ foci in the endpoint lungs; mean ± S.D, n = 6/group) of orthotopic LAPC9-GFP tumors (b) without significantly affecting tumor growth (c) and extended animal survival (d; Kaplan-Meier analysis and Log-Rank test). e,f. The fourth set of therapeutic experiment in LAPC9 cells. Shown are representative lung images (e; animal number and tumor weight indicated on top; scale bar, 100 μm) and quantification of lung metastases (f; mean ± S.D, n = 10/group).
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
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[{'image_id': 'nihms255508f3', 'image_file_name': 'nihms255508f3.jpg', 'image_path': '../data/media_files/PMC3076220/nihms255508f3.jpg', 'caption': 'miR-34a inhibits clonal and clonogenic properties of PCa cellsa. Holoclone assays in Du145 cells. Cells transfected with miR-NC (NC) or miR-34a (34a) oligos were used in three experiments (Exp. I, 100 cells/well scored on d 9; Exp. II, 100 cells/well scored on d 13; Exp. III, 500 cells/well scored on d 7). b. Clonogenic assays in Du145 cells. Cells (3,000/well) were plated in MG and colonies counted on day 13. NT, non-transfected. c. MG clonogenic assays in LAPC4 cells. Two experiments were performed (Exp. I, 1,250 cells/well scored on d 5, *P = 0.005; Exp. II, 25,000 cells/well scored on d 5, **P = 0.015). d. Sphere assays in LAPC4 cells. LAPC4 cells infected with lenti-ctl (C) or lenti-34a were plated (10,000 cells/well) for both 1° and 2° assays and spheres scored on d 15. e. Holoclone assays in PPC-1 cells. Cells transfected with miR-NC or miR-34a oligos were plated (500 cells/well) in triplicate and holoclones quantified on d 5. f. Sphere assays in HPCa101 (Gleason 9) cells. Purified HPCa101 cells infected with lenti-ctl (C) or lenti-34a were plated (20,000 cells/well) for both 1° and 2° and spheres scored 3 weeks later. g,h. Sphere assays in purified CD44+ HPCa116 (Gleason 7) cells transfected with NC or miR-34a oligos (g) or CD44− HPCa116 cells transfected with anti-NC or anti-34a oligos (h). Spheres were scored on d 15.', 'hash': '3b9657fcda67342512a6ad5494d75310839702500fb2d6618f1650fb8f10e82b'}, {'image_id': 'nihms255508f4', 'image_file_name': 'nihms255508f4.jpg', 'image_path': '../data/media_files/PMC3076220/nihms255508f4.jpg', 'caption': 'CD44 as a direct and functional target of miR-34aa. Representative CD44 IHC images in Du145 tumors from cells infected with MSCV-PIG (control) or MSCV-34a vectors (Western blot on the right) and PC3 tumors harvested from animals treated with miR-NC or miR-34a oligos. Scale bars, 10 μm. b. miR-34a downregulates CD44 in Du145 (left) and PPC-1 (right) cells. Relative levels of CD44 indicated at the bottom. c. Schematic of two putative miR-34a binding sites in the CD44 3’-UTR. d. Luciferase experiments in Du145 cells (*P <0.01). e. CD44 knockdown inhibits LAPC4 tumor regeneration (see Supplementary Fig. 12). f,g. CD44 knockdown inhibits PC3 cell metastasis evidenced by both quantification (f) and images (g; scale bar, 100 μm). h,i. Invasion assays. miR-34a oligos inhibit Matrigel invasion of CD44+ Du145 cells (h), which was partially overcome by overexpression of a human CD44 cDNA lacking the miR-34a binding sites at the 3’-UTR (i). Invasion was expressed as values relative to the corresponding controls. j. A schematic summary. The part highlighted in red refers to the novel findings made in the present study.', 'hash': '157dea3c5224f4c95fd0df7584e8aca351d7998ab70f014f455cfc3301fcf275'}, {'image_id': 'nihms255508f2', 'image_file_name': 'nihms255508f2.jpg', 'image_path': '../data/media_files/PMC3076220/nihms255508f2.jpg', 'caption': 'Therapeutic effects of miR-34aa. Tail vein-injected miR-34a inhibited orthotopic PC3 tumor growth (n = 9 each). b–d. Tail vein-injected miR-34a oligos inhibited metastasis (GFP+ foci in the endpoint lungs; mean ± S.D, n = 6/group) of orthotopic LAPC9-GFP tumors (b) without significantly affecting tumor growth (c) and extended animal survival (d; Kaplan-Meier analysis and Log-Rank test). e,f. The fourth set of therapeutic experiment in LAPC9 cells. Shown are representative lung images (e; animal number and tumor weight indicated on top; scale bar, 100 μm) and quantification of lung metastases (f; mean ± S.D, n = 10/group).', 'hash': '8788ff4255ba8b3caf01198884542ca13dba2246d9526ddd235fe81e9792ea45'}, {'image_id': 'nihms255508f1', 'image_file_name': 'nihms255508f1.jpg', 'image_path': '../data/media_files/PMC3076220/nihms255508f1.jpg', 'caption': 'Underexpression and tumor-inhibitory effects of miR-34aa. Experimental scheme. b,c. Lower miR-34a levels in CD44+ xenograft (b; PC4, LAPC4) or primary tumor (HPCa; c) cells. Results are expressed as the mean % of marker-positive over marker-negative cells. d,e. miR-34a inhibited LAPC9 (d) and HPCa58 (e; black, lenti-ctl; grey, lenti-34a; n = 10 and n = 7 for 1° and 2° experiments) tumor growth (mean ± S.D). f. CD44+ Du145 cells infected with lenti-ctl or lenti-34a were injected (10,000 cells each) s.c in NOD-SCID mice. Tumor incidence was 10/10 and mean weight was 0.6 g for lenti-ctl group whereas the incidence for lenti-34a group was 0/10. g. Purified CD44+ LAPC9 cells were transfected with miR-NC or miR-34a and s.c injected. Tumor incidence was 7/8 and mean weight was 0.5 g for miR-NC group whereas incidence was 1/8 and tumor weight was 0.03 g for miR-34a group (P = 0.016, incidence). h. Purified CD44− Du145 cells were transfected with anti-NC or anti-34a and s.c injected. Tumor incidence was 5/8 and mean weight was 0.05 g for anti-NC group whereas incidence was 6/8 and tumor weight was 0.2 g for anti-34a group (P = 0.038, weight). i. Bulk LAPC9 cells were transfected with anti-NC or anti-34a oligos and implanted (100,000 cells) in the DP. Mice were terminated at d 46. Tumor incidences were 5/8 and 7/8 for anti-NC and anti-34a groups, respectively. j. Representative microphotographs (animal number and tumor weight indicated on top; scale bar, 100 μm) showing increased lung metastasis by anti-34a (also see Supplementary Fig. 7c–d).', 'hash': 'e8aba97d6dd0f99d1cfc522f276bae71d23c9404e46e0f788a51860dca2f58f4'}]
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{'nihms255508f1': ['We used quantitative reverse transcription – polymerase chain reaction (qRT-PCR) to compare the miRNA expression25,26 of CD44+ and CD44− PCa cells. The CD44+ PCa cell population harbors tumor-initiating and metastatic cells18,19 and is enriched in self-renewal gene NANOG27. CD44+ PCa cells were purified from three xenograft models18,19,27,28: LAPC9, LAPC4, and Du145. For comparison, we also purified LAPC4 CD133+ and LAPC9 side population (SP) cells. The CD133+ PCa cells are clonogenic in vitro17 and LAPC9 SP is also enriched in tumor-initiating cells28. We first used unsorted cells to measure the levels of 324 sequence-validated human miRNAs and found that 137 miRNAs were expressed at reliably detectable levels (<xref rid="nihms255508f1" ref-type="fig">Fig. 1a</xref>). We then compared expression levels of these 137 miRNAs in marker-positive versus marker-negative PCa cell populations and found that miR-34a (1p36.22) was prominently under-expressed in all CD44). We then compared expression levels of these 137 miRNAs in marker-positive versus marker-negative PCa cell populations and found that miR-34a (1p36.22) was prominently under-expressed in all CD44+ populations (<xref rid="nihms255508f1" ref-type="fig">Fig. 1a</xref>), representing <3% of the levels in the corresponding CD44), representing <3% of the levels in the corresponding CD44− cells (<xref rid="nihms255508f1" ref-type="fig">Fig. 1b</xref>). The other two miR-34 family members, miR-34b and miR-34c (11q23.1), did not show consistent differences between CD44). The other two miR-34 family members, miR-34b and miR-34c (11q23.1), did not show consistent differences between CD44+ and CD44− PCa cells (not shown). Under-expression of miR-34a in CD44+ PCa cells was more pronounced than that of let-7b, a tumor suppressive miRNA6 and an important regulator of both normal and cancer stem cells3,4. miR-34a was also under-expressed in LAPC4 CD133+ (<xref rid="nihms255508f1" ref-type="fig">Fig. 1b</xref>) and LAPC9 SP (not shown) cells. To validate miR-34a under-expression in CD44) and LAPC9 SP (not shown) cells. To validate miR-34a under-expression in CD44+ PCa cells and to determine the clinical relevance, we purified CD44+ and CD44− PCa cells from 18 primary tumors27,29 (HPCa; Supplementary Table 1) and compared the miR-34a levels. CD44+ HPCa cells expressed miR-34a at levels ~25–70% of those in CD44− cells from the same tumors (<xref rid="nihms255508f1" ref-type="fig">Fig. 1c</xref>). Altogether, these results suggest that miR-34a is under-expressed in the CD44). Altogether, these results suggest that miR-34a is under-expressed in the CD44+ PCa cells in both xenograft and primary tumors.', 'miR-34a is regulated by p53 and induces apoptosis, cell-cycle arrest, or senescence when introduced into cancer cells20–24,30. miR-34a levels in ten prostate (cancer) cell types correlated with the p53 status (Supplementary Fig. 1) and transfection of synthetic miR-34a oligonucleotides (oligos), but not the negative control (NC) miRNA oligos, induced cell-cycle arrest, apoptosis or senescence in p53-mutant PCa cells (Supplementary Data and Supplementary Figs. 2 and 3). To determine whether miR-34a possesses tumor-inhibitory effects, we manipulated miR-34a levels (Supplementary Fig. 4) in a variety of PCa cell types and then implanted the cells subcutaneously (s.c) or orthotopically in the dorsal prostate (DP) in NOD-SCID mice (<xref rid="nihms255508f1" ref-type="fig">Fig. 1d,e</xref>; ; Supplementary Fig. 5). miR-34a transfected LAPC9 (<xref rid="nihms255508f1" ref-type="fig">Fig. 1d</xref>; ; Supplementary Fig. 5a) and HPCa58 (<xref rid="nihms255508f1" ref-type="fig">Fig. 1e</xref>) cells produced significantly smaller tumors than the same cells transfected with miR-NC oligos. LAPC9 cells are androgen-dependent whereas HPCa58 cells were from an early-generation xenograft tumor () cells produced significantly smaller tumors than the same cells transfected with miR-NC oligos. LAPC9 cells are androgen-dependent whereas HPCa58 cells were from an early-generation xenograft tumor (Supplementary Methods). miR-34a also inhibited the secondary transplantation of HPCa58 cells (<xref rid="nihms255508f1" ref-type="fig">Fig. 1e</xref>). Similar tumor-inhibitory effects of miR-34a were observed with androgen-dependent LAPC4 (). Similar tumor-inhibitory effects of miR-34a were observed with androgen-dependent LAPC4 (Supplementary Fig. 5b) and androgen-independent Du145 (Supplementary Fig. 5d) and PPC-1 (Supplementary Fig. 5g) cells. We also infected PCa cells with lentiviral or retroviral vectors encoding pre-miR-34a (Supplementary Fig. 1d) prior to implantation. The viral vector-mediated miR-34a overexpression also inhibited tumor regeneration of LAPC4 (Supplementary Fig. 5c), Du145 (Supplementary Fig. 5e,f), and LAPC9 (not shown) cells. Histological and immunohistochemical (IHC) examination of tumor sections (Supplementary Fig. 6) revealed increased necrotic areas and reduced Ki-67+ cells in miR-34a transfected tumors, which also showed increased expression of HP-1γ, a protein associated with cell-cycle arrest and senescence.', 'To evaluate whether the miR-34a-mediated tumor inhibition might be due to an effect on the CSC populations, we performed tumor experiments using purified CD44+ and CD44− PCa cells. Remarkably, when purified CD44+ Du145 cells were infected with lenti-34a, tumor development was completely blocked (<xref rid="nihms255508f1" ref-type="fig">Fig. 1f</xref>). Similarly, when CD44). Similarly, when CD44+ LAPC9 cells were transfected with miR-34a oligos (<xref rid="nihms255508f1" ref-type="fig">Fig. 1g</xref>) or infected with lenti-34a () or infected with lenti-34a (Supplementary Fig. 5h), tumor incidence was virtually abolished. Conversely, introducing an antisense inhibitor of miR-34a (i.e., anti-34a or miR-34a antagomir) into purified CD44− Du145 cells promoted tumor growth (<xref rid="nihms255508f1" ref-type="fig">Fig. 1h</xref>; ; Supplementary Fig. 5i). Likewise, bulk or CD44− LAPC9 cells transfected with anti-34a oligos generated larger tumors than those with anti-NC oligos (<xref rid="nihms255508f1" ref-type="fig">Fig. 1i</xref>; ; Supplementary Fig. 7a,b). Importantly, we observed more lung metastasis in the anti-34a transfected group (<xref rid="nihms255508f1" ref-type="fig">Fig. 1j</xref>; ; Supplementary Fig. 7c,d). Collectively, these results suggest that miR-34a possesses tumor-inhibitory effects in both bulk and purified CD44+ PCa cells.'], 'nihms255508f2': ['Subsequently, we performed 4 sets of therapeutic experiments (<xref rid="nihms255508f2" ref-type="fig">Fig. 2</xref>; see ; see Online Methods) in NOD-SCID mice bearing pre-established PCa. We first observed that repeated intratumoral injections of miR-34a into PPC-1 tumors halted tumor growth (Supplementary Fig. 5g). We then established orthotopic PC3 tumors and, 3 weeks later, injected miR-34a or miR-NC oligos complexed with a lipid-based delivery agent26 into tail veins of mice every two days. Systemically delivered miR-34a reduced PC3 tumor burden by 50% (<xref rid="nihms255508f2" ref-type="fig">Fig. 2a</xref>). In two therapeutic experiments with orthotopic LAPC9 tumors, miR-34a reduced lung metastasis (). In two therapeutic experiments with orthotopic LAPC9 tumors, miR-34a reduced lung metastasis (<xref rid="nihms255508f2" ref-type="fig">Fig. 2b,e,f</xref>; ; Supplementary Fig. 8) without affecting tumor growth (<xref rid="nihms255508f2" ref-type="fig">Fig. 2c</xref>). miR-34a also promoted survival of tumor-bearing animals (). miR-34a also promoted survival of tumor-bearing animals (<xref rid="nihms255508f2" ref-type="fig">Fig. 2d</xref>). These results indicate that miR-34a possesses therapeutic efficacy against pre-established prostate tumors.). These results indicate that miR-34a possesses therapeutic efficacy against pre-established prostate tumors.'], 'nihms255508f3': ['Since the CD44+ PCa cell population is enriched in CSCs, we performed holoclone, colonogenic, and sphere formation assays18,19,27,33,34 to determine whether miR-34a might regulate certain stem cell-associated properties. PCa cell holoclones contain self-renewing cancer cells34 and sphere-formation assays have been widely used to measure stem/progenitor cell activities1,35. We first established stringent assay conditions in which clones (i.e., holoclones formed in culture dish), colonies (formed in Matrigel or methylcellulose), and (floating) spheres were all of clonal origin (Supplementary Fig. 9). Under these conditions, miR-34a overexpression inhibited holoclone formation, clonogenic capacity, and sphere establishment in Du145 (<xref rid="nihms255508f3" ref-type="fig">Fig. 3a,b</xref>; ; Supplementary Fig. 2d,e), LAPC4 (<xref rid="nihms255508f3" ref-type="fig">Fig. 3c,d</xref>), and PPC-1 (), and PPC-1 (<xref rid="nihms255508f3" ref-type="fig">Fig. 3e</xref>; ; Supplementary Fig. 3h,i) cells. Importantly, miR-34a inhibited sphere formation in primary HPCa cells (<xref rid="nihms255508f3" ref-type="fig">Fig. 3f</xref>; ; Supplementary Fig. 10) and abrogated secondary sphere establishment (<xref rid="nihms255508f3" ref-type="fig">Fig. 3d,f</xref>). Moreover, HPCa cells infected with lenti-34a formed tiny or differentiated spheres (). Moreover, HPCa cells infected with lenti-34a formed tiny or differentiated spheres (Supplementary Fig. 10b). Of significance, miR-34a overexpression in purified CD44+ HPCa116 cells inhibited sphere formation (<xref rid="nihms255508f3" ref-type="fig">Fig. 3g</xref>) and, by contrast, anti-34a increased the inherently low sphere-forming capacity of CD44) and, by contrast, anti-34a increased the inherently low sphere-forming capacity of CD44− HPCa116 cells by several fold (<xref rid="nihms255508f3" ref-type="fig">Fig. 3h</xref>). Taken together, these observations indicate that miR-34a negatively regulates stem cell properties of PCa cells.). Taken together, these observations indicate that miR-34a negatively regulates stem cell properties of PCa cells.'], 'nihms255508f4': ['Cyclin D1, CDK4 and 6, E2F3, N-Myc, c-MET, and BCL-2 have been reported to be direct targets of miR-34a20–24,26,30–32. A survey of some of these molecules revealed that miR-34a affected the levels of cyclin D1, CDK4, CDK6 and c-MET in our PCa models (Supplementary Fig. 4d,e; Supplementary Fig. 6d,e). Interestingly, we consistently observed a strong inverse correlation between miR-34a levels and CD44 (<xref rid="nihms255508f4" ref-type="fig">Fig. 4a,b</xref>; ; Supplementary Fig. 1a,4e,11a–c; Supplementary Table 2). For example, CD44 protein and CD44+ PCa cells were reduced in miR-34a treated tumors (<xref rid="nihms255508f4" ref-type="fig">Fig. 4a</xref>). Transfected miR-34a downregulated CD44 in PCa cells (). Transfected miR-34a downregulated CD44 in PCa cells (<xref rid="nihms255508f4" ref-type="fig">Fig. 4b</xref>; ; Supplementary Fig. 11a,b). In contrast, CD44 mRNA (Supplementary Fig. 4e) and protein (Supplementary Fig. 11c) were increased in anti-34a transfected tumors. Of interest, the target-prediction program rna22 (36) revealed 2 putative miR-34a binding sites in the 3’-UTR of CD44 mRNA (<xref rid="nihms255508f4" ref-type="fig">Fig. 4c</xref>). When we cloned the 3’-UTR fragment harboring both putative miR-34a binding sites downstream of a luciferase coding sequence (). When we cloned the 3’-UTR fragment harboring both putative miR-34a binding sites downstream of a luciferase coding sequence (Supplementary Fig. 11d,e), co-transfection of the luciferase reporter and miR-34a oligos into three PCa cell types produced lower luciferase activity than cells co-transfected with the NC oligos but mutation of the seed sequence in either site, especially the distal site, partially abrogated the suppressive effect of miR-34a (<xref rid="nihms255508f4" ref-type="fig">Fig. 4d</xref>; ; Supplementary Fig. 11f,g). These results suggest that miR-34a regulates CD44 expression via two binding sites located in the 3’-UTR of the CD44 gene.', 'To determine whether CD44 represents a functionally important target of miR-34a in the context of regulating PCa development, we reduced CD44 expression using a lentiviral CD44-shRNA vector (Supplementary Fig. 1d) in LAPC4, PC3, and Du145 cells. CD44 knockdown in LAPC4 cells inhibited both orthotopic tumor regeneration (<xref rid="nihms255508f4" ref-type="fig">Fig. 4e</xref>) and lung metastasis () and lung metastasis (Supplementary Fig. 12). CD44 knockdown in PC3 cells dramatically inhibited metastasis (<xref rid="nihms255508f4" ref-type="fig">Fig. 4f,g</xref>; ; Supplementary Fig. 13) without affecting tumor regeneration (not shown). CD44 knockdown in Du145 cells inhibited tumor development in both s.c and orthotopic sites (Supplementary Fig. 14a,b) as well as metastasis (not shown). These results not only reveal a critical role of CD44 itself in determining the tumorigenic and metastatic capacity of PCa cells but also indicate that CD44 knockdown phenocopies the anti-PCa effects of miR-34a. Mechanistically, we observed that the CD44+ PCa cells demonstrated higher migratory (Supplementary Fig. 14c,d) and invasive (Supplementary Fig. 14e) capacities than CD44− cells, which were partially inhibited by miR-34a (<xref rid="nihms255508f4" ref-type="fig">Fig. 4h</xref>; ; Supplementary Fig. 14f,g). ‘Rescue’ experiments wherein CD44 was overexpressed using a cDNA that lacked the 3’-UTR containing the miR-34a binding sites abrogated miR-34a-mediated inhibition of invasion of CD44+ Du145 cells (<xref rid="nihms255508f4" ref-type="fig">Fig. 4i</xref>), reinforcing CD44 as a direct and ), reinforcing CD44 as a direct and functional target of miR-34a. In contrast, CD44 overexpression did not significantly relieve miR-34a inhibition of PCa cell proliferation (Supplementary Fig. 15).', 'We report herein underexpression of miR-34a in tumorigenic CD44+ PCa cells and demonstrate its potent anti-tumor and anti-metastasis effects. We establish miR-34a as a critical negative regulator of CD44+ PCa cells and CD44 itself as an important target of miR-34a. Our results suggest that reduced expression of miR-34a in CD44+ PCa cells contributes to PCa development and metastasis by allowing for the elevated expression of CD44 and manifestation of their migratory, invasive and metastatic properties (<xref rid="nihms255508f4" ref-type="fig">Fig. 4j</xref>). It is interesting that p53, which directly activates miR-34a, also negatively regulates CD44 through a non-canonical p53-binding site in the promoter). It is interesting that p53, which directly activates miR-34a, also negatively regulates CD44 through a non-canonical p53-binding site in the promoter37, suggesting the importance in controlling CD44 expression. Considering the widespread expression of CD44 in CSCs (7–16) and functional involvement of CD44 in mediating CSC migration and homing38 and in metastasis of many cancers including PCa, the newly identified miR-34a suppression of CD44 reveals a key role for miRNA-based gene regulation. The emerging role of miR-34a in regulating other CSC32,39 properties (<xref rid="nihms255508f4" ref-type="fig">Fig. 4j</xref>), coupled with the therapeutic effects of miR-34a on lung), coupled with the therapeutic effects of miR-34a on lung26 and prostate (this study) tumors, establishes a strong rationale for developing miR-34a as a novel therapeutic targeting tumorigenic PCa cells.']}
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Identification of miR-34a as a potent inhibitor of prostate cancer progenitor cells and metastasis by directly repressing CD44
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Nat Med
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1297843200
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Radiation is used in the treatment of a broad range of malignancies. Exposure of normal tissue to radiation may result in both acute and chronic toxicities that can result in an inability to deliver the intended therapy, a range of symptoms, and a decrease in quality of life. Radioprotectors are compounds that are designed to reduce the damage in normal tissues caused by radiation. These compounds are often antioxidants and must be present before or at the time of radiation for effectiveness. Other agents, termed mitigators, may be used to minimize toxicity even after radiation has been delivered. Herein, we review agents in clinical use or in development as radioprotectors and mitigators of radiation-induced normal tissue injury. Few agents are approved for clinical use, but many new compounds show promising results in preclinical testing.
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[
"Amifostine",
"Animals",
"Antioxidants",
"Cyclic N-Oxides",
"DNA Damage",
"Fibroblast Growth Factor 7",
"Free Radical Scavengers",
"Humans",
"Magnetic Resonance Imaging",
"Neoplasms",
"Radiation Injuries",
"Radiation Protection",
"Radiation-Protective Agents",
"Risk Factors",
"Spin Labels"
] |
other
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PMC3076220
| null |
92
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[
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"{'Citation': 'Xavier S, Piek E, Fujii M, et al. Amelioration of radiation-induced fibrosis: Inhibition of transforming growth factor-beta signaling by halofuginone. J Biol Chem. 2004;279:15167–15176.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '14732719'}}}",
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"{'Citation': 'Baum BJ, Zheng C, Cotrim AP, et al. Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction. Biochim Biophys Acta. 2006;1758:1071–1077.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16368071'}}}"
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Nat Med. 2011 Feb 16; 17(2):211-215
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Subcellular localization of RPS27L under unstressed and stressed conditionsA549 cells were cultured in cover-slide for overnight. Cells were left untreated or treated with actionmycin D (5 nM), Etoposide (25 µM), or MI-219 (10 µM) for 24 hrs, followed by immuno-fluorescent stained with indicated antibodies and photography (A). A549 cells were treated with three agents as above. Cells were harvested and whole cell extract (WCE, 100 µg of total protein) were subjected to Western blotting analysis (B) or cells were fractionated into cytoplasm and nuclear fractions and subjected (cytoplasm, 80 µg of proteins; nucleoplasm, 15–30 µg of proteins) to Western blotting using indicated antibodies (C). Purity of each fractionation was determined by Western blotting detecting cytoplasmic procaspase-3 and nuclear PARP. SE: short exposure, LE: long exposure.
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[{'image_id': 'nihms252055f4', 'image_file_name': 'nihms252055f4.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f4.jpg', 'caption': 'The in vivo binding between endogenous RPS27L or RPS27 and endogenous MDM2, and competitive binding of RPS27L/RPS27 with p53 for MDM2(A) Endogenous RPS27L or RPS27 binds to endogenous MDM2: Subconfluent p53-null H1299 cells were subjected to immunoprecipitation either with antibodies against RPS27L or RPS27L, followed by Western blotting with antibody against MDM2 (A), or with antibodies against MDM2, followed by Western blotting with antibody against RPS27L or RPS27. Immunoprecipitates or WCE (whole cell extracts) were subjected to Western blotting using indicated Abs. (C&D) The competition between RPS27L or RPS27 and p53 for MDM2 binding: 293 cells were transfected with RPS27L, RPS27, or a MDM2 double mutant K446R/C475S (446/475) alone, or in combination (C). 293 cells were infected with lenti-virus silencing control or p53, followed by transfected with MDM2 (446/475), along with the vector control (D). Cell lysates were prepared for immunoprecipitation using FLAG-beads. Immunoprecipitates were then subjected to Western blotting using indicated Abs. Direct Western blotting of WCE is shown at the bottom.', 'hash': 'de17f63167bb121baa377aea3bb9cf74d64a60311b17ac97e9a21ebe427dcd41'}, {'image_id': 'nihms252055f3', 'image_file_name': 'nihms252055f3.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f3.jpg', 'caption': 'The in vitro binding between RPS27L or RPS27 and MDM2 and the binding domain mapping(A&B) Direct binding of RPS27L or RPS27 to MDM2: The full length RPS27L, RPS27, their N-terminal fragments (AA1–36), and C-terminal fragment (AA37–84) were expressed as GST-fusion proteins. The fusion proteins were purified with GSH-beads and eluted with GSH. One portion was subjected to SDS-PAGE, followed by Coomassie blue staining (B), and the other portion was subjected to binding with His-tagged-MDM2 immobilized on beads, along with the His-Tag control, followed by Western blotting (IB) using anti-GST antibody (A). (C&D) The in vitro MDM2-RPS27L binding: GST-fused MDM2 (1–491) and its deletion mutants were expressed in bacteria and purified using GSH-coated beads. About 500 ng of purified GST or GST-fusion proteins immobilized on GSH beads were used in the GST pull-down assay using cell lysates (2 mg), prepared from p53/MDM2-double null MEFs cells. After extensive washing, the beads were boiled, and bound RPS27L was detected by Western blotting using an antibody against RPS27L (C). The levels and purity of GST-MDM2 and its deletion mutants were shown in a Coomassie staining gel (D).', 'hash': 'a7638946897163e7b12ef1528dfb17e07ceee4b0f7d07ad50b50b78bcd745e89'}, {'image_id': 'nihms252055f10', 'image_file_name': 'nihms252055f10.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f10.jpg', 'caption': 'A schematic model for the interplay among RPS27L/RPS27 and p53/MDM2DNA damage induced by ionizing radiation or chemo-drugs activates p53. Activated p53, on one hand, transactivates MDM2 and RPS27L, and on the other hand, transrepresses RPS27. Upon induction by p53, MDM2 binds to p53 as well as RPS27L and promotes their ubiquitination and degradation, whereas induced RPS27L competes with p53 for MDM2 binding, thus releasing p53 for MDM2-mediated degradation. Under overexpressed condition (*), MDM2 also binds to RPS27 and promotes its ubiquitination and degradation and at the same time, RPS27-MDM2 binding frees up p53 and reduces p53 degradation by MDM2. Through interplay with MDM2-p53 axis, RPS27L/S27 regulates cell growth and survival.', 'hash': 'dcb2b84c2285ce6a09a8b1cab64c430cefb63d4f0a77c84aa28d278521c8f861'}, {'image_id': 'nihms252055f2', 'image_file_name': 'nihms252055f2.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f2.jpg', 'caption': 'p53 represses RPS27(A) HCT116 model: Cells with different p53 background were left untreated or treated with etoposide (25 µM) for 24 hrs, followed by Western blotting using indicated Abs. (B&C) A549 model: Cells were infected with lentivirus based control siRNA or siRNA targeting p53 (Sun et al 2008) for 48 hrs. Cells were then left untreated or treated with MI-219 (10 µM) or etoposide (25 µM) for 24 hrs, followed by Western blotting analysis using indicated antibodies (B). Parental A549 cells were treated with different concentrations of MI-219 and subjected to RT-qPCR analysis. The relative level of RPS27 mRNA is shown (C, n=2). (D) H1299-p53-ts model: H1299-p53-ts cells or its control cells (H1299-neo) were grown either at 32°C or 37°C for indicated periods and subjected to Western blotting with indicated Abs.', 'hash': '1edf424e54feb0c5a59b1e5c23be53d0791513b6e86bbddaa760ffb0e2772e3b'}, {'image_id': 'nihms252055f5', 'image_file_name': 'nihms252055f5.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f5.jpg', 'caption': 'MDM2 regulation of RPS27L and RPS27 under overexpressed or physiological conditions(A&B) Ectopic expression of RPS27L (A) or RPS27 (B) is reduced by wild-type MDM2: H1299 cells were transfected with RPS27L (A), RPS27 (B) alone, or in combination with wild-type MDM2 (WT), MDM2 acidic domain delete mutant (ΔA), or MDM2 Ring E3 ligase domain delete mutant (ΔR), followed by Western blotting analysis using indicated antibodies. (C) MDM2 shortens protein half-life of RPS27L, not RPS27: H1299 cells were transiently co-transfected with RPS27L or RPS27 alone, or in combination with MDM2. Cells were treated with cycloheximide (CHX) 24 hrs post transfection for various time periods, followed by Western blotting using indicated antibodies. (D) MDM2 promotes RPS27L ubiquitination: 293 cells were transfected with indicated plasmids. At 48 hrs post-transfection, cells were treated with MG132 for 4 hrs, followed by IP with FLAG-beads, and Westen blotting with anti-HA antibody. (E&F) Regulation of endogenous RPS27L/RPS27 by endogenous MDM2: (E) The protein level of RPS27L/RPS27: The cell lysates of p53-null and p53/MDM2-double null MEFs are subjected to Western blotting using indicated Abs. (F) The protein half-life of RPS27L/RPS27: Cells were treated with cycloheximide (CHX) for various time periods, followed by Western blotting using indicated Abs.', 'hash': 'cfab1ff730a05f1da0d83f07a98b5bc78300a0747c3fc4547ccff5496529228e'}, {'image_id': 'nihms252055f6', 'image_file_name': 'nihms252055f6.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f6.jpg', 'caption': 'RPS27L or RPS27 inhibits MDM2-induced p53 ubiquitination and extends p53 half-life(A) Inhibition of p53 ubiquitination: H1299 cells were transiently transfected with indicated plasmids. At 24 hrs post-transfection, cells were treated with MG132 for 6 hrs, and cell pellets were lysed by 6 M guanidinium-HCl. His-tagged ubiquitinated proteins were purified by Ni-NTA beads, eluted with imidazole, and subjected to Western blotting. Ubiquitinated p53 was detected by anti-p53 Ab. (B) Effect on endogenous p53 level: A549 cells were transiently transfected with RPS27L or RPS27, and subjected to Western blotting using indicated Abs. (C&D) Extension of p53 protein half-life by RPS27L is dependent on the presence of MDM2: p53-null (C) and p53/MDM2-double null (D) MEFs were transiently co-transfected with p53 alone, or in combination with RPS27L. Cells were treated with cycloheximide (CHX) 48 hrs post transfection for various time periods, followed by Western blotting using indicated Abs.', 'hash': 'a7426d159d47c119b202688625d6a74f057181f322aa757b2beeb7cbb5e289ec'}, {'image_id': 'nihms252055f1', 'image_file_name': 'nihms252055f1.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f1.jpg', 'caption': 'RPS27L and RPS27, their domain structures (A) and the evolutionary conservation (B): Two family members differ in three amino acids at the N-terminus. The remainder of the molecules share a 100% identity. The zinc finger domain is indicated. The bar graphs were not drawn to scale. The % identity of protein sequence among different. (C) Specificity of antibodies against RPS27L or RPS27. Human 293 cells were transiently transfected with HA-tagged (at the C-terminus) RPS27L (S27L-HA) or RPS27 (S27-HA), respectively, along with an empty vector, followed by Western blotting analysis using antibodies against RPS27L (top panel), RPS27 (middle) or HA (bottom) with β-actin as the loading control.', 'hash': 'f16126dc5aed2b6b3b6ffb5e6fce0aa9d6b192ac77efa74929f3d5d503923fb5'}, {'image_id': 'nihms252055f8', 'image_file_name': 'nihms252055f8.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f8.jpg', 'caption': 'Subcellular localization of RPS27L under unstressed and stressed conditionsA549 cells were cultured in cover-slide for overnight. Cells were left untreated or treated with actionmycin D (5 nM), Etoposide (25 µM), or MI-219 (10 µM) for 24 hrs, followed by immuno-fluorescent stained with indicated antibodies and photography (A). A549 cells were treated with three agents as above. Cells were harvested and whole cell extract (WCE, 100 µg of total protein) were subjected to Western blotting analysis (B) or cells were fractionated into cytoplasm and nuclear fractions and subjected (cytoplasm, 80 µg of proteins; nucleoplasm, 15–30 µg of proteins) to Western blotting using indicated antibodies (C). Purity of each fractionation was determined by Western blotting detecting cytoplasmic procaspase-3 and nuclear PARP. SE: short exposure, LE: long exposure.', 'hash': '8a48e1aa01b4b85a747d791449d982da79ca05f999a932c942221fe971ddad81'}, {'image_id': 'nihms252055f9', 'image_file_name': 'nihms252055f9.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f9.jpg', 'caption': 'SiRNA silencing of RPS27L attenuates p53 induction by various stimuliA549 (A&B) or SJSA (C) cells were infected with lentivirus targeting RPS27L, along with a scrambled control lentivirus for 48 hrs. Cells were then left untreated with treated for 24 hrs with actinomycin D (5 nM) (A), ionizing radiation (6 Gy) (B) or etoposide (25 µM) (C), followed by cell lysate preparation and Western blotting analysis using indicated antibodies. A549 cells post lentivirus infection and actinomycin D treatment were harvested for cell fractionation into cytosol and nuclear fractions, followed by Western blotting analysis using indicated antibodies (D).', 'hash': 'd6addc67193209b4f8b4f38d7206708dd2b3f17bb96f5f3e68bc3ff60e84041f'}, {'image_id': 'nihms252055f7', 'image_file_name': 'nihms252055f7.jpg', 'image_path': '../data/media_files/PMC3077453/nihms252055f7.jpg', 'caption': 'siRNA silencing of RPS27L reduces p53 level, shortens p53 protein half-life, and inhibits p53 transcription activity(A&B) Reduction of p53 protein level: A549 cells were infected with lentivirus targeting RPS27L (siS27L) or RPS27 (siS27), along with scrambled siRNA as the control (siCont). Three days after infection, cells were harvested and subjected to Western blotting analysis using indicated antibodies (A) or RT-qPCR for p53 mRNA levels (B, n=2). (C) Reduction of p53 level upon RPS27L siRNA silencing is abrogated by silencing of MDM2: A549 cells were infected with lenti-virus targeting RPS27L or scrambled control siRNA, then transfected with control siRNA or siRNA targeting MDM2 (SMART pool) 24 hrs post infection. Cells were harvested 48 hrs post transfection and subjected to Western blotting using indicated Abs. (D) Shortening of p53 protein half-life: A549 cells were transfected with oligo-based siRNA targeting RPS27L or scrambled control siRNA. Cells were treated with cycloheximide (CHX) 48 hrs post transfection for various time periods, followed by Western blotting using indicated Abs. LE: long exposure; SE: short exposure. (E&F) Reduction of p53 transcription activity: H1299-p53-ts model: H1299 cells stably transfected with p53138V and BP100-luc were infected with control siRNA or siRNA targeting RPS27L for 48 hrs. Cells were then grown either at 32°C for indicated periods and subjected to Western blotting with indicated Abs (E) or for 18 hrs and subjected to luciferase reporter assay. Shown are mean ± SEM from three independent experiments (F).', 'hash': 'cb531c446d1ed8ddbf3ff4a56db79949c54296be45de39705808545ceb745d2c'}]
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{'nihms252055f1': ['RPS27 and its family member, RPS27L are 84 amino acids-containing proteins with a zinc-finger-like domain. Two family members differ from each other by only three amino acids (R5K, L12P, K17R) at the N-terminus (<xref ref-type="fig" rid="nihms252055f1">Fig 1A</xref>). We compared the protein sequences of RPS27 and RPS27L in more than 10 species. As shown in ). We compared the protein sequences of RPS27 and RPS27L in more than 10 species. As shown in <xref ref-type="fig" rid="nihms252055f1">Figure 1B</xref>, both family members are highly conserved during evolution, suggesting their functional significance. In addition to human, the R5K/L12P/K17R conservation of two family members is also found in mouse, rat, and dog. Like in humans, there are two members of RPS27/S27L family in yeast , both family members are highly conserved during evolution, suggesting their functional significance. In addition to human, the R5K/L12P/K17R conservation of two family members is also found in mouse, rat, and dog. Like in humans, there are two members of RPS27/S27L family in yeast S. cerevisiae, RPS27A (YKL156W) and RPS27B (YHR021C), differing by only one amino acid at codon 62 (I62V). The yeast with systematic deletion of either RPS27A or RPS27B is still viable, although deletion of RPS27B showed some growth defect (Giaever et al 2002) (also see www.yeastgenome.org). In C-elegans, however, there is only one family member (RPS27). SiRNA silencing lead to embryonic lethality (Sonnichsen et al 2005) (also see www.wormgenome.org). In Drosophila, one family member is identified. Its molecular function is described as structural constituent of ribosome, nucleic acid binding (see http://flybase.bio.indiana.edu/reports/FBgn0039300.html) and it is involved in the biological process of translation.', 'To facilitate the detection of endogenous RPS27L and RPS27 selectively, we generated, affinity purified, and characterized two antibodies specifically against either one of the family members. As shown in <xref ref-type="fig" rid="nihms252055f1">Figure 1C</xref>, the RPS27L antibody detected both endogenous and HA- tagged RPS27L (S27L-HA), but not HA-tagged RPS27 (S27-HA) (top panel), whereas the RPS27 antibody detected both endogenous and S27-HA, but not S27L-HA (second panel). As expected, anti-HA tag antibody detected both S27L-HA and S27-HA (third panel). Thus, two antibodies are specific for their corresponding proteins, and do not cross-react with each other., the RPS27L antibody detected both endogenous and HA- tagged RPS27L (S27L-HA), but not HA-tagged RPS27 (S27-HA) (top panel), whereas the RPS27 antibody detected both endogenous and S27-HA, but not S27L-HA (second panel). As expected, anti-HA tag antibody detected both S27L-HA and S27-HA (third panel). Thus, two antibodies are specific for their corresponding proteins, and do not cross-react with each other.', 'Cells, except for MEFs, were plated into six-well plate at 2 ×105 cells per well and transfected the following day with a variety of plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). MEF cells were plated into six-well plate at 1 ×106 cells per well and transfected with indicated plasmids using PolyJet DNA transfection reagent (Signagen Laboratories, Ijamsville, MD, USA). Cells were harvested, lysed and subjected to Western blotting analysis and immunoprecipitation, as described (Tan et al 2008), using various antibodies as follows: p53 (Ab-6, Calbiochem, San Diego, CA, USA), HA (Roche, IN, USA), FLAG and β-Actin, (Sigma, St Louis, MO, USA), MDM2 (Ab-1, Calbiochem; 2A9, gift from Jiandong Chen; N-20 and SMP14 from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p21 (BD Pharmingen, San Jose, CA, USA). RPS27L or RPS27 polyclonal rabbit antibody was raised and purified as described (He and Sun 2007). The antigenic peptides used to generate these two antibodies are RL-S27L: MPLARDLLHPSLEEC (for RPS27L) and KP-S27: MPLAKDLLHPSPEEC (for RPS27), respectively, differing in two amino acids from each other. After two runs of affinity purification and affinity absorption, the specificity of two antibodies were tested and confirmed in cells over-expressing each protein (see <xref ref-type="fig" rid="nihms252055f1">Fig. 1C</xref>).).'], 'nihms252055f2': ['We previously showed that RPS27L is a p53 target, subjecting to p53 induction (He and Sun 2007), which was later confirmed by the others (Li et al 2007). During our parallel study of p53 regulation of RPS27L and RPS27, we found that in contrast to RPS27L, RPS27 was actually repressed by p53. As shown in <xref ref-type="fig" rid="nihms252055f2">Figure 2A</xref>, treatment with etoposide, a DNA damaging agent, which induced p53 and its downstream targets p21 and RPS27L, significantly reduced the level of RPS27 in wt p53-containing HCT116 cells (lanes 1 vs. 2), but only a slight reduction of RPS27 was seen in p53-null HCT116 cells (lanes 3&4). The p53-dependent repression of RPS27 was further demonstrated in wt p53-containing A549 cells. As shown in , treatment with etoposide, a DNA damaging agent, which induced p53 and its downstream targets p21 and RPS27L, significantly reduced the level of RPS27 in wt p53-containing HCT116 cells (lanes 1 vs. 2), but only a slight reduction of RPS27 was seen in p53-null HCT116 cells (lanes 3&4). The p53-dependent repression of RPS27 was further demonstrated in wt p53-containing A549 cells. As shown in <xref ref-type="fig" rid="nihms252055f2">Figure 2B</xref>, in A549 cells infected with control siRNA, the basal level of RPS27 was very high, but remarkably reduced upon p53 activation by MI-219, a small molecule that disrupts the MDM2-p53 binding (, in A549 cells infected with control siRNA, the basal level of RPS27 was very high, but remarkably reduced upon p53 activation by MI-219, a small molecule that disrupts the MDM2-p53 binding (Shangary 2008), or etoposide (lanes 1–3). The p53-induced RPS27 repression was abrogated if p53 was silenced via siRNA (lanes 4–6). We further confirmed that p53-induced RPS27 repression occurred at the transcriptional level, as demonstrated by RT-qPCR analysis (<xref ref-type="fig" rid="nihms252055f2">Fig 2C</xref>). Finally, we used the H1299-p53 temperature sensitive model (). Finally, we used the H1299-p53 temperature sensitive model (Peng et al 2003, Robinson 2003) and showed that while MDM2 and RPS27L were induced when cells were grown at the permissive 32°C (wt p53 conformation), the RPS27 expression was repressed. No difference was seen among these proteins at 37°C with a mutant p53 confirmation (<xref ref-type="fig" rid="nihms252055f2">Fig 2D</xref>, top). Neither RPS27 reduction, nor RPS27L induction was due to cold shock, since the same treatment in p53-null H1299-neo control cells even slightly increased the RPS27 levels, but had no effect on RPS27L (, top). Neither RPS27 reduction, nor RPS27L induction was due to cold shock, since the same treatment in p53-null H1299-neo control cells even slightly increased the RPS27 levels, but had no effect on RPS27L (<xref ref-type="fig" rid="nihms252055f2">Fig 2D</xref>, bottom). Our results demonstrate that , bottom). Our results demonstrate that RPS27 is a p53 repressible gene.', 'RPS27 was found to be overexpressed in many human cancers (Atsuta et al 2002, Fernandez-Pol et al 1997, Ganger et al 1997, Ganger et al 2001, Lee et al 2004, Wang et al 2006). However, the underlying mechanism for its overexpression is unknown. Our study report here that RPS27 is subjected to transcriptional repression by wild type p53, but not mutant p53 (<xref ref-type="fig" rid="nihms252055f2">Fig 2</xref>) may provide one logical explanation. It is well known that p53 is mutated in ~50% of human cancer () may provide one logical explanation. It is well known that p53 is mutated in ~50% of human cancer (Greenblatt et al 1994). Human cancers with p53 mutation would, therefore, likely have a higher RPS27 expression due to the loss of repression by wild type p53. On the other hand, overexpressed RPS27 may in turn stabilize mutant p53, since ectopic expression of RPS27 could increase p53 levels (<xref ref-type="fig" rid="nihms252055f6">Fig. 6B</xref>). This feed-forward regulation could confer growth advantage in these cancer cells. Future study will be directed to address this interesting correlation.). This feed-forward regulation could confer growth advantage in these cancer cells. Future study will be directed to address this interesting correlation.'], 'nihms252055f3': ['A few ribosomal proteins (L11, L23, L5, L26 or S7) were previously found to bind to MDM2 and activate p53 (Bhat et al 2004, Dai and Lu 2004, Dai et al 2004, Jin et al 2004, Lohrum et al 2003, Ofir-Rosenfeld et al 2008, Zhang et al 2003, Zhu et al 2009). We, therefore, took this candidate approach and determined if RPS27L or RPS27 would directly bind to MDM2, and if so, defined the binding motif on respective protein. GST-fusion proteins were expressed and purified for full-length RPS27L 1–84, the N-terminal portion, 1–36 (where RPS27L differs from RPS27 at three amino acids, codons, 5, 12, 17), the C-terminal portion 37–84 (exactly same as RPS27), the full-length RPS27 1–84, and the N-terminal portion of RPS27 1–36, along with GST control (<xref ref-type="fig" rid="nihms252055f3">Fig 3B</xref>). Approximately equal amount of proteins were used for the pull-down assay with His-tagged full length MDM2, along with the empty His-tag vector control. As shown in ). Approximately equal amount of proteins were used for the pull-down assay with His-tagged full length MDM2, along with the empty His-tag vector control. As shown in <xref ref-type="fig" rid="nihms252055f3">Figure 3A</xref>, the full-length RPS27L or RPS27 bound to MDM2, as well as the N-terminal portion, but not the C-terminal zinc-finger containing portion, although the binding was much weaker for RPS27. Thus, both family members bind to MDM2 and the binding domain is mapped to the N-terminus (AA 1–36)., the full-length RPS27L or RPS27 bound to MDM2, as well as the N-terminal portion, but not the C-terminal zinc-finger containing portion, although the binding was much weaker for RPS27. Thus, both family members bind to MDM2 and the binding domain is mapped to the N-terminus (AA 1–36).', 'To further confirm the RPS27L-MDM2 binding at the cellular level, and to define the potential RPS27L binding region on MDM2, we performed a GST-pull-down experiment, using full-length MDM2, and its deletion mutants. GST-fused full length MDM2 (1–491) and a deletion mutant (1–301), but not the mutant 1–150, nor other deletion mutants, bind to endogenous RPS27L in MEF cells derived from p53/Mdm2 double null mice (to avoid potential interference from p53) (<xref ref-type="fig" rid="nihms252055f3">Fig 3C</xref>). Expression and purity of GST-fused MDM2 and its deletion mutants are shown in ). Expression and purity of GST-fused MDM2 and its deletion mutants are shown in <xref ref-type="fig" rid="nihms252055f3">Figure 3D</xref>. Finally, we confirmed the binding of MDM2-RPS27L and MDM2-RPS27 using GST-MDM2 to pull-down RPS27L or RPS27 produced by . Finally, we confirmed the binding of MDM2-RPS27L and MDM2-RPS27 using GST-MDM2 to pull-down RPS27L or RPS27 produced by in vitro transcription and translation (data not shown). These results clearly demonstrated that MDM2 binds to endogenous RPS27L in the absence of p53 and that the binding region on MDM2 is from codons 151–293 (<xref ref-type="fig" rid="nihms252055f3">Fig 3C</xref>, bottom panel), an acidic domain-containing region, previously shown as the second p53 binding domain (, bottom panel), an acidic domain-containing region, previously shown as the second p53 binding domain (Kulikov et al 2006), implying a potential competition between RPS27L and p53 for MDM2 binding.'], 'nihms252055f4': ['We further determined if endogenous RPS27L or RPS27 binds to endogenous MDM2 under physiological condition. Normally growing p53-null H1299 cells at the subconfluency were harvested and subjected to immunoprecipitation (IP) using antibodies against RPS27L, RPS27, or IgG control. As shown in <xref ref-type="fig" rid="nihms252055f4">Figure 4A</xref>, MDM2 can be detected in the immunoprecipitates pulled down by either RPS27L or RPS27 antibody, respectively, but not by normal IgG control. In a reciprocal experiment, RPS27L or RPS27 can be pulled down by antibody against MDM2, but not by normal IgG control (, MDM2 can be detected in the immunoprecipitates pulled down by either RPS27L or RPS27 antibody, respectively, but not by normal IgG control. In a reciprocal experiment, RPS27L or RPS27 can be pulled down by antibody against MDM2, but not by normal IgG control (<xref ref-type="fig" rid="nihms252055f4">Fig 4B</xref>). Thus, RPS27L or RPS27 forms a complex in vivo independent of p53.). Thus, RPS27L or RPS27 forms a complex in vivo independent of p53.', 'It is well-established that the major Mdm2-p53 binding occurs between their N-terminal domains (Kussie et al 1996). A second binding site at the center portion of each protein (the DNA binding domain of p53 and the acidic domain of Mdm2) was also reported (Burch et al 2000, Kulikov et al 2006, Shimizu et al 2002). Since RPS27L or RPS27 binds to Mdm2 on the acidic region, where it also binds to p53, we determined if RPS27L or RPS27 competes with p53 for Mdm2 binding, thus interfering the MDM2-p53 binding. We used a FLAG-tagged ligase-dead MDM2 mutant, MDM2-446/475 (Swaroop and Sun 2003), avoiding potential degradation of p53 or RPS27L (see below). After cotransfection with RPS27L or RPS27, FLAG-MDM2 was immunoprecipitated, along with its binding partners. As shown in <xref ref-type="fig" rid="nihms252055f4">Figure 4C</xref>, the p53 level in MDM2 complex was significantly reduced, if either RPS27L or RPS27 was cotransfected. Likewise, in a reciprocal experiment, we found that siRNA silencing of endogenous p53 significantly enhanced the binding of RPS27L or RPS27 to MDM2-446/475 (, the p53 level in MDM2 complex was significantly reduced, if either RPS27L or RPS27 was cotransfected. Likewise, in a reciprocal experiment, we found that siRNA silencing of endogenous p53 significantly enhanced the binding of RPS27L or RPS27 to MDM2-446/475 (<xref ref-type="fig" rid="nihms252055f4">Fig. 4D</xref>). Taken together, these results suggested a competition of RPS27L or RPS27 with p53 for MDM2 binding.). Taken together, these results suggested a competition of RPS27L or RPS27 with p53 for MDM2 binding.', 'Since RPS27L/S27 competes with p53 for MDM2 binding (<xref ref-type="fig" rid="nihms252055f4">Fig 4</xref>), we reasoned that their overexpression would free up p53 and reduces its ubiquitination by MDM2. Indeed, in a typical ), we reasoned that their overexpression would free up p53 and reduces its ubiquitination by MDM2. Indeed, in a typical in vivo ubiquitination assay, using p53-null H1299 cells, we found that p53 ubiquitination was remarkably promoted by MDM2 (<xref ref-type="fig" rid="nihms252055f6">Figure 6A</xref>, lanes 4 vs. 3), but significantly reduced by co-transfection with RPS27L (lane 5) or RPS27 (lane 6). Likewise, overexpression of either RPS27L or RPS27 caused the accumulation of endogenous wild type p53 in A549 cells (, lanes 4 vs. 3), but significantly reduced by co-transfection with RPS27L (lane 5) or RPS27 (lane 6). Likewise, overexpression of either RPS27L or RPS27 caused the accumulation of endogenous wild type p53 in A549 cells (<xref ref-type="fig" rid="nihms252055f6">Fig 6B</xref>). Furthermore, we found that p53, when transfected alone into p53-null MEF cells, had a half-life of less than 2 hrs. However, when p53 was cotransfected with RPS27L, the steady-state level of p53 was much higher, and p53 half-life was extended to ~ 6 hrs (). Furthermore, we found that p53, when transfected alone into p53-null MEF cells, had a half-life of less than 2 hrs. However, when p53 was cotransfected with RPS27L, the steady-state level of p53 was much higher, and p53 half-life was extended to ~ 6 hrs (<xref ref-type="fig" rid="nihms252055f6">Fig 6C</xref>). Importantly, RPS27L-induced p53 increase and half-life extension was indeed mediated by MDM2, since a similar steady-state p53 level and p53 half-life was observed when tested in p53-Mdm2 double null MEF cells, regardless of RPS27L co-transfection (). Importantly, RPS27L-induced p53 increase and half-life extension was indeed mediated by MDM2, since a similar steady-state p53 level and p53 half-life was observed when tested in p53-Mdm2 double null MEF cells, regardless of RPS27L co-transfection (<xref ref-type="fig" rid="nihms252055f6">Fig 6D</xref>). Taken together, these results strongly suggest that via binding to MDM2, thus reducing MDM2-p53 binding, RPS27L decreases MDM2-induced p53 ubiquitination, leading to increased p53 levels by extending p53 protein half-life.). Taken together, these results strongly suggest that via binding to MDM2, thus reducing MDM2-p53 binding, RPS27L decreases MDM2-induced p53 ubiquitination, leading to increased p53 levels by extending p53 protein half-life.'], 'nihms252055f5': ['Two MDM2 binding ribosomal proteins L26 (Ofir-Rosenfeld et al 2008) and S7 (Zhu et al 2009) was found to be the substrates of MDM2. We determined if RPS27L or RPS27 is subjected to MDM2-mediated degradation. As shown in <xref ref-type="fig" rid="nihms252055f5">Figure 5A</xref>, when co-transfected into p53-null H1299 cells, ectopic expression of MDM2 dramatically reduced the steady-state level of exogenous expressed RPS27L (HA-S27L, lanes 2 vs. 1). Reduction of RPS27L requires MDM2 acidic domain where two proteins bind and the RING E3 ligase domain, since the deletion of either domain abolishes MDM2 activity (lanes 3&4). Similar results were found when MDM2 was cotransfected with RPS27 (, when co-transfected into p53-null H1299 cells, ectopic expression of MDM2 dramatically reduced the steady-state level of exogenous expressed RPS27L (HA-S27L, lanes 2 vs. 1). Reduction of RPS27L requires MDM2 acidic domain where two proteins bind and the RING E3 ligase domain, since the deletion of either domain abolishes MDM2 activity (lanes 3&4). Similar results were found when MDM2 was cotransfected with RPS27 (<xref ref-type="fig" rid="nihms252055f5">Fig 5B</xref>). We further determined the potential effect of MDM2 on the protein half-life of RPS27L or RPS27. Transfected RPS27L has a half-life of ~60 min which is shortened to ~30 min by MDM2-cotransfection. In contrast, RPS27 has a much longer protein half-life up to 180 min and was only moderately affected, if any, by MDM2 within the 3-hrs testing periods (). We further determined the potential effect of MDM2 on the protein half-life of RPS27L or RPS27. Transfected RPS27L has a half-life of ~60 min which is shortened to ~30 min by MDM2-cotransfection. In contrast, RPS27 has a much longer protein half-life up to 180 min and was only moderately affected, if any, by MDM2 within the 3-hrs testing periods (<xref ref-type="fig" rid="nihms252055f5">Fig 5C</xref>). Finally, we showed, using an immunoprecipitation assay, that MDM2 remarkably promotes polyubiquitination of RPS27L, but with a minor effect on RPS27 (). Finally, we showed, using an immunoprecipitation assay, that MDM2 remarkably promotes polyubiquitination of RPS27L, but with a minor effect on RPS27 (<xref ref-type="fig" rid="nihms252055f5">Fig 5D</xref>). The results strongly suggested that although both family members bind to MDM2 and are subjected to MDM2 degradation under overexpressed conditions, RPS27L with a much shorter protein half-life is more likely regulated by MDM2 under physiological condition.). The results strongly suggested that although both family members bind to MDM2 and are subjected to MDM2 degradation under overexpressed conditions, RPS27L with a much shorter protein half-life is more likely regulated by MDM2 under physiological condition.', 'We tested this hypothesis by determining if endogenous RPS27L or RPS27 is subject to regulation by endogenous MDM2. We compared the levels of the two family members in p53-null and p53/Mdm2-double null MEFs. As shown in <xref ref-type="fig" rid="nihms252055f5">Figure 5E</xref>, the levels of RPS27L, but not of RPS27, were higher in p53/Mdm2-double null MEFs than that in p53-null MEFs, suggesting that only RPS27L is subjected to Mdm2 regulation. We followed up this finding by measuring the protein half-life of two proteins in these two types of MEFs. As shown in , the levels of RPS27L, but not of RPS27, were higher in p53/Mdm2-double null MEFs than that in p53-null MEFs, suggesting that only RPS27L is subjected to Mdm2 regulation. We followed up this finding by measuring the protein half-life of two proteins in these two types of MEFs. As shown in <xref ref-type="fig" rid="nihms252055f5">Figure 5F</xref>, the protein half-life of endogenous RPS27L is about 8 hrs in p53-null cells, but was significantly extended up to 24 hrs in p53/Mdm2 double null cells. In contrast, the half-life of RPS27 is much longer, up to 24 hrs, regardless of the presence of Mdm2. Taken together, these results clearly suggested that RPS27L is a physiological substrate of MDM2, whereas RPS27, with a much longer protein half-life, can be ubiquitinated and degraded by MDM2 under overexpressed conditions., the protein half-life of endogenous RPS27L is about 8 hrs in p53-null cells, but was significantly extended up to 24 hrs in p53/Mdm2 double null cells. In contrast, the half-life of RPS27 is much longer, up to 24 hrs, regardless of the presence of Mdm2. Taken together, these results clearly suggested that RPS27L is a physiological substrate of MDM2, whereas RPS27, with a much longer protein half-life, can be ubiquitinated and degraded by MDM2 under overexpressed conditions.'], 'nihms252055f7': ['We further determined if siRNA silencing of RPS27L or RPS27 would change the levels of endogenous p53. As shown in <xref ref-type="fig" rid="nihms252055f7">Figure 7A</xref>, lentivirus-based siRNA silencing caused a complete elimination of endogenous RPS27L and a nearly complete elimination RPS27 in wt p53-containing A549 cells, respectively. Accompanying the silencing of RPS27L, but not RPS27, the basal level of p53 was significantly decreased (, lentivirus-based siRNA silencing caused a complete elimination of endogenous RPS27L and a nearly complete elimination RPS27 in wt p53-containing A549 cells, respectively. Accompanying the silencing of RPS27L, but not RPS27, the basal level of p53 was significantly decreased (<xref ref-type="fig" rid="nihms252055f7">Fig 7A</xref>), which appeared to occur at the protein levels, since no change at the p53 mRNA level was observed (), which appeared to occur at the protein levels, since no change at the p53 mRNA level was observed (<xref ref-type="fig" rid="nihms252055f7">Fig 7B</xref>). We, therefore, focused our study on RPS27L silencing and found that p53 reduction, upon RPS27L silencing, was MDM2 dependent, since simultaneous silencing RPS27L and MDM2 restored the p53 level (). We, therefore, focused our study on RPS27L silencing and found that p53 reduction, upon RPS27L silencing, was MDM2 dependent, since simultaneous silencing RPS27L and MDM2 restored the p53 level (<xref ref-type="fig" rid="nihms252055f7">Fig 7C</xref>, lanes 3 vs. 4). Furthermore, in wild-type p53-containing A549 cells, siRNA silencing of RPS27L shortened the p53 half-life from ~1 hr to 30 min (, lanes 3 vs. 4). Furthermore, in wild-type p53-containing A549 cells, siRNA silencing of RPS27L shortened the p53 half-life from ~1 hr to 30 min (<xref ref-type="fig" rid="nihms252055f7">Fig 7D</xref>). Finally, using a p53-temperature sensitive H1299-p53ts line (). Finally, using a p53-temperature sensitive H1299-p53ts line (Peng et al 2003, Robinson 2003), we found that p53 induction of p21, at the permissive temperature of 32°C where p53 is active, was significantly reduced upon RPS27L silencing (<xref ref-type="fig" rid="nihms252055f7">Fig 7E</xref>). Likewise, p53 transcription activity, as reflected by luciferase activity in a p53-driven luciferase reporter assay, was also significantly decreased upon RPS27L silencing (). Likewise, p53 transcription activity, as reflected by luciferase activity in a p53-driven luciferase reporter assay, was also significantly decreased upon RPS27L silencing (<xref ref-type="fig" rid="nihms252055f7">Fig 7F</xref>). Taken together, RPS27L, a p53 inducible protein and an MDM2 substrate, can positively regulate p53 activity through MDM2 binding, establishing a three-way interplay among p53, MDM2, and RPS27L.). Taken together, RPS27L, a p53 inducible protein and an MDM2 substrate, can positively regulate p53 activity through MDM2 binding, establishing a three-way interplay among p53, MDM2, and RPS27L.', 'Our siRNA silencing study revealed that knockdown of RPS27L, but not RPS27, caused destabilization of basal p53 in A549 and SJSA cells (<xref ref-type="fig" rid="nihms252055f7">Fig. 7A</xref> & & <xref ref-type="fig" rid="nihms252055f9">Fig. 9</xref>), suggesting under normal physiological condition, RPS27L is the one involving in the interplay with MDM2/p53 axis. It is noteworthy, however, that in some human cancer lines with wild type p53 (e.g. HCT116 and U2OS), RPS27L knockdown did not destabilize p53 (data not shown). This is likely due to a higher sensitivity of these lines to unknown stresses triggered by RPS27L knockdown to cause p53 induction, which counteracts direct modulation of MDM2/p53 axis by RPS27L. On the other hand, it is unclear why RPS27 silencing did not destabilize p53 in A549 cells, although it binds to MDM2 and inhibits MDM2-induced p53 degradation under overexpressed condition. One possibility is that RPS27 is a house-keeping ribosomal protein whose knockdown may trigger ribosomal stress which could induce p53. It is conceivable that, compared to RPS27L, house-keeping RPS27 has a long protein half-life and is resistant to MDM2-mediated degradation under physiological or even stressed conditions. This RPS27 unique feature could confer more flexibility for cells to deal with harsh environmental conditions without stopping essential protein synthesis.), suggesting under normal physiological condition, RPS27L is the one involving in the interplay with MDM2/p53 axis. It is noteworthy, however, that in some human cancer lines with wild type p53 (e.g. HCT116 and U2OS), RPS27L knockdown did not destabilize p53 (data not shown). This is likely due to a higher sensitivity of these lines to unknown stresses triggered by RPS27L knockdown to cause p53 induction, which counteracts direct modulation of MDM2/p53 axis by RPS27L. On the other hand, it is unclear why RPS27 silencing did not destabilize p53 in A549 cells, although it binds to MDM2 and inhibits MDM2-induced p53 degradation under overexpressed condition. One possibility is that RPS27 is a house-keeping ribosomal protein whose knockdown may trigger ribosomal stress which could induce p53. It is conceivable that, compared to RPS27L, house-keeping RPS27 has a long protein half-life and is resistant to MDM2-mediated degradation under physiological or even stressed conditions. This RPS27 unique feature could confer more flexibility for cells to deal with harsh environmental conditions without stopping essential protein synthesis.'], 'nihms252055f8': ['Several MDM2 binding ribosomal proteins were found to be localized in nucleolus, such as L11 (Bhat et al 2004, Lohrum et al 2003) and S7 (Zhu et al 2009) or in nucleoplasm, such as L23 (Dai et al 2004, Jin et al 2004). We determined the subcellular localization of RPS27L and its co-localization with MDM2 under unstressed or stressed conditions by immune-fluorescent staining. As shown in <xref ref-type="fig" rid="nihms252055f8">Figure 8A</xref>, under unstressed condition, endogenous RPS27L as well as RPS27 (not shown) are mainly localized in the cytoplasm in A549 cells or in SJSA cells (data not shown), whereas Mdm2 was mainly localized in nucleoplasm with a very low, but detectable level in cytoplasm. Upon exposure of cells to different types of stress inducers, including etoposide, MI-219 (, under unstressed condition, endogenous RPS27L as well as RPS27 (not shown) are mainly localized in the cytoplasm in A549 cells or in SJSA cells (data not shown), whereas Mdm2 was mainly localized in nucleoplasm with a very low, but detectable level in cytoplasm. Upon exposure of cells to different types of stress inducers, including etoposide, MI-219 (Shangary et al 2008), or low dose of actionmycin D that triggers ribosomal stress, a portion of RPS27L was translocated into nucleoplasm, where it co-localized with Mdm2. These stress inducers also increased the cytoplasmic Mdm2 level. Western blotting analysis confirmed the induction by these stress inducers of p53 and its downstream targets, Mdm2, p21 and to a less extent, RPS27L (except actinomycin D which failed to induce RPS27L) (<xref ref-type="fig" rid="nihms252055f8">Fig 8B</xref>). Cell fractionation experiment showed that upon exposure to p53-activating signals, both cytoplasmic and nuclear p53, Mdm2 and RPS27L (except by actinomycin D) were induced, whereas p21 induction was only detectable in the cytoplasm (). Cell fractionation experiment showed that upon exposure to p53-activating signals, both cytoplasmic and nuclear p53, Mdm2 and RPS27L (except by actinomycin D) were induced, whereas p21 induction was only detectable in the cytoplasm (<xref ref-type="fig" rid="nihms252055f8">Fig 8C</xref>). These results indicate that RSP27L is mainly a cytoplasmic protein and can be translocated into nucleoplasm in response to p53 inducing signals where it co-localized with Mdm2, and likely competed with p53 for Mdm2 binding.). These results indicate that RSP27L is mainly a cytoplasmic protein and can be translocated into nucleoplasm in response to p53 inducing signals where it co-localized with Mdm2, and likely competed with p53 for Mdm2 binding.', 'Our finding that RPS27L or RPS27 binds to MDM2 via its central acidic domain add them into the list of other ribosomal proteins, including L5, L11, L23, and S7 (Dai and Lu 2004, Dai et al 2004, Zhu et al 2009), known to regulate p53 activity via binding to MDM2 at the same acidic domain or adjacent zinc finger region. However, among all MDM2-binding ribosomal proteins that inhibit MDM2-induced p53 polyubiquitination for p53 stabilization, RPS27L and RPS27 have several unique features. First, they are the only known MDM2-binding ribosomal or ribosomal-like proteins directly subjecting to p53 transcriptional regulation, thus are directly involved in feedback regulation of p53/MDM2 axis. Second, unlike other MDM2-binding ribosomal proteins which are localized either in nucleolus (e.g. L11 or S7) (Bhat et al 2004, Lohrum et al 2003, Zhu et al 2009) or in nucleoplasm, (e.g. L23) (Dai et al 2004, Jin et al 2004), RPS27L (or RPS27) is mainly localized in cytoplasm under physiological unstressed condition where very low level of cytoplasmic MDM2 exists. Under stressed conditions that activate p53, a portion of RPS27L is shuttled to nucleoplasm where it co-localized with induced MDM2. Since RPS27L and MDM2 in both cytoplasm and nucleus are induced upon p53 activating signals (<xref ref-type="fig" rid="nihms252055f8">Fig 8C</xref>), induced RPS27L could compete with p53 for Mdm2 binding, thus releasing/protecting p53 from MDM2-mediated degradation in both subcellular locations, as evidenced by a reduction of p53 levels at both cytoplasm and nucleus upon actinomycin D treatment, if RPS27L is silenced (), induced RPS27L could compete with p53 for Mdm2 binding, thus releasing/protecting p53 from MDM2-mediated degradation in both subcellular locations, as evidenced by a reduction of p53 levels at both cytoplasm and nucleus upon actinomycin D treatment, if RPS27L is silenced (<xref ref-type="fig" rid="nihms252055f9">Fig 9D</xref>). Finally, our study showing that RPS27L is a physiological substrate of MDM2 added RPS27L into the list of ribosomal proteins, including L26 (). Finally, our study showing that RPS27L is a physiological substrate of MDM2 added RPS27L into the list of ribosomal proteins, including L26 (Ofir-Rosenfeld et al 2008) and S7 (Zhu et al 2009), that directly bind to MDM2 and serve as the substrates of MDM2. It is very likely that MDM2 in different sub-cellular compartments is responsible for the degradation of these ribosomal or ribosomal-like proteins under physiological condition or after stress stimuli.'], 'nihms252055f9': ['Finally, we determined if RPS27L silencing would reduce p53 levels induced by these p53-activating signals. Indeed, RPS27L silencing remarkably inhibited the basal and induced levels of p53 and p21 upon exposure to actinomycin D (<xref ref-type="fig" rid="nihms252055f9">Fig 9A</xref>) or radiation () or radiation (<xref ref-type="fig" rid="nihms252055f9">Fig 9B</xref>). Induction of p53 and its downstream targets, p21 and MDM2 by etoposide was also remarkably inhibited (). Induction of p53 and its downstream targets, p21 and MDM2 by etoposide was also remarkably inhibited (<xref ref-type="fig" rid="nihms252055f9">Fig 9C</xref>). Blockage of induction of p53 and its downstream targets was not due to an overall inhibition of protein expression upon RPS27L silencing, since the expression of many other short-lived proteins, including p27, c-JUN, WEE1, IκBα, and β-catenin was not changed under the same experimental conditions (data not shown). We also observed in a cell fractionation study that RPS27L silencing reduced the levels of p53 and MDM2 in both cytoplasm and nucleus upon actinomycin D treatment (). Blockage of induction of p53 and its downstream targets was not due to an overall inhibition of protein expression upon RPS27L silencing, since the expression of many other short-lived proteins, including p27, c-JUN, WEE1, IκBα, and β-catenin was not changed under the same experimental conditions (data not shown). We also observed in a cell fractionation study that RPS27L silencing reduced the levels of p53 and MDM2 in both cytoplasm and nucleus upon actinomycin D treatment (<xref ref-type="fig" rid="nihms252055f9">Fig 9D</xref>). Thus, the presence of RPS27L ensures the full activation of p53, consistent with our previous observation that RPS27L, upon induced by p53, amplifies p53 signals (). Thus, the presence of RPS27L ensures the full activation of p53, consistent with our previous observation that RPS27L, upon induced by p53, amplifies p53 signals (He and Sun 2007). This is likely achieved by competition of RPS27L with p53 for Mdm2 binding, thus counteracting the Mdm2-p53 negative feedback loop.'], 'nihms252055f10': ['In summary, our current work, along with our previous finding (He and Sun 2007), revealed a multi-level interplay between RPS27L and RPS27 family members and p53/MDM2 axis. p53 is activated upon DNA damage to transactivate MDM2 and RPS27L, and to transrepress RPS27 as well. On one hand, induced MDM2 binds to p53 and RPS27L and promotes their ubiquitination and degradation. On the other hand, induced RPS27L competes with p53 for MDM2 binding, thus releasing p53 from MDM2-mediated degradation, resulting in p53 stabilization. Through interacting with MDM2-p53 axis, RPS27L/S27 could regulate cell growth and survival (<xref ref-type="fig" rid="nihms252055f10">Fig 10</xref>).).']}
|
Ribosomal protein S27-like and S27 interplay with p53-MDM2 axis s a target, a substrate, and a regulator
|
[
"MDM2",
"p53",
"protein interplays",
"ribosomal proteins",
"ubiquitination"
] |
Oncogene
|
1302764400
|
We describe a phage display methodology for engineering synthetic antigen binders (sABs) that recognize either the apo or the ligand-bound conformation of maltose-binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by substantially increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for this allosteric effect. We show that sABs that recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Furthermore, the sABs can enhance maltose binding in vivo, as they provide a growth advantage to bacteria under low-maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations.
|
[
"Allosteric Regulation",
"Ligands",
"Models, Molecular",
"Mutation",
"Protein Binding",
"Protein Conformation",
"Protein Engineering"
] |
other
|
PMC3077453
| null |
35
|
[
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] |
Oncogene. 2011 Apr 14; 30(15):1798-1811
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NO-CC CODE
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Validation of the PlxnD1 mutant.(a) Negative stain 2D class averages of PlxnD1 WT were obtained by classifying 1305 particles into 10 classes. Scale bar represents 10 nm. (b) Negative stain 2D class averages of PlxnD1 mutant were obtained by classifying 1357 particles into 10 classes. (c) The double mutant of PlxnD1 was labelled with a thiol-reactive fluorescent dye, Alexa Fluor 488 C5 maleimide. The degree of labelling shows that the vast majority of PlxnD1 mutant molecules form the disulphide linked bond and thus the ring of the majority of PlxnD1 mutant molecules appears to be locked by the covalent bond. The degree of labelling for the hen egg ovalbumin, which we used as a positive control, is close to the number of free cysteines in ovalbumin; n=3. The data represent mean±SEM. P values were calculated by two-tailed t-test in Graphpad Prism, ***p<0.001
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EMS85132-f014
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2
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9b83cb1a935d131ad3f6e21e42155e2611c07b73cd146b2d746b8bb4713a2777
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EMS85132-f014.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
1258
] |
[{'image_id': 'EMS85132-f015', 'image_file_name': 'EMS85132-f015.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f015.jpg', 'caption': "An open conformation of PlxnD1 is required for force-dependent signalling.Mouse lung ECs in which endogenous PlxnD1 was knocked down were infected with adenoviruses expressing β-galactosidase (Ad.LacZ), WT or Mutant PlxnD1 and incubated with anti-PlxnD1 paramagnetic beads, followed by force application for 5 minutes before lysing and assaying vinculin phosphorylation by Western blotting; n=3 biological repeats. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism; *p<0.05.", 'hash': '1e7b6ec3985c3502fcfc5d32997d82dc3739284e770efe677d3477315c35ac7b'}, {'image_id': 'EMS85132-f012', 'image_file_name': 'EMS85132-f012.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f012.jpg', 'caption': "Relationship of PlxnD1 with other established mechanosensors(a) PECAM+/+ and PECAM-/- ECs were incubated with anti-PlxnD1-coated beads and subjected to force application for 5 minutes before examining phosphorylation of vinculin (n=3; *p < 0.05). (b,c) Mouse ECs were treated with siRNAs to Piezo1 and Gαq/11, then incubated with anti-PlxnD1-coated beads and subjected to force application for 5 minutes before examining phosphorylation of vinculin (n=3; *p < 0.05). The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism", 'hash': 'b64ce87fa5a9f833641f3b4e4ec6d66feae32687f453126502ac5f75a49e9729'}, {'image_id': 'EMS85132-supplement-Extended_Fig_11', 'image_file_name': 'EMS85132-supplement-Extended_Fig_11.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_11.jpg', 'caption': None, 'hash': '118dacdccd1cee08e6269b8250dcaa194e60ea05cfbec98090307bb0f2cce5a9'}, {'image_id': 'EMS85132-f003', 'image_file_name': 'EMS85132-f003.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f003.jpg', 'caption': "PlxnD1, NRP1 and VEGFR2 mechano-complex functions upstream of known mechanosensory hotspots and is sufficient for responsiveness to shear stress(a) Schematic showing signalling at the junctional complex and integrins. (b) Mouse ECs were transfected with Scr or PlxnD1 siRNA, exposed to shear stress for 2min before immunoprecipitating VEGFR2 and examining its phosphorylation and association with the p85 subunit of PI3K and VE-Cadherin, n=3. (c) Mouse ECs were transfected with Scr or PlxnD1 siRNA, exposed to shear stress for 30min before immunoprecipitating integrin αVβ3 and examining its association with Shc, n=3. (d) ECs treated with VEGFR2 kinase inhibitor SU1498, transfected with siRNA to NRP1 or treated with NRP1 blocking antibody, then incubated with anti-PlxnD1-coated beads and subjected to force for 5min before examining phosphorylation of vinculin. (n=3; *p < 0.05) The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism. (e) Mouse ECs were exposed to shear stress before immunoprecipitating VEGFR2 and examining its phosphorylation and association with PlxnD1, NRP1 and Src; n=3. (f) Mouse ECs were exposed to shear stress before immunoprecipitating NRP1 and examining its association with PlxnD1 and VEGFR2; n=3. (g) Mouse ECs transfected with either Scr or NRP1 siRNA were exposed to shear stress before immunoprecipitating VEGFR2 and examining its association with PlxnD1; n=3. (h) Schematic showing that reconstitution of PlxnD1, VEGFR2 and NRP1 in Cos7 cells confers shear stress sensitivity to these cells. (i) Cos7 cells were left untransfected or transfected with NRP1 and VEGFR2, with or without PlxnD1 before being subjected to shear stress for 2min and VEGFR2 was immunoprecipitated. Shear stress sensitivity was assessed by examining phospho-VEGFR2 levels, complex formation between VEGFR2 and Src and complex formation of PlxnD1, VEGFR2 and NRP1, n=3. All shear stress experiments were at 12 dynes/cm2 using a parallel plate system.", 'hash': '2643a2189acf1f649c7491ba6e4ffe734a110679496942f402f3745f2a465494'}, {'image_id': 'EMS85132-supplement-Extended_Fig_6', 'image_file_name': 'EMS85132-supplement-Extended_Fig_6.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_6.jpg', 'caption': None, 'hash': '7a3bd33c270451898f38d769ba07ede95fa8bb8c6cfb3378450c9da63ce31b43'}, {'image_id': 'EMS85132-f004', 'image_file_name': 'EMS85132-f004.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f004.jpg', 'caption': "PlxnD1 flexion is required for mechanotransduction.(a) Schematic domain organisation of PlxnD1 spanning amino acids 1-1925. SS, signal sequence; TM, transmembrane region; c, cytoplasmic region. (b) Representative negative stain class averages of the PlxnD1 ectodomain and corresponding structural models showing the ring-like and open conformations, scale bar 10nm. 2D class averages were obtained by classifying 1357 particles into 10 classes. (c) Model of opening the ring-like ectodomain which confers PlxnD1 mechanosensory functions. (d) Design of PlxnD1 mutant with an intramolecular disulphide bond to lock the ring-like structure. Zoom-in view shows the disulphide bond between the sema domain (domain 1) and IPT5 domain (domain 9). (e,f) ECs in which endogenous PlxnD1 was knocked down were infected with adenoviruses expressing WT or Mutant PlxnD1, treated with Sema3E for 30min or incubated with anti-PlxnD1 paramagnetic beads followed by force application (10pN; 30min). Cells were immunostained with anti-vinculin antibodies. Focal adhesion number was quantified using ImageJ; n=30 cells over either 4 (in e) or 3 (in f) biological replicates ****p<0.0001; scale bar 10 μm. (g) Cos7 cells were transfected with WT or mutant PlxnD1, NRP1 and VEGFR2 before shear stress application for 2mins and VEGFR2 was immunoprecipitated. Shear stress sensitivity was assessed by examining phospho-VEGFR2 levels, complex formation between VEGFR2 and Src and complex of PlxnD1, VEGFR2 and NRP1, n=3. (h) ECs in which endogenous PlxnD1 was knocked down were infected with adenoviruses expressing WT or mutant PlxnD1 and incubated with anti-PlxnD1 paramagnetic beads followed by force application (10pN). Phosphorylation of Akt, ERK 1/2 and VEGFR2 was determined, n=3; *p<0.05 relative to “no force” condition; #p<0.05 relative to the respective WT force time point. (i) ECs in which endogenous PlxnD1 was knocked down were infected with adenoviruses expressing WT or mutant PlxnD1 and subjected to fluid shear stress. Phosphorylation of Akt, ERK 1/2 and eNOS was determined, n=3 biological repeats; *p<0.05 relative to static condition; #p<0.05 relative to the respective WT shear time point. All data are mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism.", 'hash': 'adc712c881ce623945f4b8c9621247888fb4c27c418856e1f44bc8c2c0695f08'}, {'image_id': 'EMS85132-supplement-Extended_Fig_1', 'image_file_name': 'EMS85132-supplement-Extended_Fig_1.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_1.jpg', 'caption': None, 'hash': '4c70eb5f6316c8386541c2d312c10b17430ebd85e45868981273927d8a44e9c6'}, {'image_id': 'EMS85132-supplement-Extended_Fig_8', 'image_file_name': 'EMS85132-supplement-Extended_Fig_8.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_8.jpg', 'caption': None, 'hash': '6fb7f3447eb08a28f3f31675d54439adaddc3241b20928bcb4d858d72e98f973'}, {'image_id': 'EMS85132-supplement-Extended_Fig_10', 'image_file_name': 'EMS85132-supplement-Extended_Fig_10.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_10.jpg', 'caption': None, 'hash': '977346e891a3dfc7cf0caae88cde1edf47d5eb246b0c5f0fca33bd611b6ed95b'}, {'image_id': 'EMS85132-f013', 'image_file_name': 'EMS85132-f013.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f013.jpg', 'caption': 'Force application on other members of the PlxnD1 mechano-complex does not elicit a mechanotransduction responseMouse ECs were incubated with (a) anti-VEGFR2 or (b) anti-NRP1 antibody-coated beads and subjected to force (10pN) for the indicated time periods. Phosphorylation of Akt and ERK1/2 was determined by western blotting and quantified using Image Studio Lite Ver 5.2, n=3 biological repeats. The data represent mean±SEM.', 'hash': '1a947db92084931a737b34d8b3510027ed95f28e9ac1f1ef7ce0aab0d8f8d6ed'}, {'image_id': 'EMS85132-f014', 'image_file_name': 'EMS85132-f014.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f014.jpg', 'caption': 'Validation of the PlxnD1 mutant.(a) Negative stain 2D class averages of PlxnD1 WT were obtained by classifying 1305 particles into 10 classes. Scale bar represents 10 nm. (b) Negative stain 2D class averages of PlxnD1 mutant were obtained by classifying 1357 particles into 10 classes. (c) The double mutant of PlxnD1 was labelled with a thiol-reactive fluorescent dye, Alexa Fluor 488 C5 maleimide. The degree of labelling shows that the vast majority of PlxnD1 mutant molecules form the disulphide linked bond and thus the ring of the majority of PlxnD1 mutant molecules appears to be locked by the covalent bond. The degree of labelling for the hen egg ovalbumin, which we used as a positive control, is close to the number of free cysteines in ovalbumin; n=3. The data represent mean±SEM. P values were calculated by two-tailed t-test in Graphpad Prism, ***p<0.001', 'hash': '9b83cb1a935d131ad3f6e21e42155e2611c07b73cd146b2d746b8bb4713a2777'}, {'image_id': 'EMS85132-supplement-Extended_Fig_9', 'image_file_name': 'EMS85132-supplement-Extended_Fig_9.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_9.jpg', 'caption': None, 'hash': '14c768a91aa94156dbfe281eeb15025012fa37db3b202202a6f4e4556125d99c'}, {'image_id': 'EMS85132-f005', 'image_file_name': 'EMS85132-f005.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f005.jpg', 'caption': 'Knockout/knockdown of PlxnD1 and other genes in ECs(a-f) ECs were either isolated from PlxnD1fl/fl and PlxnD1iECKO mice, or treated with siRNAs to knockdown PlxnD1, NRP1, Piezo1 and Gαq/11. Knockdowns/knockouts were confirmed by western blotting, using GAPDH as a loading control (g) PlxnD1 was knocked down in mouse ECs using a pool of siRNAs, followed by infection with an adenovirus expressing either β-galactosidase (LacZ), WT or mutant PlxnD1. Protein levels were normalised against the housekeeping gene GAPDH. KD=Mean knockdown efficiency based on n=3; KO= Mean knockout efficiency based on n=3.', 'hash': '1329b8fa30fd6665beacef779eccbc598b698291ebcbf81750af495689780b53'}, {'image_id': 'EMS85132-f002', 'image_file_name': 'EMS85132-f002.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f002.jpg', 'caption': "PlxnD1 is a mechanosensor that mediates the EC response to force(a) Mouse ECs were incubated with anti-PlxnD1 or C44 (negative control) antibody-coated beads and subjected to force (10pN) for the indicated time periods. Phosphorylation of VEGFR2, Akt and ERK1/2 was determined by western blotting and quantified using Image Studio Lite Ver 5.2, n=3 biological repeats; *p<0.05 relative to no force condition, #p<0.05 relative to the respective force application time point with PlxnD1. (b) BAECs were loaded with Fluo-8AM dye and then incubated with beads coated with an antibody to the extracellular domain of PlxnD1 or Poly L-lysine (negative control). The beads were then subjected to force (1nN). Calcium responses were measured by calculating the fluorescent intensity of individual cells before (10 seconds), during (20 seconds), and after (30 seconds) stimulation. Representative images are shown along with quantification. n=18 cells for PlxnD1 and n=19 cells for control over 3 independent biological replicates. ***p < 0.001 relative to unstimulated controls, scale bar represents 10 μm. Representative trace for calcium influx response over time has also been shown. The arrow marks the start of the stimulation. (c) BAECs were incubated with anti-PlxnD1-coated beads and subjected to force (10pN) for 30min. ECs were fixed and stained with an anti-vinculin antibody to mark focal adhesions. Focal adhesion number was quantified using ImageJ software. Values were normalised to the “no force” condition. Location of the beads are highlighted in yellow circles (n=50 cells for each condition from 3 independent biological replicates;****p<0.0001; scale bar represents 10 μm (d) Mouse ECs were incubated with anti-PlxnD1 or C44 coated beads and subjected to 10pN force for the indicated time periods. Phosphorylation of vinculin was determined by western blotting and quantified using Image Studio Lite Ver 5.2, n=3 biological repeats; *p<0.05 relative to no force condition, #p<0.05 relative to the respective force application time point with PlxnD1. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism.", 'hash': '5a76cdbee14737a4799c01a2d7ea4b03da23d848873da74ae036c03e8134c46a'}, {'image_id': 'EMS85132-supplement-Extended_Fig_7', 'image_file_name': 'EMS85132-supplement-Extended_Fig_7.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_7.jpg', 'caption': None, 'hash': 'e21aa70e7bfc88792c47553f5c205d4f6140802fd055919f831ec279eedcacde'}, {'image_id': 'EMS85132-f008', 'image_file_name': 'EMS85132-f008.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f008.jpg', 'caption': "Lipid profile analysis and expression of inflammatory markers in the aortic arch(a) Bodyweights and lipid profile analysis of PlxnD1fl/fl; ApoE-/- and PlxnD1iECKO; ApoE-/- mice after 10 weeks of high fat diet feeding (at 16-17 weeks of age); n=8. The data represent mean±SEM. (b) Representative en face preparations of aortic arches immunostained for VCAM-1 (n=3) and MCP-1 (n=5) from PlxnD1fl/fl; ApoE-/- and PlxnD1iECKO, ApoE-/- mice with quantification of fluorescence intensity in fold change; 3-5 images taken from the inner curvature of aortic arches of each mice. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism, *p<0.05", 'hash': 'bc948fdbb33c83ea0d0cb727c10b16ccda7116e25b8bc334324143e1e9061911'}, {'image_id': 'EMS85132-f001', 'image_file_name': 'EMS85132-f001.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f001.jpg', 'caption': "PlxnD1 mediates the endothelial cell response to fluid shear stress and regulates the site-specific distribution of atherosclerosis(a.b) Mouse ECs were transfected with either Scr or PlxnD1 siRNA and exposed to either atheroprotective or atheroprone flow for 24 hours, using a cone and plate viscometer. Q-PCR was performed for KLF2 and KLF4 on samples subjected to atheroprotective flow, and inflammatory markers MCP-1 and VCAM-1 on samples subjected to atheroprone flow; n=4 biological replicates (c) The descending thoracic aorta was isolated and prepared en face from PlxnD1fl/fl and PlxnD1iECKO mice and stained for β-catenin, phalloidin and DAPI to visualise the cell junctions, actin stress fibres and nuclei. Quantification of alignment was performed using ImageJ; 3-5 images (each image has n≤100 cells) taken from three regions along the length of descending aorta collected from 5 animals of each genotype (for exact n, please refer to source data). (d) Representative en face preparations of whole aortas showing atherosclerosis in PlxnD1fl/fl; ApoE-/- and PlxnD1iECKO; ApoE-/- mice after 10 weeks of high fat diet feeding, visualised by Oil Red O staining. (e) Quantification of lesion area in whole aortas and aortic arches from PlxnD1fl/fl; ApoE-/- and PlxnD1iECKO; ApoE-/- mice; n=8 (f) Aortic arches from PlxnD1fl/fl; ApoE-/- and PlxnD1iECKO, ApoE-/- mice were isolated and Q-PCR was performed for inflammatory markers VCAM-1 and MCP-1; n=5. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; scale bar represents 20 μm", 'hash': 'ea450bf750e1fcb58e472411301f7e3268ffecdcd233e4243d4c433522717762'}, {'image_id': 'EMS85132-supplement-Extended_Fig_4', 'image_file_name': 'EMS85132-supplement-Extended_Fig_4.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_4.jpg', 'caption': None, 'hash': '54e38b05228fded000d00408b04880d395b42635312e95aaf6e7ce300d4adbc5'}, {'image_id': 'EMS85132-f006', 'image_file_name': 'EMS85132-f006.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f006.jpg', 'caption': "PlxnD1 mediates the endothelial cell response to fluid shear stress(a) Bovine aortic ECs (BAECs) were transfected with Scr or PlxnD1 siRNA and exposed to laminar fluid shear stress (12 dynes/cm2) using a parallel plate system for the indicated time periods. Phosphorylation of Akt (n=6), ERK1/2 (n=5) and eNOS (n=8) was determined by western blotting and quantified using Image Studio Lite Ver 5.2. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism.*p<0.05 relative to static condition; #p<0.05 relative to the respective siScr shear time point. (b) BAECs were transfected with Scr or PlxnD1 siRNA and exposed to atheroprotective shear stress for 24 hours. Cells were fixed and stained with phallodin and DAPI as well as antibodies to β-catenin to visualise actin stress fibres, nuclei and cell junctions, respectively. Quantification of alignment was performed using ImageJ; n>50cells over 4 biological replicates (for exact n, please refer to source data). The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism; ****p<0.0001", 'hash': '98a8f445c1af255bdc4bd840aced51a226c37d12fc0d9ca8a8c58e53b809d8ca'}, {'image_id': 'EMS85132-supplement-Extended_Fig_3', 'image_file_name': 'EMS85132-supplement-Extended_Fig_3.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_3.jpg', 'caption': None, 'hash': '6149e5ba9c595ede5fa929dc2948a6cdc6b745eba0f2e5105eb7829bff518f00'}, {'image_id': 'EMS85132-f010', 'image_file_name': 'EMS85132-f010.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f010.jpg', 'caption': "Mechanical force on PlxnD1 results in integrin activation, but ligand stimulation causes ECs to collapse(a) BAECs were incubated with anti-PlxnD1-coated beads and subjected to force (10pN) for 5 minutes. ECs were fixed and stained with HUTS4 antibody to mark ligated β1 integrin. Mean fluorescence intensity was quantified using ImageJ software. Values were normalised to the “no force” condition. Location of the beads are highlighted in yellow circles. n=50 cells/condition from 3 independent experiments. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism ****p<0.0001, scale bar represents 10 μm. (b) BAECs were incubated with Sema3E or vehicle, fixed and stained with anti-vinculin antibody to mark focal adhesions. Focal adhesion number was quantified using ImageJ software. Values were normalised to the “vehicle” condition. n=30 cells/condition from 3 independent experiments. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism ****p<0.0001, scale bar represents 10 μm.", 'hash': 'a8c66653125b34fc1e45e4ff948342f15f97b20951edb0ef53c89eff5f4afdb5'}, {'image_id': 'EMS85132-f007', 'image_file_name': 'EMS85132-f007.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f007.jpg', 'caption': 'Mechanotransduction via PlxnD1 is independent of its ligand binding functions(a) BAECs were treated with Sema3E function blocking antibody or control antibody (1ug/ml) and exposed to fluid shear stress for the indicated times. Phosphorylation of eNOS, Akt and ERK1/2 was determined by western blotting and quantified using Image Studio Lite Ver 5.2, n=3 biological repeats. The data represent mean±SEM. (b) BAECs were treated with Sema3E blocking antibody or control antibody for 1 hour before being exposed to Sema3E for 30 minutes at the indicated concentrations. Cells were fixed and probed with anti-vinculin, then stained with phalloidin and DAPI to visualise focal adhesions, actin stress fibres and nuclei, respectively. EC collapse was quantified by measuring cell area using ImageJ. The data represent mean±SEM. Significance was determined by ANOVA with a Tukey post hoc test in Graphpad Prism; ****p<0.0001. n=59-82 cells over 3 independent experiments (for exact n, please refer to source data); scale bar represents 50 μm.', 'hash': '825fad91cb3cf2ddafc6c02f4823135c79ed8ca5effd6ec18caca02966c98cb2'}, {'image_id': 'EMS85132-supplement-Extended_Fig_2', 'image_file_name': 'EMS85132-supplement-Extended_Fig_2.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_2.jpg', 'caption': None, 'hash': '8dd7878665130db2dcbd3840bd3d990d3697d2bb20383b42f1f7148d6a69e344'}, {'image_id': 'EMS85132-supplement-Extended_Fig_5', 'image_file_name': 'EMS85132-supplement-Extended_Fig_5.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-supplement-Extended_Fig_5.jpg', 'caption': None, 'hash': '52611f7498c558b33857d86a3bff91aae8329d81d8e402af22f79407f8d9fe14'}, {'image_id': 'EMS85132-f009', 'image_file_name': 'EMS85132-f009.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f009.jpg', 'caption': "Atherosclerosis in the descending aorta(a) Representative en face preparations of the whole aorta showing atherosclerosis in PlxnD1fl/fl; ApoE-/- and Plxn D1iECKO; ApoE-/- mice after 20 weeks of high fat diet feeding, visualised by Oil red O staining. (b) Quantification of lesion area in the thoracic aortas, abdominal aortas and whole descending aortas (thoracic aorta+abdominal aorta) from PlxnD1fl/fl; ApoE-/- and PlxnD1iECKO; ApoE-/- mice; n=9 PlxnD1fl/fl; ApoE-/- and 8 PlxnD1iECKO; ApoE-/-. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism *p<0.05, **p<0.01, ****p<0.0001", 'hash': '117c859694961c02c5e21369323efc3fe08069c8eacaf51d5b48225ab6aa224e'}, {'image_id': 'EMS85132-f011', 'image_file_name': 'EMS85132-f011.jpg', 'image_path': '../data/media_files/PMC7025890/EMS85132-f011.jpg', 'caption': "PlxnD1 co-localises and associates with members of the junctional mechanosensory complex, and its levels are not regulated by flow, unlike Sema3E(a) The descending thoracic aorta or the inner curvature of aortic arches were isolated and prepared en face from wild-type mice and stained for PlxnD1, PECAM-1 and DAPI. Quantification of PlxnD1 levels was performed by fluorescence intensity measurement on ImageJ; 4-6 images were taken on tissue collected from n=4 animals. The data represent mean±SEM. Scale bar represents 20 μm (b) The descending thoracic aorta was isolated and prepared en face from PlxnD1iECKO mice and stained for PlxnD1 to assess the specificity of the PlxnD1 immunostain, n=3 animals all showing similar result. (c) The descending thoracic aorta or the inner curvature of aortic arches were isolated and prepared en face from wild-type mice and stained for Sema3E, PECAM-1 and DAPI. Quantification of Sema3E levels was performed by fluorescence intensity measurement on ImageJ; 4-6 images were taken on tissue collected from n=3 animals. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism *p<0.05; scale bar represents 20 μm (d) Mouse ECs were exposed to shear stress for the indicated times or left as static controls before immunoprecipitating PlxnD1 and examining its association with the junctional mechanosensory complex (PECAM, VE-cadherin and VEGFR2) as well as PI3K/p85, n=3", 'hash': '2ebf89eccebb4d212132e1af86830fd674025a22d597bd74fca7202012d69feb'}]
|
{'EMS85132-f005': ['To determine the role of PlxnD1 under flow conditions, we transfected bovine aortic ECs (BAECs) with either Scrambled (Scr) or PlxnD1 siRNAs (<xref ref-type="fig" rid="EMS85132-f005">Extended Fig. 1a</xref>), and subjected them to shear stress. Knockdown of PlxnD1 attenuated shear stress-induced activation of key signalling mediators Akt, ERK1/2 and eNOS (), and subjected them to shear stress. Knockdown of PlxnD1 attenuated shear stress-induced activation of key signalling mediators Akt, ERK1/2 and eNOS (<xref ref-type="fig" rid="EMS85132-f006">Extended Fig. 2a</xref>). PlxnD1-dependent mechanotransduction is independent of its ligand Sema3E, as incubation with a Sema3E function blocking antibody did not affect the flow-induced activation of signalling cascades (). PlxnD1-dependent mechanotransduction is independent of its ligand Sema3E, as incubation with a Sema3E function blocking antibody did not affect the flow-induced activation of signalling cascades (<xref ref-type="fig" rid="EMS85132-f007">Extended Fig. 3</xref>). Next, we examined the role of PlxnD1 in the hallmark response to atheroprotective shear stress by examining alignment in the direction of flow. EC alignment with flow direction is highly correlated with atheroresistant regions of arteries and plays an important role in the activation of anti-inflammatory pathways. PlxnD1-depleted ECs showed a striking failure to align in response to shear stress and displayed fewer and more disorganised actin stress fibres (). Next, we examined the role of PlxnD1 in the hallmark response to atheroprotective shear stress by examining alignment in the direction of flow. EC alignment with flow direction is highly correlated with atheroresistant regions of arteries and plays an important role in the activation of anti-inflammatory pathways. PlxnD1-depleted ECs showed a striking failure to align in response to shear stress and displayed fewer and more disorganised actin stress fibres (<xref ref-type="fig" rid="EMS85132-f006">Extended Fig.2b</xref>). Quantification of alignment by measuring the orientation angle and the elongation factor indicate that PlxnD1 is required for EC alignment with flow. We also examined levels of Kruppel-like factors KLF2 and KLF4, key anti-inflammatory transcription factors which are known to be upregulated by atheroprotective shear stress.). Quantification of alignment by measuring the orientation angle and the elongation factor indicate that PlxnD1 is required for EC alignment with flow. We also examined levels of Kruppel-like factors KLF2 and KLF4, key anti-inflammatory transcription factors which are known to be upregulated by atheroprotective shear stress.8,9 Congruently, we found that knockdown of PlxnD1 attenuated flow-induced upregulation of both these genes compared to control cells. (<xref ref-type="fig" rid="EMS85132-f001">Fig. 1a</xref>). We then asked if PlxnD1 could mediate the endothelial response to disturbed shear stress. We subjected ECs to atheroprone flow for 24h and examined mRNA levels of pro-inflammatory genes Monocyte Chemoattractant Protein-1 (MCP-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1)). We then asked if PlxnD1 could mediate the endothelial response to disturbed shear stress. We subjected ECs to atheroprone flow for 24h and examined mRNA levels of pro-inflammatory genes Monocyte Chemoattractant Protein-1 (MCP-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1)10. We noted that knockdown of PlxnD1 in ECs with siRNA significantly reduced the upregulation of both genes in response to atheroprone shear stress (<xref ref-type="fig" rid="EMS85132-f001">Fig. 1b</xref>). Combined, these data demonstrate that PlxnD1 is a critical mediator of key shear stress responses in ECs.). Combined, these data demonstrate that PlxnD1 is a critical mediator of key shear stress responses in ECs.', 'To explore the biological relevance of our findings, we used a transgenic mouse model to enable endothelial-specific inducible deletion of PlxnD1 (PlxnD1iECKO) (<xref ref-type="fig" rid="EMS85132-f005">Extended Fig.1b</xref>, , <xref ref-type="fig" rid="EMS85132-f011">7b</xref>). Confocal imaging of the endothelial actin filaments and staining for the junctional marker β-catenin revealed reduced EC elongation and intensity of actin stress fibres in the absence of PlxnD1 (). Confocal imaging of the endothelial actin filaments and staining for the junctional marker β-catenin revealed reduced EC elongation and intensity of actin stress fibres in the absence of PlxnD1 (<xref ref-type="fig" rid="EMS85132-f001">Fig.1c</xref>), consistent with ), consistent with in vitro observations (<xref ref-type="fig" rid="EMS85132-f006">Extended Fig.2b</xref>).).', 'To investigate molecular mechanisms even further, we examined the role of the PlxnD1 co-receptor neuropilin-1 (NRP1). NRP1 is a cell surface transmembrane protein that acts as a Sema3 and VEGF co-receptor for PlxnD1 and VEGFR2, respectively24 and its presence in neurons switches the Sema3E signal from repulsion to attraction25. We found a requirement for NRP1 in the PlxnD1 force response, as both knockdown (<xref ref-type="fig" rid="EMS85132-f005">Extended Fig. 1d</xref>) and inhibition of NRP1 abolished force-induced phosphorylation of vinculin () and inhibition of NRP1 abolished force-induced phosphorylation of vinculin (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3d</xref>). We also observed that shear stress induced the formation of a complex between PlxnD1, VEGFR2 and NRP1 (). We also observed that shear stress induced the formation of a complex between PlxnD1, VEGFR2 and NRP1 (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3e, f</xref>) and this complex was dependent on NRP1 () and this complex was dependent on NRP1 (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3g</xref>). Taken together, these data show that PlxnD1 associates with NRP1 and VEGFR2 in response to flow and operates upstream of both the junctional complex and integrins.). Taken together, these data show that PlxnD1 associates with NRP1 and VEGFR2 in response to flow and operates upstream of both the junctional complex and integrins.'], 'EMS85132-f001': ['Given the decrease in inflammatory gene expression in response to atheroprone shear stress in vitro observed with loss of PlxnD1 (<xref ref-type="fig" rid="EMS85132-f001">Fig.1b</xref>), we assessed the role of endothelial PlxnD1 in a pathophysiogical setting. Atherosclerotic lesions are known to occur in regions of the vasculature with low/disturbed blood flow, flow reversal and other complex spatial/temporal flow patterns), we assessed the role of endothelial PlxnD1 in a pathophysiogical setting. Atherosclerotic lesions are known to occur in regions of the vasculature with low/disturbed blood flow, flow reversal and other complex spatial/temporal flow patterns1. Systemic risk factors, such as hypercholesterolaemia, interact with local biomechanical factors to initiate and advance atherosclerotic plaque deposition. To assess whether endothelial deletion of PlxnD1 affected atherosclerosis in vivo, we crossed PlxnD1fl/fl and PlxnD1iECKO into the hypercholesterolaemic Apolipoprotein E deficient (ApoE-/-) background11 and subjected them to a high-fat diet for 10 weeks. Although animal body weights and lipid levels were unaffected by loss of PlxnD1 (<xref ref-type="fig" rid="EMS85132-f008">Extended Fig. 4a</xref>), quantification revealed a significant decrease in plaque burden for both the whole aorta and the arch in PlxnD1), quantification revealed a significant decrease in plaque burden for both the whole aorta and the arch in PlxnD1iECKO /ApoE-/- animals (<xref ref-type="fig" rid="EMS85132-f001">Fig. 1d, e</xref>). To explore these differences further, we examined expression of inflammatory markers in the inner curvature of the aortic arch. Immunostaining and qPCR analysis showed reduced levels of MCP-1 and VCAM-1 in PlxnD1). To explore these differences further, we examined expression of inflammatory markers in the inner curvature of the aortic arch. Immunostaining and qPCR analysis showed reduced levels of MCP-1 and VCAM-1 in PlxnD1iECKO/ApoE-/- compared to PlxnD1fl/fl/ApoE-/- mice (<xref ref-type="fig" rid="EMS85132-f001">Fig. 1f</xref>, , <xref ref-type="fig" rid="EMS85132-f008">Extended Fig. 4b</xref>). Given the atheroprotective role of laminar shear stress and the reduced alignment with loss of PlxnD1, we examined the effects in the atheroprotected descending aorta. After high-fat feeding for an extended period of 20 weeks, we observed increased plaque burden in the descending aortas of PlxnD1). Given the atheroprotective role of laminar shear stress and the reduced alignment with loss of PlxnD1, we examined the effects in the atheroprotected descending aorta. After high-fat feeding for an extended period of 20 weeks, we observed increased plaque burden in the descending aortas of PlxnD1iECKO /ApoE-/- (<xref ref-type="fig" rid="EMS85132-f009">Extended Fig. 5</xref>); these plaques also appeared to correlate with intercostal branch points that have flow disturbances. Together, these results show that endothelial PlxnD1 is required for the endothelial response to fluid shear stress and the site-specific distribution of atherosclerosis.); these plaques also appeared to correlate with intercostal branch points that have flow disturbances. Together, these results show that endothelial PlxnD1 is required for the endothelial response to fluid shear stress and the site-specific distribution of atherosclerosis.', 'The datasets generated during and/or analysed during this study are either included within the manuscript or are available from the corresponding author on reasonable request. Source data for <xref ref-type="fig" rid="EMS85132-f001">Fig.1</xref>--<xref ref-type="fig" rid="EMS85132-f004">4</xref> and and <xref ref-type="fig" rid="EMS85132-f006">Extended Data Fig 2</xref>--<xref ref-type="fig" rid="EMS85132-f015">11</xref> are included in the online version of the paper. Gel Source Data can be found in are included in the online version of the paper. Gel Source Data can be found in Supplementary Fig. 1.'], 'EMS85132-f002': ['The requirement for PlxnD1 in flow-mediated responses in vitro and in vivo, prompted us to ask if this is because PlxnD1 is simply a player in mechanochemical signalling cascades, or functions as a mechanoreceptor, capable of detecting mechanical force. We applied tensional forces, using a magnetic system12, to paramagnetic beads coated with an antibody that recognises the extracellular domain of PlxnD1 and examined force responses using 4 different readouts. First, force on PlxnD1 induced activation of the same signalling cascades (ERK1/2, Akt, VEGFR2) (<xref ref-type="fig" rid="EMS85132-f002">Fig. 2a</xref>), as those induced by shear stress (), as those induced by shear stress (<xref ref-type="fig" rid="EMS85132-f006">Extended Fig. 2a</xref>))13. Second, we observed robust transient increase in intracellular calcium levels in ECs when force was applied on PlxnD1 (<xref ref-type="fig" rid="EMS85132-f002">Fig. 2b</xref>), similar to the response observed for other recently discovered mechanosensors), similar to the response observed for other recently discovered mechanosensors14,15. Third, we examined cytoskeletal responses12: ECs responded to application of force on PlxnD1 by exhibiting a robust increase in both vinculin-positive focal adhesions (<xref ref-type="fig" rid="EMS85132-f002">Fig. 2c</xref>) and ligated integrin β1 staining () and ligated integrin β1 staining (<xref ref-type="fig" rid="EMS85132-f010">Extended Fig. 6a</xref>). Notably, the mechanotransduction response was not restricted to the vicinity of the magnetic bead under tension, but was a global cell-wide phenomenon. Fourth, we examined phosphorylation of vinculin at Y822, a site known to be phosphorylated when force is applied on E-cadherin). Notably, the mechanotransduction response was not restricted to the vicinity of the magnetic bead under tension, but was a global cell-wide phenomenon. Fourth, we examined phosphorylation of vinculin at Y822, a site known to be phosphorylated when force is applied on E-cadherin16, and observed a significant increase in its activation following force on PlxnD1 (<xref ref-type="fig" rid="EMS85132-f002">Fig. 2d</xref>). These mechanoresponses were specific to PlxnD1 because ECs incubated with beads coated with another transmembrane receptor CD44 or Poly-L-lysine did not respond to force. These data clearly demonstrate that PlxnD1 is a direct force sensor that can elicit robust and global mechanical signalling in ECs.). These mechanoresponses were specific to PlxnD1 because ECs incubated with beads coated with another transmembrane receptor CD44 or Poly-L-lysine did not respond to force. These data clearly demonstrate that PlxnD1 is a direct force sensor that can elicit robust and global mechanical signalling in ECs.'], 'EMS85132-f003': ['Cell-cell and cell-matrix adhesions represent two highly mechanically active sites within ECs. These include the junctional mechanosensory complex comprising PECAM-1, VEGFR2 and VE-cadherin and integrins at the cytoskeleton-extracellular matrix interface13,17 (<xref ref-type="fig" rid="EMS85132-f003">Fig.3a</xref>). We interrogated the relationship between PlxnD1 and these mechanical ‘hotspots’ in ECs. ). We interrogated the relationship between PlxnD1 and these mechanical ‘hotspots’ in ECs. En face confocal imaging revealed robust and similar expression of PlxnD1 in ECs in both arch and descending aorta and co-localisation with PECAM-1 at cell-cell junctions (<xref ref-type="fig" rid="EMS85132-f011">Extended Fig. 7a</xref>). Staining was specific as it was not observed in PlxnD1). Staining was specific as it was not observed in PlxnD1iECKO aortas (<xref ref-type="fig" rid="EMS85132-f011">Extended Fig. 7b</xref>). Sema3E was also observed in ). Sema3E was also observed in en face sections with its expression being lower in the arch (<xref ref-type="fig" rid="EMS85132-f011">Extended Fig. 7c</xref>). Co-immunoprecipitation experiments showed flow-induced association of PlxnD1 with components of the junctional mechanosensory complex (PECAM-1, VEGFR2, VE-cadherin and PI3K/p85) (). Co-immunoprecipitation experiments showed flow-induced association of PlxnD1 with components of the junctional mechanosensory complex (PECAM-1, VEGFR2, VE-cadherin and PI3K/p85) (<xref ref-type="fig" rid="EMS85132-f011">Extended Fig. 7d</xref>). To explore if PlxnD1 is just another component of the junctional complex or whether it operates upstream, we used immunoprecipitation to interrogate complex formation both at the junctional mechanosensory complex and integrin-matrix adhesions. We found that responses at the junctional complex, such as shear stress-induced phosphorylation of VEGFR2 and association of the p85 subunit of PI3K and VE-cadherin with VEGFR2). To explore if PlxnD1 is just another component of the junctional complex or whether it operates upstream, we used immunoprecipitation to interrogate complex formation both at the junctional mechanosensory complex and integrin-matrix adhesions. We found that responses at the junctional complex, such as shear stress-induced phosphorylation of VEGFR2 and association of the p85 subunit of PI3K and VE-cadherin with VEGFR213, were all abrogated by knockdown of PlxnD1 (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3b</xref>). In agreement, both inhibition of VEGFR2 receptor kinase (). In agreement, both inhibition of VEGFR2 receptor kinase (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3d</xref>) and deletion of PECAM-1 abrogated force-induced signalling, suggesting that junctional mechanosensory components are necessary intermediates for the PlxnD1 force response () and deletion of PECAM-1 abrogated force-induced signalling, suggesting that junctional mechanosensory components are necessary intermediates for the PlxnD1 force response (<xref ref-type="fig" rid="EMS85132-f012">Extended Fig. 8a</xref>). Similarly, flow-induced complex formation at integrin-matrix adhesions (as assayed by association of Shc with integrin α). Similarly, flow-induced complex formation at integrin-matrix adhesions (as assayed by association of Shc with integrin αvβ318,19) was also strongly reduced with loss of PlxnD1 (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3c</xref>). Recent work has highlighted a role for Piezo1 and G). Recent work has highlighted a role for Piezo1 and Gq/G11-mediated mechanosignalling, although there are conflicting reports as to whether these pathways are linked20,21 or independent of each other22,23. Force application on PlxnD1 showed that loss of Gq/G11 abolished the PlxnD1 force response, while knockdown of Piezo1 had no effect (<xref ref-type="fig" rid="EMS85132-f005">Extended Fig. 1e,f</xref>; ; <xref ref-type="fig" rid="EMS85132-f012">Extended Fig., 8b,c</xref>).).', 'To test if PlxnD1 (and its molecular partners) is sufficient to confer mechanosensitivity in a heterologous cell line, we transfected Cos7 cells with plasmid constructs expressing PlxnD1, NRP1 and/or VEGFR2 and applied shear stress (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3h</xref>). These cells do not express any of the components of the junctional complex (i.e. PECAM-1 or VE-cadherin) and, thus, constitute an ideal system to monitor mechanical responses specifically due to PlxnD1. Cos7 cells expressing all three proteins (VEGFR2, NRP1 and PlxnD1) showed activation of early signalling responses, including phosphorylation of VEGFR2, association of VEGFR2 with Src tyrosine kinase, and PlxnD1/VEGFR2/NRP1 complex formation in response to shear stress. Importantly, none of these responses occurred in the absence of PlxnD1, thus providing further evidence that PlxnD1 is a specific and direct force sensor (). These cells do not express any of the components of the junctional complex (i.e. PECAM-1 or VE-cadherin) and, thus, constitute an ideal system to monitor mechanical responses specifically due to PlxnD1. Cos7 cells expressing all three proteins (VEGFR2, NRP1 and PlxnD1) showed activation of early signalling responses, including phosphorylation of VEGFR2, association of VEGFR2 with Src tyrosine kinase, and PlxnD1/VEGFR2/NRP1 complex formation in response to shear stress. Importantly, none of these responses occurred in the absence of PlxnD1, thus providing further evidence that PlxnD1 is a specific and direct force sensor (<xref ref-type="fig" rid="EMS85132-f003">Fig. 3i</xref>). Overall, these data provide evidence for a necessary and sufficient role of PlxnD1 in the shear stress-induced response. To demonstrate that PlxnD1 operates as a specific force sensor even further, we applied force on other elements of the complex. As shown in ). Overall, these data provide evidence for a necessary and sufficient role of PlxnD1 in the shear stress-induced response. To demonstrate that PlxnD1 operates as a specific force sensor even further, we applied force on other elements of the complex. As shown in <xref ref-type="fig" rid="EMS85132-f013">Extended Fig. 9</xref>, application of force on either NRP1 or VEGFR2 failed to elicit downstream responses. Taken together, these data unambiguously show that PlxnD1 is a specific and direct mechanosensor., application of force on either NRP1 or VEGFR2 failed to elicit downstream responses. Taken together, these data unambiguously show that PlxnD1 is a specific and direct mechanosensor.'], 'EMS85132-f010': ['The mechanical response of PlxnD1 is in stark contrast to the ligand response, as force on PlxnD1 increases focal adhesions, whereas Sema3E treatment reduces focal adhesions and leads to collapse of the actin cytoskeleton 4 (<xref ref-type="fig" rid="EMS85132-f010">Extended Fig. 6b</xref>). Structure-function studies on Semaphorins, Plexins and their cognate complexes, have established that the ligand-binding response requires a dimeric semaphorin to engage the N-terminal sema domains of two plexin receptors). Structure-function studies on Semaphorins, Plexins and their cognate complexes, have established that the ligand-binding response requires a dimeric semaphorin to engage the N-terminal sema domains of two plexin receptors26. Recent crystal structures and negative stain electron microscopy (EM) analyses of entire, ten domain, class A plexin (PlxnA) ectodomains revealed a distinctive ring-like conformation that is suitable for coupling extracellular semaphorin-based dimerization through to the transmembrane and cytoplasmic regions to transduce the ligand-binding response 7,27. However, the negative stain EM studies also revealed that the PlxnA ectodomain is capable of flexion, with distinctive minor populations of more open conformations. We carried out negative stain EM analysis of the PlxnD1 ectodomain and found evidence that it can flex to a more open conformation although the dominant state is ring-like (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4a, 4b</xref> and and <xref ref-type="fig" rid="EMS85132-f014">Extended Data Fig. 10a</xref>). We speculated that the ability to have flexion and switch between these two conformation states might provide an explanation for the binary nature of PlxnD1 function (). We speculated that the ability to have flexion and switch between these two conformation states might provide an explanation for the binary nature of PlxnD1 function (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4c</xref>). To examine this, we generated a double mutant of PlxnD1, Y517C A1135C, designed to promote formation of an intramolecular disulphide bond between domain 1 and domain 9 of the PlxnD1 ectodomain (). To examine this, we generated a double mutant of PlxnD1, Y517C A1135C, designed to promote formation of an intramolecular disulphide bond between domain 1 and domain 9 of the PlxnD1 ectodomain (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4d</xref>). Based on structural analysis, we predicted that the introduction of this disulphide bridge would lock the receptor ectodomain into the ring-like conformation, still allowing triggering of the ligand-binding reponse by Sema3E, but preventing switching to the “open” and putative mechanosensory conformation. Purification of the protein and subsequent quantitative assay using a thiol-reactive fluorescent dye, as well as negative stain EM, demonstrated that the protein did indeed contain the desired covalent disulphide linkage (). Based on structural analysis, we predicted that the introduction of this disulphide bridge would lock the receptor ectodomain into the ring-like conformation, still allowing triggering of the ligand-binding reponse by Sema3E, but preventing switching to the “open” and putative mechanosensory conformation. Purification of the protein and subsequent quantitative assay using a thiol-reactive fluorescent dye, as well as negative stain EM, demonstrated that the protein did indeed contain the desired covalent disulphide linkage (<xref ref-type="fig" rid="EMS85132-f014">Extended Fig. 10b, 10c</xref>).).'], 'EMS85132-f004': ['PlxnD1-depleted ECs were infected with adenovirus expressing either WT or mutant PlxnD1 and were assayed for their ability to respond to the ligand Sema3E or mechanical force. Treatment with Sema3E resulted in a decrease in focal adhesions in both WT and mutant PlxnD1 expressing cells (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4e</xref>), showing that the PlxnD1 ectodomain, when locked into a ring-like conformation, maintains its ability to bind Sema3E and signal to cause the disassembly of the cytoskeleton. We then tested if trapping the PlxnD1 in the semaphorin binding ring-like conformation was permissive of its mechanosensory function. We found that cells expressing the mutant PlxnD1 did not respond to mechanical force, as assayed by activation of early signalling responses (pVEGFR2, pAkt and pERK1/2 in ), showing that the PlxnD1 ectodomain, when locked into a ring-like conformation, maintains its ability to bind Sema3E and signal to cause the disassembly of the cytoskeleton. We then tested if trapping the PlxnD1 in the semaphorin binding ring-like conformation was permissive of its mechanosensory function. We found that cells expressing the mutant PlxnD1 did not respond to mechanical force, as assayed by activation of early signalling responses (pVEGFR2, pAkt and pERK1/2 in <xref ref-type="fig" rid="EMS85132-f004">Fig. 4h</xref>), cytoskeleton signalling (pVinculin in ), cytoskeleton signalling (pVinculin in <xref ref-type="fig" rid="EMS85132-f015">Extended Fig. 11</xref>) and focal adhesion maturation () and focal adhesion maturation (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4f</xref>). To further determine the requirement for PlxnD1 flexion in mechanotransduction, we examined the effects of mutant PlxnD1 in shear stress signalling. In contrast to ECs expressing WT PlxnD1, ECs expressing mutant PlxnD1 were unable to activate Akt, ERK1/2 or eNOS in response to shear stress (). To further determine the requirement for PlxnD1 flexion in mechanotransduction, we examined the effects of mutant PlxnD1 in shear stress signalling. In contrast to ECs expressing WT PlxnD1, ECs expressing mutant PlxnD1 were unable to activate Akt, ERK1/2 or eNOS in response to shear stress (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4i</xref>). Additionally, reconstitution of mutant PlxnD1 in Cos7 cells blocked early shear stress responses, including phosphorylation of VEGFR2, association of VEGFR2 with Src tyrosine kinase and shear stress-induced VEGFR2 and NRP1 complex formation (). Additionally, reconstitution of mutant PlxnD1 in Cos7 cells blocked early shear stress responses, including phosphorylation of VEGFR2, association of VEGFR2 with Src tyrosine kinase and shear stress-induced VEGFR2 and NRP1 complex formation (<xref ref-type="fig" rid="EMS85132-f004">Fig. 4g</xref>). Collectively, these results demonstrate that trapping PlxnD1 in its ring-like conformation maintains its ligand-dependent signalling function but compromises its ability to sense and respond to mechanical force.). Collectively, these results demonstrate that trapping PlxnD1 in its ring-like conformation maintains its ligand-dependent signalling function but compromises its ability to sense and respond to mechanical force.']}
|
The Guidance Receptor Plexin D1 Moonlights as an Endothelial Mechanosensor
| null |
Nature
|
1580889600
|
Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature.
|
[
"Animals",
"Atherosclerosis",
"Endothelial Cells",
"Female",
"Integrins",
"Intracellular Signaling Peptides and Proteins",
"Mechanotransduction, Cellular",
"Membrane Glycoproteins",
"Mice",
"Neuropilin-1",
"Pliability",
"Receptors, Cell Surface",
"Semaphorins",
"Stress, Mechanical",
"Vascular Endothelial Growth Factor Receptor-2"
] |
other
|
PMC7025890
| null |
38
|
[
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] |
Nature. 2020 Feb 5; 578(7794):290-295
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NO-CC CODE
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p53R270H/+ organoids have a selective growth advantage in the setting of dietary carcinogen exposure.(a) Immunoblot showing expression of mutant p53R270H in whole cell lysates derived from p53LSL-R270H/+ and p53LSL-R270H/R270H gastric organoids with or without AdenoCre induction; immunofluorescent images of p53LSL-R270H/+;mtmG gastric organoids before and after AdenoCre induction. Organoids without induction remain red indicating that recombination has not occurred, whereas those with AdenoCre induction convert to green indicating that recombination has occurred. This experiment was repeated once with similar results.(b) Flow cytometry of eGFP+ Mist-p53R270H/+ and td+ Mist-p53+/− organoids cultured in DMSO or 50ng/mL MNU. Quantification of eGFP+ Mist-p53R270H/+ and td+ Mist-p53+/− organoids at passage 1 (P1) and 3 (P3).(c) Phase contrast and immunofluorescent images of eGFP+ Mist-p53R270H/+ and td+ Mist-p53+/− organoids cultured in DMSO or 50ng/mL MNU. This experiment was repeated twice with similar results.(d) Schematic depicting competitive growth advantage of p53-altered gastric organoids in the setting of MNU
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nihms-1546989-f0011
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2
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cc8e6c2c47a08bc5ce0533159f5f4d57f28f13654a837d105535c7b978dd5d67
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nihms-1546989-f0011.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
750,
408
] |
[{'image_id': 'nihms-1546989-f0013', 'image_file_name': 'nihms-1546989-f0013.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0013.jpg', 'caption': 'Interferon signaling is upregulated in dysplastic Lgr5-p53KO gastric organoids(a) mRNA expression of Trp53 in p53KO, p53WT, Lgr5-p53KO, Lgr5-p53WT, dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids. Data presented as mean ± s.d. of n = 2 (nondysplastic) and n = 3 (dysplastic) cell culture replicates.(b) mRNA expression levels of Csf3, Cxcl10, and Ccl5 in p53KO, p53WT, Lgr5-p53KO, Lgr5-p53WT, dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids by RT-PCR. Data presented as mean ± s.d. of n = 3 cell culture replicates.(c) Volcano plot of differential expressed gene-sets in p53KO gastric organoids plotted as normalized enrichment scores (NES) by -log10(p-value), where p-values for gene set enrichment scores were determined by randomly permuting genes (n=100,000 permutations). Individual inflammation gene sets are described with corresponding NES.(d) Scatter plot of single sample GSEA (ssGSEA) of interferon pathway and Ccl5 mRNA expression in gastric cancer cell lines annotated by TP53 mutation status (n = 26 for TP53 mutant and n = 10 for TP53 wildtype). Gray shaded region represents 95% confidence interval of the linear regression fit. Pearson’s correlation value R = 0.6 and p-value computed based on a two-sided t-distribution.(e) Schematic of proposed vicious feedback cycle in gastric premalignancy: chronic inflammation and carcinogen exposure selects for p53 mutant gastric cells, which in turn stimulate inflammatory cytokines, particularly Csf3, Cxcl10, Ccl5', 'hash': 'd7f70bcfd914c6e232c94b000657f208a2eb319ddc614f5319a610ef788d871f'}, {'image_id': 'nihms-1546989-f0014', 'image_file_name': 'nihms-1546989-f0014.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0014.jpg', 'caption': 'WNT pathway is upregulated in dysplastic Lgr5-p53KO gastric organoids(a) WNT-reporter activity in adherent culture of dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric cells following transient transfection. Data presented as mean ± s.d. of n = 4 cell culture replicates; p-value calculated by two-sided Mann Whitney test.(b) Phase contrast images of dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids two days following the first and second passage in WNT independent media (DMEM + 10%FBS). This experiment was repeated twice with similar results.(c) Gene network of significantly mutated genes in dysplastic gastric lesions from DCA/MNU-treated Lgr5-p53WT mice (n = 8) using GeNets (adjusted p-value = 0.00065 using Bonferroni correction) (Li et al., 2018).', 'hash': '7a93fe1fe6144fb81a0779d43cf4909646ef875a87c492598064d198b3db75a7'}, {'image_id': 'nihms-1546989-f0005', 'image_file_name': 'nihms-1546989-f0005.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0005.jpg', 'caption': 'Dysregulation of inflammatory, stem cell, and cell cycle pathways in dysplastic Lgr5-p53KO gastric organoids(a) Schematic showing methodology for gene expression profiling of p53KO(n = 2), p53WT(n = 2), Lgr5-p53KO (n = 2), Lgr5-p53WT (n = 2), dys-Lgr5-p53KO (n = 6) and dys-Lgr5-p53WT (n = 6) gastric organoids.(b) Volcano plot of gene-set enrichment pathways in premalignant dLgr5-p53KO (n = 5 organoids from 2 mice) versus dLgr5-p53WT (n = 6 organoids from 2 mice) gastric organoids plotted as normalized enrichment scores (NES) by -log10(p-value), where p-values for gene set enrichment scores were determined by randomly permuting genes (n=100,000 permutations) (left). Individual inflammation gene sets are described with corresponding Normalized Enrichment Scores (NES). Gene-set enrichment plot of inflammatory response pathway; associated heat map of scoring genes in gene-set (right).(c) Scatter plot of NES of gene-sets in dys-Lgr5-p53KO/dys-Lgr5-p53WT (y-axis) versus nondysplastic Lgr5-p53KO/Lgr5-p53WT (x-axis) gastric organoids; Select pathways downregulated in both comparisons are indicated in blue; upregulated in both comparisons indicated in red; and only upregulated in dys-Lgr5-p53KO comparison indicated in orange.(d) Immunoblot showing protein levels of WNT target Sox9, total β-catenin, and vinculin (loading control) in p53WT, p53KO , Lgr5-p53WT, Lgr5-p53KO, dys-Lgr5-p53WT, dys-Lgr5-p53KO gastric organoids. This experiment was repeated once.(e) Histopathology of nondysplastic gastric antrum of untreated Lgr5-p53WT and Lgr5-p53KO mice and dysplastic gastric lesions from treated dys-Lgr5-p53WT and dys-Lgr5-p53KO mice; H&E staining (top panel) and Sox9 immunohistochemistry (bottom panel) of gastric antrum. Scale bar = 250μM', 'hash': 'ca8296dbe30d4be7d2d749520757a67a872ab7c5f3f815e24f847bb11868b253'}, {'image_id': 'nihms-1546989-f0002', 'image_file_name': 'nihms-1546989-f0002.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0002.jpg', 'caption': 'p53KO human nondysplastic premalignant cells have a selective advantage in the setting of MNU exposure(a) dsDNA damage as indicated by γH2Ax immunofluorescence (top panel), phase-contrast microscopy (middle panel) and DNA content flow cytometry (bottom panel) of CP-A cells treated with indicated doses of MNU. This experiment was repeated three times.(b) Immunoblot showing protein levels of p53 in CP-A cells treated with DMSO or indicated concentrations of MNU. This experiment was repeated three times.(c) Relative proliferation of control (n = 3) or p53 KO CP-A cells (n = 3/sgRNA) treated with MNU by Celtiter glo assay. Data presented as mean ± s.d.; p-values calculated by two-sided Student’s t-test.(d) DNA content flow cytometry of CP-A cells expressing control orTP53 shRNA#2 CP-A cells treated with indicated MNU concentrations(e) Colony formation of CP-A cells stably expressing control or p53 targeting sgRNAs/Cas9 treated with indicated MNU concentrations (left). Quantification of colony formation assay using acetic acid treatment and subsequent absorbance measurement (right). Data presented as mean ± s.d. of three culture replicates p-value calculaged byusing two-sided Student’s t-test.(f) Gene set enrichment analysis showing relative enrichment of DNA repair pathway in control (n = 2) and p53KD CP-A cells (n = 4) (Fasle Discovery Rate q-value = 0.0046). Genes associated with DNA repair are indicated in purple.', 'hash': '8e1613b11ad1cb3c5061d59cc3b0206c9388361cf2565d70a0ec4239789e448a'}, {'image_id': 'nihms-1546989-f0015', 'image_file_name': 'nihms-1546989-f0015.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0015.jpg', 'caption': 'CDKN2A is upregulated in p53KO premalignant lesions and co-altered with TP53 in human gastric cancer(a) Scatter plot of single sample GSEA (ssGSEA) of p53 pathway and CDKN2A mRNA expression in gastric cancer cell lines (n = 36). Gray shaded region represents 95% confidence interval of the linear regression fit. Pearson’s correlation value R = −0.36 and p-value computed based on a two-sided t-distribution.(b) Heatmap showing mRNA expression of CDKN2B, CDKN2C, CDKN2D, CDKN1B, CDKN1C in CP-A cell line genetically engineered to express two p53 targeting shRNA under doxycycline inducible conditions.(c) Oncoporint from cbioportal showing alterations and mRNA expression in CDKN2A and TP53 in human gastric adenocarcinomas from TCGA (n = 478). Deep deletions (solid blue); amplifications (solid red); missense mutations in the COSMIC repository (solid green); nonsense or frameshift mutations (black); elevated mRNA expression > 2.0 (hollow red). Table showing number of cases with double, single, or no alteration in CDKN2A and TP53 in human gastric adenocarcinomas from TCGA (right)(d) Oncoprint from cbioportal showing alterations in CDKN2A and TP53 in human esophageal adenocarcinomas from TCGA and (Dulak et al., 2013) combined data-set (n = 337). Deep deletions (solid blue); amplifications (solid red); missense mutations in the COSMIC repository (solid green); nonsense or frameshift mutations (black). Table showing number of cases with double, single, or no alteration in CDKN2A and TP53 in combined esophageal adenocarcinoma data set.(e) Table showing number of cases with double, single, or no alteration in CDKN2A and TP53 in combined gastric and esophageal adenocarcinomas from patients treated at DFCI (n = 1299). Q-value determined by one-sided Fischer Exact Test with Benjamin-Hochberg FDR correction.', 'hash': '650f0dfc083bf0160835cb61365bb85964dfc7bc014a9a593023be3db4c8af94'}, {'image_id': 'nihms-1546989-f0012', 'image_file_name': 'nihms-1546989-f0012.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0012.jpg', 'caption': 'Dysplastic Lgr5-p53KO gastric organoids capture properties of premalignant lesions(a) Recombination specific PCR of DNA extract from premalignant dys-Lgr5-p53WT and dys-Lgr5-p53KO gastric organoids. Data presented as mean ± s.d of n = 3 technical replicates per group.(b) Quantification of proliferation by CellTiter-Glo and phase contrast images of nondysplastic and dysplastic Lgr5-p53WT and Lgr5-p53KO gastric organoids in the presence or absence of Nutlin-3 (30uM for 72 hours). Data presented as mean ± s.d. of n = 6 cell culture replicates.(c) Representative images of phase contrast and GFP-immunofluorescent images from dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoid cultures. The experiment was repeated once with similar results.(d) Karyotype analysis of premalignant Lgr5-p53KO (n = 2 mice), Lgr5-p53WT (n = 2 mice), and Lgr5-p53WT + AdenoCre gastric organoids; chromosome count of 10 cells per group. Data presented as mean ± s.d.; p-values calculated by Ordinary One-way ANOVA with Sidak’s multiple comparisons adjustment.(e) Copy number analysis of a patient specimen of a high-grade dysplastic Barrett’s metaplasia harboring TP53R175H mutation and genome doubling.(f) Histopathology of primary tumor xenograft of Lgr5-p53KO gastric organoid showing features of dysplasia. This experiment was repeated twice with similar results.(g) DAPI staining and GFP (Lgr5+) immunofluorescence of primary xenograft from Lgr5-p53KO gastric organoids. This experiment was repeated twice with similar results.(h) Schematic of low-pass whole genome sequencing (LP-WGS) experiment of cultured and xenograft dys-Lgr5-p53KO gastric organoids. Common and distinct broad somatic copy number alterations that were only found in xenograft tissue are displayed.', 'hash': '99ee4a367fef9d9b1e621020f0670bf8095e7bbaa5e754f157dbe203cfe55d3c'}, {'image_id': 'nihms-1546989-f0003', 'image_file_name': 'nihms-1546989-f0003.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0003.jpg', 'caption': 'p53KO murine gastric organoids have a competitive advantage in the setting of dietary carcinogenesis(a) Phase contrast images and quantification of p53WT and p53KO organoids in the setting of DMSO, 50ng/mL MNU, or 200ng/mL MNU (n = 3/group). Data presented as mean ± s.d. of three culture replicates; p-values calculated by two-sided Student’s t-test. (b) Flow cytometry of GFP+ Mist-p53WT and GFP+ Mist-p53KO relative to RFP+ p53WT organoids cultured in DMSO or 50ng/mL MNU. Quantification of GFP+ Mist-p53WT, GFP+ Mist-p53KO , and RFP+ p53WT organoids at passage 1 (P1) and 3 (P3). The experiment was repeated once.(c) Quantification of percent GFP+ Mist-p53WT (mouse #1437) and GFP+ Mist-p53KO (mouse #1449) relative to RFP+ p53WT organoids cultured in DMSO, 50ng/mL MNU, or 200ng/mL MNU. There are between 5-7 cell culture replicates per condition.', 'hash': '8aa9e619d84228a8300f78d9b0f72d453ba37d18ff86fd62dc57362714d6ffe7'}, {'image_id': 'nihms-1546989-f0004', 'image_file_name': 'nihms-1546989-f0004.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0004.jpg', 'caption': 'Dysplastic and metaplastic Lgr5-p53KO gastric organoids demonstrate genome doubling and capacity to transform(a) Schematic showing isolation of dysplastic organoids from premalignant dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids from experiments shown in Figure 1. Phase contrast images of Nutlin-3 treated dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids(b) Quantification of colony forming ability in ultra-low attachment culture of dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids. Data presented as mean ± s.d. from three biological replicates; p-values calculated by two-sided Student’s t-test.(c) Quantification of percent Lgr5+ cells in dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoid culture as measured by GFP+. Data presented as mean ± s.d. from three biological replicates; p-values calculated by two-sided Student’s t-test.(d) Histopathological analysis of gastric dysplasia in dys-Lgr5-p53KO and dys-Lgr5-p53WT mice: H&E stain (top), Alcian blue stain (middle), anti-GFP/Lgr5 IHC (bottom)(e) Karyotype analysis of dys-Lgr5-p53KO (n = 2 mice) and dys-Lgr5-p53WT (n = 2 mice) gastric organoids; chromosome count of 10 cells per group. Representative images shown to the right; inserts are higher magnification images of chromosomes. Data presented as mean ± s.e.m.; p-values calculated by two-sided Student’s t-test.(f) Primary xenograft growth of dys-Lgr5-p53KO (n = 10 mice) and dys-Lgr5-p53WT (n = 10 mice) gastric organoids in nude mice', 'hash': 'fb667b635514a734c51bb69a0dcb9bd93befc879640b7f721b295e61e514ff00'}, {'image_id': 'nihms-1546989-f0007', 'image_file_name': 'nihms-1546989-f0007.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0007.jpg', 'caption': 'Co-deletion of CDKN2A/p16 and TP53 promotes gastric premalignancy, induces replication stress, and sensitizes to DNA damage response pathway blockade(a) Immunoblot showing protein levels of p53, p16, and vinculin (loading control) in dys-Lgr5-p53WT control or Trp53/Cdkn2a DKO (top panel) and dys-Lgr5-p53KO control or p16 KO gastric organoids. This experiment was repeated once.(b) Quantification and representative dissection microscope images of dys-Lgr5-p53WT control or Trp53/Cdkn2a DKO gastric organoid colonies formed under ultra-low attachment culture conditions in WRN media after 21 days.(c) Quantification and representative dissection microscope images of dys-Lgr5-p53KO control or Cdkn2a DKO gastric organoid colonies formed under low-attachment culture conditions in indicated media after 7 days. Data presented as mean ± s.d. of three culture replicates; p-values calculated by two-sided Student’s t-test.(d) Primary xenograft growth of dys-Lgr5-p53WT gastric organoids expressing either a control sgRNA, Trp53 sgRNA, Cdkn2a sgRNA, or Trp53/Cdkn2a sgRNA (DKO) in nude mice (n=6 per group).(e) Immunoblot showing protein levels of phospho-CHK1, total-CHK1, phospho-RPA32, p53, p16, and vinculin (loading control) in dys-Lgr5-p53WT expressing either a control sgRNA, Trp53 sgRNA, Cdkn2a sgRNA, or Trp53/Cdkn2a sgRNA (DKO). This experiment was repeated once.(f) Heatmap showing relative sensitivity of CHEK1/2 inhibitor AZD7762 in 30 human gastric cancer cell lines. Solid red rectangles indicate cell-lines with co-disruption of TP53/CDKN2A; p-value = 0.0051, FDR = 0 (Top panel); Box plot showing quantiles of sensitivity to CHEK1/2 inhibitor AZD77622 in 30 human gastric cancer cell lines (black dots) stratified into four molecular groups: CDKN2AWT/TP53WT (n = 8), CDKN2Aaltered/TP53WT (n = 1), CDKN2AWT/TP53altered (n = 15), and CDKN2Aaltered/TP53altered (n = 6). Drug response was measured as Area Under the Curve (AUC). Differences in sensitivity were determined by two-sided Student’s t-test with *p-value <0.05 and **p-value < 0.01 (Bottom Panel)(g) Dose-response curve of isogenic CP-A cell lines expressing either control sgRNA, p53 sg#1, or p53 sg#2 to indicated concentrations of Prexasertib. IC50 scores are displayed; Data presented as mean ± s.d. of six culture replicates at each indicated dose; Comparison of Fits, p value = 0.011 based on differences in IC50 (right panel). Immunoblot phospho-CHK1, phospho-ATR, total-ATR, and B-actin loading control in CP-A control or p53KO BE cells treated with indicated concentrations of Prexasertib (left panel). This experiment was repeated once. (h) Dose-response curve of isogenic dys-Lgr5-p53WT-control and dys-Lgr5-p53WT-DKO gastric organoids to indicated concentrations of Prexasertib (Top). Comparison of Fits, p = 0.0001 based on bottom value. Dose-response curve of isogenic dys-Lgr5-p53KO-control and dys-Lgr5-p53KO-sgCDKN2A gastric organoids to indicated concentrations of Prexasertib. Comparison of Fits, p = 0.0047 based on IC50. Data presented as mean ± s.d. of six culture replicates at each indicated dose(i) Representative phase contrast images of dys-Lgr5-p53WT-control, dys-Lgr5-p53WT-DKO, dys-Lgr5-p53KO-control, dys-Lgr5-p53WT-p16KO gastric organoids treated for 36 hours with DMSO or indicated concentration of CHK1/2 inhibitor Prexasertib. This experiment was repeated once.(j) Quantification of organoid size from each group in experiment (F). Data presented as mean ± s.d. of seven technical replicates; p-values calculated by two-sided Student’s t-test.(k) Schematic of functional and therapeutic role of CDKN2A and TP53 alterations in gastric premalignancy', 'hash': 'effa29b0e4648280059da6c46e56c108f9d1697157242a1fc5468b08dd0d92d3'}, {'image_id': 'nihms-1546989-f0009', 'image_file_name': 'nihms-1546989-f0009.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0009.jpg', 'caption': 'Lgr5-p53KO cells have a selective advantage in the setting of dietary carcinogen to promote premalignant gastric lesions(a) Recombination specific PCR of DNA extract from gastric lesions of Lgr5-p53WT and Lgr5-p53KO mice. Data presented as mean ± s.d of three technical replicates.(b) Schematic showing Lgr5-p53LSL-R270H experimental design; DCA/MNU treatment and tamoxifen injection schedule during indicated duration depicted below. (c) Kaplan-Meier survival curve of Lgr5-p53R270H experiments; table shows frequency of dysplasia in DCA/MNU treated mice of indicated genotype.(d) Schematic showing Lgr5-p53KO experimental design; DCA alone, MNU alone, or DCA/MNU combination as well as tamoxifen injection schedule during indicated duration depicted below. Table shows frequency of dysplasia in Lgr5-p53KO mice with indicated treatment after 12 months. *1/6 Cre-negative, tamoxifen-induced control mice treated with DCA/MNU developed dysplasia.', 'hash': '2a3561d32e9e56f14baf16a098e745e8fd39cf00159e66cb04d719fdb32ee835'}, {'image_id': 'nihms-1546989-f0011', 'image_file_name': 'nihms-1546989-f0011.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0011.jpg', 'caption': 'p53R270H/+ organoids have a selective growth advantage in the setting of dietary carcinogen exposure.(a) Immunoblot showing expression of mutant p53R270H in whole cell lysates derived from p53LSL-R270H/+ and p53LSL-R270H/R270H gastric organoids with or without AdenoCre induction; immunofluorescent images of p53LSL-R270H/+;mtmG gastric organoids before and after AdenoCre induction. Organoids without induction remain red indicating that recombination has not occurred, whereas those with AdenoCre induction convert to green indicating that recombination has occurred. This experiment was repeated once with similar results.(b) Flow cytometry of eGFP+ Mist-p53R270H/+ and td+ Mist-p53+/− organoids cultured in DMSO or 50ng/mL MNU. Quantification of eGFP+ Mist-p53R270H/+ and td+ Mist-p53+/− organoids at passage 1 (P1) and 3 (P3).(c) Phase contrast and immunofluorescent images of eGFP+ Mist-p53R270H/+ and td+ Mist-p53+/− organoids cultured in DMSO or 50ng/mL MNU. This experiment was repeated twice with similar results.(d) Schematic depicting competitive growth advantage of p53-altered gastric organoids in the setting of MNU', 'hash': 'cc8e6c2c47a08bc5ce0533159f5f4d57f28f13654a837d105535c7b978dd5d67'}, {'image_id': 'nihms-1546989-f0016', 'image_file_name': 'nihms-1546989-f0016.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0016.jpg', 'caption': 'Co-deletion of CDKN2A and TP53 sensitizes to DNA damage response pathway blockade(a) Dose-response curves of CHK1 inhibitor AZD7762 of two gastric cancer cell lines wildtype for either TP53 or CDKN2A and two gastric cancer cell lines with co-alteration of TP53 and CDKN2A.(b) Relative dependency of 23 gastric cancer cell lines to CHEK1, CHEK2, and WEE1 shRNA-mediated knockdown as shown by CERES dependency score. Black rectangles indicate gastric cancer cell lines with simultaneous disruption of TP53 and CDKN2A.(c) Dose-response curve of KE39 (TP53MUT, CDKN2AWT) and GSU (TP53MUT, CDKN2AMUT) gastric cancer cell lines to Prexasertib. Data presented as mean ± s.d. of n = 6 cell culture replicates at each dose; p-value calculated by Comparison of Fits based on differences in IC50.(d) Dose-response curve of KE39 (TP53MUT, CDKN2AWT) and GSU (TP53MUT, CDKN2AMUT) gastric cancer cell lines to WEE1 inhibitor AZD1775. Data presented as mean ± s.d. of n = 6 cell culture replicates at each dose; p-value calculated by Comparison of Fits based on differences in IC50.(e) Quantification by manual counting of number of organoids in experiment from Figure 7f-g.(f) Quantification of organoid size and number from dys-Lgr5-p53KO-control and dys-Lgr5-p53WT-p16KO gastric organoids treated for 36 hours with DMSO or indicated concentration of ATR inhibitor AZD6738 or WEE1 inhibitor AZD1775. Data presented as mean ± s.d. of n = 6-8 technical replicates at each dose(g) Representative phase contrast images from experiment (f)', 'hash': 'ee48c05fb5df5266947bf98780bf7b2a98680839af142922a5223ad2abddcf06'}, {'image_id': 'nihms-1546989-f0008', 'image_file_name': 'nihms-1546989-f0008.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0008.jpg', 'caption': 'Dietary carcinogen exposure promotes premalignant and malignant gastric lesions(a) Schematic of central hypothesis: Chronic inflammation and carcinogen exposure collaborate with early genomic alterations (e.g. TP53 mutations) to enable the development of premalignant gastric lesions and eventual invasive cancer(b) Schematic of p53 WT mice treated with DCA, MNU, or DCA+MNU combination for 18 months.(c) Dissection microscope images of lower esophagus and stomach flayed open along greater curvature (top panel) and histopathological H&E staining (bottom panel) of gastric antrum. *A subset of mice died before the endpoint of the experiment due to other carcinogen-induced cancers (e.g. thymomas). Scale bar = 125μM(d) Mutation signature analysis shows C→T changes characteristic of alkylating agent associated Signature 11. center line: median, lower hinge: the first quartile (Q1), upper hinge: the third quartile (Q3), extreme of the lower whisker: Q1 – 1.58 * (Q3 – Q1)/sqrt(n), extreme of the upper whisker: Q3 + 1.58 * (Q3 – Q1)/sqrt(n), n = 5.(e) Mutation burden and copy number analysis of gastric lesions (n=5).', 'hash': '715639e0678b8be4f3fe6f185022139367410b1edc31bee3146805a6628c92c5'}, {'image_id': 'nihms-1546989-f0001', 'image_file_name': 'nihms-1546989-f0001.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0001.jpg', 'caption': 'Lgr5-p53KO promotes dysplastic and metaplastic premalignant lesions in the setting of dietary carcinogen exposure.(a) Schematic showing Lgr5-p53 experimental design and outcomes; percentages represent frequency of mice with antral dysplastic lesions. DCA/MNU treatment and tamoxifen injection schedule indicated on schematic below.(b) Representative H&E staining of gastric lesions from untreated and DCA/MNU treated Lgr5-p53 mice. Scale bar = 250μM.(c) Quantification of frequency of dysplasia in mice at 12 months in DCA/MNU treated Lgr5-p53WT (n = 11) and Lgr5-p53KO (n = 19) mice. This experiment was repeated once with similar results.(d) Mutation burden in the form of insertions and deletions (indels) in dysplastic gastric lesions from Lgr5-p53WT (n = 3) and Lgr5-p53KO (n = 10) mice using whole-exome sequencing. Data is presented as mean ± s.e.m.; two-sided Kolmogorov-smirnov p = 0.084.(e) Schematic showing Mist-p53;mtmg experimental design and outcomes; percentages represent frequency of dysplastic lesions found in the antrum or corpus of mice. Same DCA/MNU treatment and tamoxifen injection schedule as shown in (a)(f) Quantification of mice with antral or corpus dysplasia in DCA/MNU treated Mist-p53WT (n = 11) and Mist-p53KO (n = 15) mice at 12 months. This experiment was performed once.(g) Representative images from DCA/MNU-treated Mist-p53WT and Mist-p53KO mice. Dissection microscope images of lower esophagus and stomach flayed open along greater curvature; arrow indicates antral lesion (left panel). H&E staining and GFP immunohistochemistry of regions of dysplasia and antral polyps. Scale bar = 125μM.', 'hash': '6f165cc195f006a9be808ea4e6b590bf7efe5f9b54bb3b07f9fe876ed69d871a'}, {'image_id': 'nihms-1546989-f0006', 'image_file_name': 'nihms-1546989-f0006.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0006.jpg', 'caption': 'CDKN2A/p16 is upregulated in Lgr5-p53KO premalignant gastric lesions and co-altered with TP53 in a subset of human gastric cancers(a) Volcano plot of differentially expressed genes in dys-Lgr5-p53KO (n = 6, three cell culture replicates from 2 different mice) relative to dys-Lgr5-p53WT (n = 6, three cell culture replicates from 2 different mice) gastric organoids show as log fold-change (logFC, x-axis) by -log10(p-value), where p-values were estimated from empirical-Bayes moderated t-statistics. Dots colored red have FDR < 0.1, where FDR-adjusted p-values are estimated using the Benjamini-Hochberg method.(b) mRNA expression of CDKN2A in p53WT, p53KO, Lgr5-p53WT, Lgr5-p53KO, dys-Lgr5-p53WT and dys-Lgr5-p53KO gastric organoids(c) Immunoblot showing protein levels of p53, p16, and vinculin (loading control) in dys-Lgr5-p53WT expressing either a control sgRNA or Trp53 sgRNA. This experiment was repeated once.(d) Localization and expression levels of p16 (red) in dysplastic lesions of DCA/MNU-treated Lgr5-p53WT and Lgr5-p53KO mice. Nuclear staining by DAPI (blue). Scale bar = 125μM(e) Localization and expression levels of p16 (red) in dysplastic lesions of DCA/MNU-treated Mist-p53WT and Mist-p53KO mice. GFP+ represents conditionally induced Mist+ cells in indicated genetic mice. Nuclear staining by DAPI (blue). Scale bar = 125μM(f) Oncoprint from cbioportal73 showing alterations in CDKN2A and TP53 as well as CDKN2A mRNA expression levels in the CIN-subtype of human gastric (n = 223) and esophageal (n = 74) adenocarcinomas from TCGA. Deep deletions (solid blue); missense mutations in the COSMIC repository (solid green); nonsense or frameshift mutations (black); promoter methylation (magenta). CDKN2A mRNA expression from Z-scores of −3 to 3.', 'hash': '22a7af16966ac6448d725b0fd92bb70db1474dafd91781f1e57f78b864b17edf'}, {'image_id': 'nihms-1546989-f0010', 'image_file_name': 'nihms-1546989-f0010.jpg', 'image_path': '../data/media_files/PMC7031028/nihms-1546989-f0010.jpg', 'caption': 'TP53-deleted human premalignant cells have a selective growth advantage in the setting of MNU(a) Relative proliferation of CP-A cells treated with indicated concentrations of DCA; phase contrast images of CP-A cells treated with DMSO or 100μM DCA.(b) Immunoblot showing protein levels of p53 in CP-A cells treated with DMSO or 50μM DCA. This experiment was repeated once with similar results.(c) DNA content flow cytometry of CP-A cells expressing control orTP53 shRNA#2 CP-A cells treated with indicated MNU concentrations. This experiment was repeated once with similar results.(d) Relative proliferation of CP-A cells treated with DMSO or indicated concentrations of MNU by Celltiter Glo. Data presented as mean ± s.d of three culture replicates; p-value calculated by two-sided Student’s t-test.(e) Immunoblot showing protein levels of p53 in genetically modified CP-A cells; top panel shows protein from CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNAs/Cas9 after treatment with MNU 200ng/mL; bottom panel shows protein from CP-A cells expressing a scrambled or two targeting p53 shRNAs treated with DMSO, Doxorubicin, or Nutlin in the presence or absence of doxycycline. This experiment was repeated once.(f) Relative proliferation of CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNA/Cas9 (top panel); expressing vector control, scramble control, or two p53 targeting shRNAs. Data presented as mean ± s.d of three culture replicates(g) Quantification of dsDNA breaks in CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNA/Cas9 in the setting of indicated concentrations of MNU by γH2AX immunofluorescence. Data presented as mean ± s.d of four technical and two cell culture replicates; p-value calculated by two-sided Student’s t-test of cell culture replicates.(h) FPKM gene counts of TP53 in CP-A cells stably expressing two p53 shRNAs(i) Ratio of TP53 pathway target gene expression levels in CP-A cells expressing indicated p53 shRNA relative to scramble control (cell culture replicates shown)', 'hash': '4d2c05422216710e5b706b81235c1d3eb2c5da5b41f776ede613d7ef0635c593'}]
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{'nihms-1546989-f0008': ['TP53 is the most common recurrent mutation in gastric and esophageal adenocarcinoma23-25. It is now clear that premalignant lesions also incur early enabling mutations as evident from clonal hematopoiesis26,27 and intestinal metaplasia, the most recognized precursor lesion to GE adenocarcinoma28,29. By comparing mutation patterns from matched patient-derived premalignant Barrett’s esophagus (BE) and esophageal adenocarcinoma lesions, we found that TP53 is mutated early in the progression of GE malignancy, often occurring before dysplasia24. Deep sequencing of noncancerous gastric epithelium from patients with gastritis showed that just under half harbored TP53 mutations30. Furthermore, we found that TP53 is preferentially mutated in the subset of nondysplastic BE patients who progress to cancer31. This sequence of genomic events is notably different than other gastrointestinal cancers, such as colorectal or pancreatic, in which TP53 is mutated relatively late in cancer development32,33. Based upon these observations, we hypothesized that chronic inflammation and carcinogenic exposures enable selection of TP53 altered cells to promote premalignant lesions (<xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1a</xref>). To test this hypothesis, we designed a new, integrative mouse model that combines disease-relevant exposures with tissue-specific ). To test this hypothesis, we designed a new, integrative mouse model that combines disease-relevant exposures with tissue-specific TP53 alterations to study the development of gastric premalignancy.', 'Prior to studying the impact of Trp53 (mouse TP53) in premalignancy, we established a gastric adenocarcinoma model in wildtype (WT) C57BL/6J mice by exposing them to drinking water containing deoxycholic acid (DCA) and N-methyl-N-nitrosourea (MNU)15 (<xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1b</xref>). MNU was selected based on the following: (a) it is a nitrosamide related to dietary nitrosamines implicated in human disease). MNU was selected based on the following: (a) it is a nitrosamide related to dietary nitrosamines implicated in human disease34, (b) when compared to six other nitroso-compounds, it preferentially led to gastric disease35, and (c) it promoted premalignant GE lesions in transgenic mice with endogenous inflammation5,36. Mice from this experiment were sacrificed after 18 months to evaluate gastric phenotypes. While untreated (n = 5) and DCA-alone treated (n = 7) mice did not develop premalignant lesions, 39% of MNU (n = 8) or DCA/MNU (n = 10) treated mice developed adenocarcinomas along the stomach lesser curvature (<xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1c</xref>, Fisher’s exact p-value = 0.03). Whole exome sequencing (WES) analyses in these lesions showed mutation signatures enriched for CT substitutions consistent with exposure to MNU, which cause the formation of O, Fisher’s exact p-value = 0.03). Whole exome sequencing (WES) analyses in these lesions showed mutation signatures enriched for CT substitutions consistent with exposure to MNU, which cause the formation of O6-alkylguanines37 (<xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1d</xref>). Mutational (single-nucleotide variants/insertion-deletions) and copy number (amplifications) analyses showed an inverse relationship (). Mutational (single-nucleotide variants/insertion-deletions) and copy number (amplifications) analyses showed an inverse relationship (<xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1e</xref>), consistent with a recent report using MNU-induced tumors), consistent with a recent report using MNU-induced tumors37. Overall, these results indicate that dietary exposures can directly promote the development of gastric adenocarcinomas in C57BL/6J mice.', 'Our central hypothesis is that mutant p53 gastric cells promote premalignant lesions when subjected to disease-relevant exposures (<xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1a</xref>). To test this hypothesis, we conditionally manipulated ). To test this hypothesis, we conditionally manipulated Trp53 in distinct cell populations of the stomach. Our first model built upon the observation that Lgr5 marks antral gastric stem cells 38. Transgenic mice with conditionally deleted Trp53 or activated missense mutant (Trp53R270H) in Lgr5+ cells were exposed to drinking water containing DCA and MNU as described previously (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1a</xref>) and sacrificed at 12 months to study premalignant lesions () and sacrificed at 12 months to study premalignant lesions (Supplementary Note#1).', 'To activate conditional alleles, experimental mice aged 6–8 weeks were injected intraperitoneally with five consecutive daily 200 ml doses of tamoxifen in sunflower oil at 10 mg/ml to activate. For experiments with Lgr5-EGFP-Ires-CreERT2, control mice did not receive tamoxifen; a subset of control mice were Cre-negative and did receive tamoxifen. For experiments with Mist1-CreERT2; R26-mtmG mice, control mice without Trp53 allele received tamoxifen. All treated mice were subjected to drinking water containing 240ppm of N-methyl-N-nitrosourea (MNU; biokemix) scheduled every other week for 6 weeks and/or 0.3 % deoxycholic acid (DCA; sigma) continuously for 1 year (or 1.5 year for p53WT experiment in <xref rid="nihms-1546989-f0008" ref-type="fig">Extended Data Fig. 1</xref>. Mice were euthanized at the endpoint of the experiment, and stomachs were harvested for histopathological and immunohistochemical analyses as well as organoid generation.. Mice were euthanized at the endpoint of the experiment, and stomachs were harvested for histopathological and immunohistochemical analyses as well as organoid generation.'], 'nihms-1546989-f0001': ['Deletion of Trp53 in Lgr5+ cells of untreated mice did not lead to detectable premalignant lesions, suggesting that p53 loss alone is not sufficient to promote dysplasia (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1a</xref>--<xref rid="nihms-1546989-f0001" ref-type="fig">b</xref>). When treated with DCA/MNU, however, Lgr5-p53). When treated with DCA/MNU, however, Lgr5-p53KO mice demonstrated a 3.5-fold increase in dysplastic lesions compared to Lgr5-p53WT mice (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1b</xref>--<xref rid="nihms-1546989-f0001" ref-type="fig">c</xref>). Dysplastic lesions occurred along the stomach antrum lesser curvature, consistent with the highest density of Lgr5+ cells). Dysplastic lesions occurred along the stomach antrum lesser curvature, consistent with the highest density of Lgr5+ cells38. Recombination-specific PCR demonstrated that Lgr5-p53KO premalignant lesions lacked p53 (<xref rid="nihms-1546989-f0009" ref-type="fig">Extended Data Fig. 2a</xref>). WES showed that dysplastic lesions from treated Lgr5-p53). WES showed that dysplastic lesions from treated Lgr5-p53KO mice harbored a greater burden of mutations compared to Lgr5-p53WT mice, consistent with p53 function in preserving the integrity of the genome (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1d</xref>). We also asked whether DCA or MNU alone could promote premalignant lesions in Lgr5-p53). We also asked whether DCA or MNU alone could promote premalignant lesions in Lgr5-p53KO mice. Only MNU containing regimens developed premalignant lesions in Lgr5-p53KO mice, demonstrating the importance of carcinogens in this model (<xref rid="nihms-1546989-f0009" ref-type="fig">Extended Data Fig. 2d</xref>). These results indicate that p53 deletion in gastric stem cells engages carcinogenic exposures to promote premalignant lesions.). These results indicate that p53 deletion in gastric stem cells engages carcinogenic exposures to promote premalignant lesions.', 'To test whether p53 deletion in a distinct gastric compartment can promote premalignancy, we utilized Mist1-CreERT2 mice. Mist1 marks chief cells, which have been shown to respond to inflammation39 and undergo neoplastic transformation following genetic alterations19. We crossed Mist1-CreERT2; Trp53F/F mice with R26-mTmG reporter mice to mark p53KO with eGFP. Mist1-CreERT2; R26-mtmG mice served as controls in which Mist1+ p53WT epithelial cells were marked by eGFP (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1e</xref>). Untreated Mist1-p53). Untreated Mist1-p53KO mice did not develop dysplastic lesions for up to two years of monitoring, consistent with our results in Lgr5-p53KO mice. Mist1-p53KO mice, however, developed over 2.5 times as many dysplastic lesions as Mist1-p53WT mice when subjected to DCA/MNU (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1f</xref>). Indeed, invasive gastric adenocarcinomas arose only in DCA/MNU treated Mist1-p53). Indeed, invasive gastric adenocarcinomas arose only in DCA/MNU treated Mist1-p53KO mice (n = 2) at this early time-point. Immunohistochemical analysis for GFP demonstrated greater expansion of Mist1-p53KO cells in dysplastic and polypoid lesions (<xref rid="nihms-1546989-f0001" ref-type="fig">Fig. 1g</xref>). These results demonstrate that p53 deletion in a distinct subpopulation of stomach cells promotes premalignancy when subjected to carcinogenic exposures.). These results demonstrate that p53 deletion in a distinct subpopulation of stomach cells promotes premalignancy when subjected to carcinogenic exposures.', '(a) Schematic showing isolation of dysplastic organoids from premalignant dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids from experiments shown in <xref rid="nihms-1546989-f0001" ref-type="fig">Figure 1</xref>. Phase contrast images of Nutlin-3 treated dys-Lgr5-p53. Phase contrast images of Nutlin-3 treated dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids'], 'nihms-1546989-f0010': ['To investigate the direct impact of DCA and MNU on premalignant cells, we optimized an in vitro culture system using human nondysplastic BE CP-A cells WT for TP53. Treatment with graded concentrations of DCA led to nonspecific toxicity without impacting p53 expression or cell cycle activity (<xref rid="nihms-1546989-f0010" ref-type="fig">Extended Data Fig 3a</xref>--<xref rid="nihms-1546989-f0010" ref-type="fig">c</xref>). MNU treatment, however, led to dose-dependent proliferation defects and G2/M growth arrest (). MNU treatment, however, led to dose-dependent proliferation defects and G2/M growth arrest (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2a</xref>, , <xref rid="nihms-1546989-f0010" ref-type="fig">Extended Data Fig. 3d</xref>). MNU also led to p53 induction (). MNU also led to p53 induction (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2b</xref>) with dsDNA damage as shown by γH2Ax immunofluorescence () with dsDNA damage as shown by γH2Ax immunofluorescence (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2a</xref>), consistent with the carcinogenic effects of dietary nitrosamines. Together, these studies suggest that dietary nitrates can lead to growth arrest secondary to p53 induction and dsDNA damage.), consistent with the carcinogenic effects of dietary nitrosamines. Together, these studies suggest that dietary nitrates can lead to growth arrest secondary to p53 induction and dsDNA damage.', 'We next asked whether MNU-induced phenotypes were dependent on p53 function in vitro. We engineered p53 knockdown (KD) and knockout (KO) CP-A cells using short-hairpin RNA (shRNA) and CRISPR/Cas9, respectively (<xref rid="nihms-1546989-f0010" ref-type="fig">Extended Data Fig. 3e</xref>). Inhibiting p53 did not affect proliferation of untreated CP-A cells (). Inhibiting p53 did not affect proliferation of untreated CP-A cells (<xref rid="nihms-1546989-f0010" ref-type="fig">Extended Data Fig. 3f</xref>). By contrast, CP-A p53). By contrast, CP-A p53KO cells exposed to MNU avoided G2/M arrest and gained a proliferative advantage relative to CP-A p53WT cells (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2c</xref>--<xref rid="nihms-1546989-f0002" ref-type="fig">e</xref>). CP-A p53). CP-A p53KO cells displayed greater γH2Ax positivity with MNU treatment (<xref rid="nihms-1546989-f0010" ref-type="fig">Extended Data Fig. 3g</xref>), demonstrating the ability to tolerate greater DNA damage. To determine which genes/pathways enable these phenotypes, we performed mRNA sequencing (RNA-seq) of CP-A p53), demonstrating the ability to tolerate greater DNA damage. To determine which genes/pathways enable these phenotypes, we performed mRNA sequencing (RNA-seq) of CP-A p53KD cells. In addition to the p53 pathway (<xref rid="nihms-1546989-f0009" ref-type="fig">Extended Data Fig. 2</xref>h-i), gene-set enrichment analysis (GSEA) showed downregulation of the DNA repair response in CP-A p53h-i), gene-set enrichment analysis (GSEA) showed downregulation of the DNA repair response in CP-A p53KD cells (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2f</xref>). These findings may explain the selective advantage of p53 altered premalignant cells in the setting of dietary carcinogens (). These findings may explain the selective advantage of p53 altered premalignant cells in the setting of dietary carcinogens (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2</xref>g).g).'], 'nihms-1546989-f0003': ['To evaluate the impact of MNU using another model system, we isolated gastric organoids from Trp53F/F mice and generated isogenic p53KO derivatives. p53KO gastric organoids displayed a growth advantage in MNU-containing media (<xref rid="nihms-1546989-f0003" ref-type="fig">Fig. 3a</xref>). We next performed competition assays using gastric organoids derived from dual-labelled R26-mtmG mice. eGFP+ Mist1-p53). We next performed competition assays using gastric organoids derived from dual-labelled R26-mtmG mice. eGFP+ Mist1-p53WT and Mist1-p53KO gastric organoids were isolated from their respective untreated, tamoxifen-induced mice. eGFP+ Mist1-p53WT gastric cells represented 1.3% of early passage cultures, which remained a stable relative proportion when passaged in DMSO control and MNU-containing media (<xref rid="nihms-1546989-f0003" ref-type="fig">Fig. 3b</xref>). By contrast, eGFP+ Mist1-p53). By contrast, eGFP+ Mist1-p53KO gastric organoids, which initially represented 0.3% of early passage cultures, expanded by approximately 13-fold to 6.7% when passaged in MNU-containing media (<xref rid="nihms-1546989-f0003" ref-type="fig">Fig. 3b</xref>--<xref rid="nihms-1546989-f0003" ref-type="fig">c</xref>). We also evaluated p53). We also evaluated p53R270H/+ gastric organoids, where eGFP+ Mist1-p53R270H/+ represented 14.8% of an early passage culture whereas td+ Mist1-p53+/− occupied 80.2% (<xref rid="nihms-1546989-f0011" ref-type="fig">Extended Data Fig. 4a</xref>--<xref rid="nihms-1546989-f0011" ref-type="fig">b</xref>). The proportion of GFP+, Mist1-p53). The proportion of GFP+, Mist1-p53R270H/+ expanded by over 3-fold to 53.1%, whereas td+, Mist1-p53+/− decreased by 2-fold to ~40% when exposed to MNU (<xref rid="nihms-1546989-f0010" ref-type="fig">Extended Data Fig. 3b</xref>--<xref rid="nihms-1546989-f0010" ref-type="fig">c</xref>). These findings demonstrate a competitive advantage of p53-altered gastric organoids when exposed to dietary carcinogens (). These findings demonstrate a competitive advantage of p53-altered gastric organoids when exposed to dietary carcinogens (<xref rid="nihms-1546989-f0011" ref-type="fig">Extended Data Fig. 4d</xref>).).', 'To determine whether upregulation in these pathways is a direct consequence of p53 loss, we performed RNA-seq on isogenic p53KO organoids (<xref rid="nihms-1546989-f0003" ref-type="fig">Fig. 3a</xref>). We reasoned that this comparison would reflect gene-expression changes in response to immediate p53 inactivation (). We reasoned that this comparison would reflect gene-expression changes in response to immediate p53 inactivation (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5a</xref>). Unexpectedly, most of the pathways found upregulated in dys-Lgr5-p53). Unexpectedly, most of the pathways found upregulated in dys-Lgr5-p53KO organoids were downregulated in p53KO organoids. In addition to the p53 pathway, epithelial-to-mesenchymal transition (EMT), inflammation-associated, hypoxia, and stem cell pathways were downregulated in p53KO organoids compared to p53WT (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6c</xref>). This experiment suggested that either the duration of p53 loss or context of dysplasia contributed to the upregulation of these pathways in dys-Lgr5-p53). This experiment suggested that either the duration of p53 loss or context of dysplasia contributed to the upregulation of these pathways in dys-Lgr5-p53KO premalignant lesions (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6e</xref>).).'], 'nihms-1546989-f0004': ['We next derived organoids from dysplastic lesions to characterize and functionally study premalignant gastric epithelium. Gastric organoids were generated from the antrum of untreated and DCA/MNU-treated Lgr5-p53WT and Lgr5-p53KO mice at the 12-month end-point (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4a</xref>). ). Trp53 deletion was validated in organoids derived from Lgr5-p53KO mice using recombination-specific PCR (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5a</xref>) and Nutlin treatment, which induces apoptosis in p53) and Nutlin treatment, which induces apoptosis in p53WT cells (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5b</xref>).).', 'To investigate phenotypic properties, we cultured these organoids under ultra-low attachment conditions. dys-Lgr5-p53KO organoids formed 5-fold larger colonies compared to dys-Lgr5-p53WT (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4b</xref>). Since colony-forming ability may be a surrogate marker for renewal capacity). Since colony-forming ability may be a surrogate marker for renewal capacity32, we examined whether dys-Lgr5-p53KO organoids contained a greater percentage of eGFP+ Lgr5-expressing stem cells (Supplementary Note#2), finding a modest increase in Lgr5+ stem cells in dys-Lgr5-p53KO organoids (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4c</xref>, , <xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5c</xref>). To corroborate these findings ). To corroborate these findings in vivo, we performed GFP IHC to measure location and quantity of Lgr5+ cells in dysplastic lesions from DCA/MNU-treated mice. Only dysplastic lesions from Lgr5-p53KO mice demonstrated ectopic expression of Lgr5+ stem cells, extending beyond the mucous gland base (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4d</xref>). These findings suggest that early p53 loss may confer renewal properties in gastric premalignancy.). These findings suggest that early p53 loss may confer renewal properties in gastric premalignancy.', 'Genome doubling is considered an intermediate state that often precedes aneuploidy33,40,41 and chromosomal instability (CIN)42,43. Alterations in p53 are strongly associated with genome doubling and the CIN subtype in human gastric cancer. To test whether chromosome-level alterations were associated with p53 loss in gastric premalignancy, we evaluated chromosome counts of dys-Lgr5-p53KO to dys-Lgr5-p53WT organoids. Karyotype analysis of dys-Lgr5-p53KO organoids demonstrated greater genome doubling (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4e</xref>). To test whether deletion of p53 in dysplastic Lgr5-p53). To test whether deletion of p53 in dysplastic Lgr5-p53WT gastric organoids lead to genome doubling, we treated dys-Lgr5-p53WT organoids with AdenoCre. Ex vivo p53 deletion did not lead to a significant increase in genome doubling, suggesting that genomic evolution toward a tetraploid state developed over time in vivo (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5d</xref>). In agreement with previous literature). In agreement with previous literature44,45, whole genome doubling is often observed in premalignant lesions with TP53 mutations (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5e</xref>). These results suggest that p53-null dysplastic gastric lesions from our mouse model capture the tetraploid state, an intermediate in cancer formation.). These results suggest that p53-null dysplastic gastric lesions from our mouse model capture the tetraploid state, an intermediate in cancer formation.', 'To determine whether dysplastic organoids were capable of xenograft growth, we injected them into the flanks of nude mice. Only organoids derived from dys-Lgr5-p53KO mice were capable of outgrowth, with an inverse relationship between degree of genome doubling and latency to exponential xenograft growth (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4f</xref>). Histopathology showed cellular atypia with prominent nucleoli and disorganized glands with stratified multilayer epithelium (). Histopathology showed cellular atypia with prominent nucleoli and disorganized glands with stratified multilayer epithelium (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5f</xref>) and abundant Lgr5+ cells by immunofluorescence () and abundant Lgr5+ cells by immunofluorescence (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Fig. 5g</xref>). To evaluate somatic copy number (SCN) alterations, we performed low-pass whole-genome sequencing (LP-WGS) on dys-Lgr5-p53). To evaluate somatic copy number (SCN) alterations, we performed low-pass whole-genome sequencing (LP-WGS) on dys-Lgr5-p53KO cultured organoids and their derivative xenografts. Although SCN alterations were not detected in cultured organoids, their derivative xenografts demonstrated broad SCN gains and losses (<xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data Figure 5h</xref>), unlike the focal events observed in human gastric cancer), unlike the focal events observed in human gastric cancer43(Supplementary Note#3). The experiment did not distinguish between (1) the emergence of a clone that already existed in culture below the detection threshold for SCN assessment by LP-WGS or (2) the development of the chromosome instability in a dominant clone during xenograft growth. These data suggest that the premalignant gastric organoid system may enable the study of structural genomic alteration evolution.'], 'nihms-1546989-f0002': ['We next investigated the molecular mechanisms underlying the phenotypes observed in dysplastic Lgr5-p53KO gastric premalignancy. Given the higher mutational burden in Lgr5-p53KO dysplastic lesions (<xref rid="nihms-1546989-f0002" ref-type="fig">Fig. 2f</xref>), we first queried whole exome sequencing data for the presence of genes significantly mutated in human cancer. These analyses did not yield a clear pattern of mutations in Lgr5-p53), we first queried whole exome sequencing data for the presence of genes significantly mutated in human cancer. These analyses did not yield a clear pattern of mutations in Lgr5-p53KO dysplastic gastric lesions (Supplementary Table 1). We therefore examined transcriptional profiles of dys-Lgr5-p53KO compared to dys-Lgr5-p53WT organoids (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5a</xref>). As expected, ). As expected, Trp53 expression was attenuated in dys-Lgr5-p53KO organoids (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6a</xref>). GSEA identified pathways enriched in dys-Lgr5-p53). GSEA identified pathways enriched in dys-Lgr5-p53KO organoids (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5b</xref>), including inflammation, WNT/stem cell, and cell cycle regulation. Among the inflammation pathways, interferon (IFN), TNFα, IL-6/Stat3, and IL-2/Stat5 signaling pathways were strongly upregulated (), including inflammation, WNT/stem cell, and cell cycle regulation. Among the inflammation pathways, interferon (IFN), TNFα, IL-6/Stat3, and IL-2/Stat5 signaling pathways were strongly upregulated (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5b</xref>). Four cytokines, ). Four cytokines, Csf3, Cxcl10, Crfl1 and Ccl5, were consistently elevated in dLgr5-p53KO organoids (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6b</xref>))'], 'nihms-1546989-f0005': ['To distinguish between these scenarios, we examined gene expression profiles of organoids derived from nondysplastic gastric antrum tissue of untreated Lgr5-p53KO and Lgr5-p53WT mice. This comparison reflects gene expression changes in response to p53 inactivation that have occurred over a year in vivo without DCA/MNU (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5a</xref>). We plotted pathways enriched in dys-Lgr5-p53). We plotted pathways enriched in dys-Lgr5-p53KO (relative to dys-Lgr5-p53WT) against nondysplastic Lgr5-p53KO (relative to nondysplastic Lgr5-p53WT) organoids to identify gene-sets selectively enriched with p53 loss in dysplasia (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5c</xref>). As anticipated, gene-sets associated with p53 signaling were downregulated in both dysplastic and nondysplastic Lgr5-p53). As anticipated, gene-sets associated with p53 signaling were downregulated in both dysplastic and nondysplastic Lgr5-p53KO groups (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5c</xref>, blue). Pathways associated with mitotic spindle, cell cycle checkpoints, and DNA replication stress were preferentially upregulated in dys-Lgr5-p53, blue). Pathways associated with mitotic spindle, cell cycle checkpoints, and DNA replication stress were preferentially upregulated in dys-Lgr5-p53KO organoids (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5c</xref>, orange), which may be expected in dysplasia. Although elevated in nondysplastic Lgr5-p53, orange), which may be expected in dysplasia. Although elevated in nondysplastic Lgr5-p53KO organoids, IFN signaling pathways were more potently upregulated in dys-Lgr5-p53KO (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5c</xref>; red). Consistently, p53 mutant human gastric cancers demonstrated a significant correlation between ; red). Consistently, p53 mutant human gastric cancers demonstrated a significant correlation between CCL5 mRNA expression levels and IFN signaling by single-sample GSEA (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6d</xref>--<xref rid="nihms-1546989-f0013" ref-type="fig">e</xref>).).', 'dys-Lgr5-p53KO organoids also demonstrated greater enrichment in stem cell pathways, especially WNT (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5c</xref>, green). By contrast, the WNT pathway was downregulated in p53, green). By contrast, the WNT pathway was downregulated in p53KO organoids (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6b</xref>). These findings agree with recent literature). These findings agree with recent literature46 and earlier results that implicated increased stem cell properties in p53KO dysplasia (<xref rid="nihms-1546989-f0004" ref-type="fig">Fig. 4b</xref>--<xref rid="nihms-1546989-f0004" ref-type="fig">d</xref>; ; <xref rid="nihms-1546989-f0012" ref-type="fig">Extended Data 5c</xref>,,<xref rid="nihms-1546989-f0012" ref-type="fig">g</xref>). To extend these results, we directly examined WNT pathway status in dysplastic gastric organoids. dys-Lgr5-p53). To extend these results, we directly examined WNT pathway status in dysplastic gastric organoids. dys-Lgr5-p53KO gastric cells demonstrated a modest 2-fold increase in WNT reporter activity (<xref rid="nihms-1546989-f0014" ref-type="fig">Extended Data Fig. 7a</xref>). Moreover, dys-Lgr5-p53). Moreover, dys-Lgr5-p53KO gastric organoids demonstrated elevated protein expression of WNT-target and stem cell transcription factor Sox9 by immunoblot (<xref rid="nihms-1546989-f0005" ref-type="fig">Figure 5d</xref>), which was not observed with p53 inactivation ), which was not observed with p53 inactivation ex vivo or in nondysplastic organoids (<xref rid="nihms-1546989-f0005" ref-type="fig">Figure 5d</xref>), suggesting that the context of dysplasia is required. Consistently, only dysplastic lesions from Lgr5-p53), suggesting that the context of dysplasia is required. Consistently, only dysplastic lesions from Lgr5-p53KO mice demonstrated overexpression of Sox9, whereas nondysplastic and dysplastic Lgr5-p53WT lesions demonstrated normal Sox9 nuclear expression restricted to mucous gland bases (<xref rid="nihms-1546989-f0005" ref-type="fig">Figure 5e</xref>). To test whether WNT activation serves a functional advantage, organoids were grown in WNT-independent media. Only dys-Lgr5-p53). To test whether WNT activation serves a functional advantage, organoids were grown in WNT-independent media. Only dys-Lgr5-p53KO organoids were able to grow in media without WNT, R-spondin, and Noggin (<xref rid="nihms-1546989-f0014" ref-type="fig">Extended Data Fig. 7c</xref>), suggesting that elevated endogenous WNT activity can confer niche-independent growth (), suggesting that elevated endogenous WNT activity can confer niche-independent growth (Supplementary Note#4). Taken together, these data indicate that dysplastic p53-null gastric lesions may develop increased renewal properties and elevated WNT signaling, possibly explaining the lower frequency of WNT pathway alterations in human gastric cancer compared to colorectal cancer43.', 'The third gene-set consistently enriched in dys-Lgr5-p53KO organoids was cell cycle regulation (<xref rid="nihms-1546989-f0005" ref-type="fig">Fig. 5b</xref>--<xref rid="nihms-1546989-f0005" ref-type="fig">c</xref>), with ), with Cdkn2a among the most significantly upregulated genes (<xref rid="nihms-1546989-f0006" ref-type="fig">Fig. 6a</xref>). mRNA levels of ). mRNA levels of Cdkn2a were elevated two- to three-fold in p53KO and nondysplastic Lgr5-p53KO gastric organoids compared to their respective controls. However, dys-Lgr5-p53KO gastric organoids displayed a greater than 10-fold increase in Cdkn2a mRNA levels relative to dys-Lgr5-p53WT (<xref rid="nihms-1546989-f0006" ref-type="fig">Fig. 6b</xref>). ). CDKN2A expression and p53 activity is also inversely correlated in human gastric cancer (<xref rid="nihms-1546989-f0015" ref-type="fig">Extended Data Fig. 8a</xref>). We next asked whether p53 deletion in p53). We next asked whether p53 deletion in p53WT gastric premalignancy induces CDKN2A expression. Although CDKN2A is absent in CP-A cells, we observed upregulation of other CDKN1 and CDKN2 gene family members in CP-A p53KD cells (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6b</xref>). Deletion of p53 in dys-Lgr5-p53). Deletion of p53 in dys-Lgr5-p53WT organoids led to elevated protein expression of p16INK4A, a protein product of CDKN2A (<xref rid="nihms-1546989-f0006" ref-type="fig">Fig. 6c</xref>). To investigate whether p53 negatively regulates p16 expression ). To investigate whether p53 negatively regulates p16 expression in vivo, we examined nuclear p16INK4a staining in Lgr5-p53KO and Lgr5-p53WT dysplastic lesions. Greater nuclear p16INK4A staining (red) was observed in Lgr5-p53KO dysplasia (focal areas marked by dotted lines) relative to Lgr5-p53WT controls (<xref rid="nihms-1546989-f0006" ref-type="fig">Fig. 6d</xref>). Using tissue from the Mist-p53). Using tissue from the Mist-p53KO mouse model, we observed greater nuclear p16 staining co-localized with eGFP+ Mist-p53KO cells in dysplastic lesions (<xref rid="nihms-1546989-f0006" ref-type="fig">Fig. 6e</xref>). These data indicate that inactivation of p53 leads to p16). These data indicate that inactivation of p53 leads to p16INK4A induction in gastric premalignancy.'], 'nihms-1546989-f0006': ['We next examined the relationship between CDKN2A and TP53 in human cancer genomic data 42. TP53Altered, CDKN2AWT GE cancers, especially the CIN subtype, showed increased mRNA expression of CDKN2A (<xref rid="nihms-1546989-f0006" ref-type="fig">Fig. 6f</xref>, , <xref rid="nihms-1546989-f0015" ref-type="fig">Extended Data Fig. 8c</xref>). There was also a subset of human gastric cancers with alterations in both ). There was also a subset of human gastric cancers with alterations in both CDKN2A and TP53 (<xref rid="nihms-1546989-f0015" ref-type="fig">Fig. Extended Data 8c</xref>). Analysis of genomic alterations in human esophageal adenocarcinoma). Analysis of genomic alterations in human esophageal adenocarcinoma23,47 also showed significant co-occurrence of TP53 and CDKN2A alterations (<xref rid="nihms-1546989-f0015" ref-type="fig">Extended Data Fig. 8d</xref>). A combined data-set containing genomic alterations in patients with GE adenocarcinoma treated at our institution (). A combined data-set containing genomic alterations in patients with GE adenocarcinoma treated at our institution (<xref rid="nihms-1546989-f0013" ref-type="fig">Extended Data Fig. 6e</xref>, n = 1,299, q < 0.001) confirmed this pattern of , n = 1,299, q < 0.001) confirmed this pattern of TP53 and CDKN2A co-alteration. In aggregate, these data support a model whereby CDKN2A/p16INK4A is upregulated in p53-altered dysplasia and subsequently inactivated during cancer progression.'], 'nihms-1546989-f0007': ['We hypothesize that CDKN2A/p16INK4A, a cell cycle negative regulator, may attenuate progression of p53-altered gastric premalignancy. A correlate to this hypothesis is that abrogating CDKN2A/p16INK4A may enable progression of p53-altered gastric premalignancy. To investigate this, Cdkn2a and Trp53 were co-deleted in dys-Lgr5-p53WT organoids to generate double knockouts (DKO). In parallel, Cdkn2a alone was inactivated in dys-Lgr5-p53KO gastric organoids (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7a</xref>). dys-Lgr5-p53). dys-Lgr5-p53WT-DKO gastric organoids demonstrated increased colony-forming ability in ultra-low attachment culture (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7b</xref>). Deletion of ). Deletion of Cdkn2a improved colony-forming ability of dys-Lgr5-p53KO gastric organoids grown in media with or without WNT (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7c</xref>). Furthermore, only dys-Lgr5-p53). Furthermore, only dys-Lgr5-p53WT-DKO gastric organoids were capable of xenograft growth in the flanks of nude mice compared to dys-Lgr5-p53WT-control, -sgP53KO, and -sgP16KO gastric organoids, although the latency before exponential growth was longer compared to dys-Lgr5-p53KO organoids (~5.5 months vs. <3 months; <xref rid="nihms-1546989-f0007" ref-type="fig">Figure 7d</xref> vs vs <xref rid="nihms-1546989-f0004" ref-type="fig">Figure 4f</xref>). These data provide functional evidence that, following ). These data provide functional evidence that, following TP53 loss in dysplastic gastric lesions, CDKN2A can serve as a checkpoint to block further progression.', 'We suspected that abrogation of Cdkn2a and Trp53 may impart strain on cellular replication. CHK1 and CHK2 are downstream components of the ATR and ATM DNA-damage response (DDR) pathways, respectively, and are activated during replication stress. Indeed, phospho-CHK1 and phospho-RPA32, another indicator of replication stress, were induced in dys-Lgr5-p53WT-DKO but not dys-Lgr5-p53WT-control, -sgP53KO, and -sgP16KO gastric organoids (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7e</xref>). Furthermore, phospho-CHK1 is elevated when p53 is deleted in CP-A cells, which are ). Furthermore, phospho-CHK1 is elevated when p53 is deleted in CP-A cells, which are CDKN2A deficient (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7g</xref>). These results indicated that premalignant lesions with co-alterations in ). These results indicated that premalignant lesions with co-alterations in Cdkn2a and Trp53 activate checkpoint responses associated with replication stress.', 'These findings also implicated a potential for therapeutic sensitivity. Specifically, we hypothesized that if the CHK1 DDR pathway is activated in Cdkn2a and Trp53-co-altered dysplastic lesions, then eliminating this checkpoint may promote mitotic catastrophe and cell death. To test this hypothesis, we queried data from an unbiased pharmacogenomic screen of multiple cancer types (Ferrer-Luna, Ramkissoon, Ramkissoon, Homberg, Verreault et al., unpublished). Gastric cancer cell lines with co-disruption of CDKN2A/TP53 showed significantly greater sensitivity to the CHK1/2 inhibitor AZD7762 compared to those with only one or neither gene altered (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7f</xref>, , <xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9a</xref>). Consistent with the drug-response, genetic knockdown of ). Consistent with the drug-response, genetic knockdown of CHK1 or WEE1, a downstream mediator of ATR-CHK1 DDR, but not CHK2 showed preferential dependency in CDKN2A/TP53 co-disrupted gastric cancer (<xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9b</xref>). To validate this dependency, we tested the sensitivity of two additional gastric cancer cell lines to a more potent CHK1/2 inhibitor, Prexasertib. KE39 is ). To validate this dependency, we tested the sensitivity of two additional gastric cancer cell lines to a more potent CHK1/2 inhibitor, Prexasertib. KE39 is TP53 mutant and CDKN2A wildtype, whereas GSU is TP53 and CDKN2A co-disrupted. Consistently, GSU gastric cancer cells were significantly more sensitive to Prexasertib than KE39 cells (<xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9c</xref>). Furthermore, GSU was also significantly more sensitive to AZD1775, a potent WEE1 inhibitor, compared to KE39 (). Furthermore, GSU was also significantly more sensitive to AZD1775, a potent WEE1 inhibitor, compared to KE39 (<xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9d</xref>). Together, these data suggest that co-disruption of ). Together, these data suggest that co-disruption of CDKN2A and TP53 may serve as a predictive biomarker for sensitivity to CHK1-WEE1 DDR inhibitors.', 'We next investigated whether co-inactivating CDKN2A/TP53 in premalignant CP-A cells confers increased sensitivity to CHK1 inhibition. Deletion of p53 in p16INK4A-deficient CP-A cells led to a significant increase in Prexasertib sensitivity at doses corresponding to phopho-CHK1 inhibition (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7g</xref>). We also examined DDR pathway inhibition using our murine dysplastic organoids. dys-Lgr5-p53). We also examined DDR pathway inhibition using our murine dysplastic organoids. dys-Lgr5-p53WT-DKO gastric organoids demonstrated greater sensitivity to Prexasertib than dys-Lgr5-p53WT-control (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7h</xref>--<xref rid="nihms-1546989-f0007" ref-type="fig">j</xref>, , <xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9e</xref>). Moreover, dys-Lgr5-p53). Moreover, dys-Lgr5-p53KO-p16KO demonstrated even greater sensitivity to Prexasertib than dys-Lgr5-p53KO-control gastric organoids (<xref rid="nihms-1546989-f0007" ref-type="fig">Fig. 7h</xref>--<xref rid="nihms-1546989-f0007" ref-type="fig">j</xref>, , <xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9e</xref>). To investigate other DDR pathway members, we tested ATR inhibitor AZD6738 and WEE1 inhibitor AZD1775. Although dys-Lgr5-p53). To investigate other DDR pathway members, we tested ATR inhibitor AZD6738 and WEE1 inhibitor AZD1775. Although dys-Lgr5-p53KO-p16KO were not preferentially sensitive to ATR inhibition, they did show increased sensitivity to WEE1 inhibition (<xref rid="nihms-1546989-f0016" ref-type="fig">Extended Data Fig. 9f</xref>--<xref rid="nihms-1546989-f0016" ref-type="fig">g</xref>). Overall, these data suggest that co-disruption of ). Overall, these data suggest that co-disruption of CDKN2A and TP53 in gastric premalignancy not only promotes cancer progression but also confers sensitivity to inhibition of the CHK1-WEE1 axis of the DDR pathway.', 'p16INK4A induction in our model may represent a response to DNA damage67 in the absence of p53. Inactivation of p16INK4A appeared to increase replication stress (<xref rid="nihms-1546989-f0007" ref-type="fig">Figure 7d</xref>,,<xref rid="nihms-1546989-f0007" ref-type="fig">g</xref>), rendering the DDR even more critical. DDR and replication stress are inappropriately activated in premalignant lesions and postulated to protect against progression to malignancy), rendering the DDR even more critical. DDR and replication stress are inappropriately activated in premalignant lesions and postulated to protect against progression to malignancy68,69. These DDR checkpoints and unscheduled replication may also generate new sensitivities in specific genomic contexts. p53-deficient cancer cells have exhibited a selective sensitivity to CHK1 inhibition when treated with cytotoxic agents or gamma-radiation70. Upstream of CHK1, ATR inhibition imparted selective toxicity in ATM- and p53-deficient cancer cells71. Inactivation of CDKN2A in head and neck cancer induced replication stress and conferred sensitivity to CHK1 inhibition72. Therefore, inactivation of p16INK4A and p53, two critical regulators of cell cycle progression and DDR, may increase sensitivity of gastric cancer to CHK1 and WEE1 inhibitors. CIN- gastric cancers may particularly be susceptible to these inhibitors.', '(e) Quantification by manual counting of number of organoids in experiment from <xref rid="nihms-1546989-f0007" ref-type="fig">Figure 7f</xref>--<xref rid="nihms-1546989-f0007" ref-type="fig">g</xref>..'], 'nihms-1546989-f0009': ['A subset of mice unexpectedly developed thymic lymphomas when treated with MNU. These events were restricted to Trp53LSL-R270H mice, which harbors the conditional LSL-p53R270H allele at the endogenous locus of p53 to facilitate expression under endogenous genomic conditions once activated by Cre/tamoxifen. An untoward effect however is that the endogenous wildtype p53 allele is knocked out. As a result, the mutant LSL-p53R270H mice are heterozygous for p53 (+/−) in every tissue and can only conditionally express p53R270H wherever Cre is activated. Nevertheless, in the setting of MNU exposure, the p53 +/− thymic tissue appears to be susceptible to losing the remaining wildtype copy and developing thymic lymphomas reliably within 3-4 months of carcinogen exposure initiation. Therefore, the development of thymic lymphomas in these mice is unrelated to Lgr5-Cre expression, which, to our knowledge, is not expressed in T-cells or other immune cells. As shown in <xref rid="nihms-1546989-f0009" ref-type="fig">Extended Data Fig. 2c</xref>, all MNU-treated mice, irrespective of tamoxifen induction, began to die around 100 days after the experiment was initiated. In other words, MNU-treated mice in which mutant p53, all MNU-treated mice, irrespective of tamoxifen induction, began to die around 100 days after the experiment was initiated. In other words, MNU-treated mice in which mutant p53R270H was not activated (green) or Lgr5-Cre was not present (orange), still died due to thymic lymphoma complications.']}
|
Early TP53 Alterations Engage Environmental Exposures to Promote Gastric Premalignancy in an Integrative Mouse Model
| null |
Nat Genet
|
1580889600
|
Cancer genomes contain large numbers of somatic mutations but few of these mutations drive tumor development. Current approaches either identify driver genes on the basis of mutational recurrence or approximate the functional consequences of nonsynonymous mutations by using bioinformatic scores. Passenger mutations are enriched in characteristic nucleotide contexts, whereas driver mutations occur in functional positions, which are not necessarily surrounded by a particular nucleotide context. We observed that mutations in contexts that deviate from the characteristic contexts around passenger mutations provide a signal in favor of driver genes. We therefore developed a method that combines this feature with the signals traditionally used for driver-gene identification. We applied our method to whole-exome sequencing data from 11,873 tumor-normal pairs and identified 460 driver genes that clustered into 21 cancer-related pathways. Our study provides a resource of driver genes across 28 tumor types with additional driver genes identified according to mutations in unusual nucleotide contexts.
|
[
"Cluster Analysis",
"Computational Biology",
"Humans",
"Mutation",
"Neoplasms",
"Nucleotides",
"Proteins",
"Exome Sequencing"
] |
other
|
PMC7031028
| null |
5
|
[
"Error: 'list' object has no attribute 'get'\nPubMed Dictionary: {'MedlineCitation': {'@Status': 'MEDLINE', '@Owner': 'NLM', 'PMID': {'@Version': '1', '#text': '31959989'}, 'DateCompleted': {'Year': '2020', 'Month': '04', 'Day': '21'}, 'DateRevised': {'Year': '2021', 'Month': '01', 'Day': '19'}, 'Article': {'@PubModel': 'Print-Electronic', 'Journal': {'ISSN': {'@IssnType': 'Electronic', '#text': '1546-170X'}, 'JournalIssue': {'@CitedMedium': 'Internet', 'Volume': '26', 'Issue': '2', 'PubDate': {'Year': '2020', 'Month': 'Feb'}}, 'Title': 'Nature medicine', 'ISOAbbreviation': 'Nat Med'}, 'ArticleTitle': 'Fecal dysbiosis in infants with cystic fibrosis is associated with early linear growth failure.', 'Pagination': {'StartPage': '215', 'EndPage': '221', 'MedlinePgn': '215-221'}, 'ELocationID': {'@EIdType': 'doi', '@ValidYN': 'Y', '#text': '10.1038/s41591-019-0714-x'}, 'Abstract': {'AbstractText': {'sup': ['1', '1', '2,3', '4,5', '6,7'], '#text': 'Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age. Nevertheless, most infants with CF continue to have poor linear growth during their first year of life. Although this early linear growth failure is associated with worse long-term respiratory function and survival, the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis. As GI microbiome activities are known to affect endocrine functions, the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF.'}}, 'AuthorList': {'@CompleteYN': 'Y', 'Author': [{'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Hayden', 'ForeName': 'Hillary S', 'Initials': 'HS', 'AffiliationInfo': {'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Eng', 'ForeName': 'Alexander', 'Initials': 'A', 'AffiliationInfo': {'Affiliation': 'Department of Genome Sciences, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Pope', 'ForeName': 'Christopher E', 'Initials': 'CE', 'AffiliationInfo': {'Affiliation': 'Department of Pediatrics, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Brittnacher', 'ForeName': 'Mitchell J', 'Initials': 'MJ', 'AffiliationInfo': {'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Vo', 'ForeName': 'Anh T', 'Initials': 'AT', 'AffiliationInfo': {'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Weiss', 'ForeName': 'Eli J', 'Initials': 'EJ', 'AffiliationInfo': {'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Hager', 'ForeName': 'Kyle R', 'Initials': 'KR', 'AffiliationInfo': {'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Martin', 'ForeName': 'Bryan D', 'Initials': 'BD', 'AffiliationInfo': {'Affiliation': 'Department of Statistics, University of Washington, Seattle, WA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Leung', 'ForeName': 'Daniel H', 'Initials': 'DH', 'AffiliationInfo': {'Affiliation': 'Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Heltshe', 'ForeName': 'Sonya L', 'Initials': 'SL', 'AffiliationInfo': {'Affiliation': \"Cystic Fibrosis Foundation Therapeutics Development Network Coordinating Center, Seattle Children's Research Institute, Seattle, WA, USA.\"}}, {'@ValidYN': 'Y', 'LastName': 'Borenstein', 'ForeName': 'Elhanan', 'Initials': 'E', 'AffiliationInfo': [{'Affiliation': 'Department of Genome Sciences, University of Washington, Seattle, WA, USA. elbo@uw.edu.'}, {'Affiliation': 'Department of Computer Science and Engineering, University of Washington, Seattle, WA, USA. elbo@uw.edu.'}, {'Affiliation': 'Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel. elbo@uw.edu.'}, {'Affiliation': 'Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. elbo@uw.edu.'}, {'Affiliation': 'Santa Fe Institute, Santa Fe, NM, USA. elbo@uw.edu.'}]}, {'@ValidYN': 'Y', 'LastName': 'Miller', 'ForeName': 'Samuel I', 'Initials': 'SI', 'AffiliationInfo': [{'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA. millersi@uw.edu.'}, {'Affiliation': 'Department of Genome Sciences, University of Washington, Seattle, WA, USA. millersi@uw.edu.'}, {'Affiliation': 'Department of Medicine, University of Washington, Seattle, WA, USA. millersi@uw.edu.'}]}, {'@ValidYN': 'Y', 'LastName': 'Hoffman', 'ForeName': 'Lucas R', 'Initials': 'LR', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-8864-6751'}, 'AffiliationInfo': [{'Affiliation': 'Department of Microbiology, University of Washington, Seattle, WA, USA. lhoffm@uw.edu.'}, {'Affiliation': 'Department of Pediatrics, University of Washington, Seattle, WA, USA. lhoffm@uw.edu.'}, {'Affiliation': \"Pulmonary and Sleep Medicine, Seattle Children's Hospital, Seattle, WA, USA. lhoffm@uw.edu.\"}]}]}, 'Language': 'eng', 'GrantList': {'@CompleteYN': 'Y', 'Grant': [{'GrantID': 'P30 DK089507', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}, {'GrantID': 'K24 HL141669', 'Acronym': 'HL', 'Agency': 'NHLBI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 DK095869', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 DK095738', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}]}, 'PublicationTypeList': {'PublicationType': [{'@UI': 'D016428', '#text': 'Journal Article'}, {'@UI': 'D016448', '#text': 'Multicenter Study'}, {'@UI': 'D064888', '#text': 'Observational Study'}, {'@UI': 'D052061', '#text': 'Research Support, N.I.H., Extramural'}, {'@UI': 'D013485', '#text': \"Research Support, Non-U.S. Gov't\"}]}, 'ArticleDate': {'@DateType': 'Electronic', 'Year': '2020', 'Month': '01', 'Day': '20'}}, 'MedlineJournalInfo': {'Country': 'United States', 'MedlineTA': 'Nat Med', 'NlmUniqueID': '9502015', 'ISSNLinking': '1078-8956'}, 'CitationSubset': 'IM', 'MeshHeadingList': {'MeshHeading': [{'DescriptorName': {'@UI': 'D049628', '@MajorTopicYN': 'N', '#text': 'Body Size'}}, {'DescriptorName': {'@UI': 'D016022', '@MajorTopicYN': 'N', '#text': 'Case-Control Studies'}}, {'DescriptorName': {'@UI': 'D003550', '@MajorTopicYN': 'N', '#text': 'Cystic Fibrosis'}, 'QualifierName': {'@UI': 'Q000382', '@MajorTopicYN': 'Y', '#text': 'microbiology'}}, {'DescriptorName': {'@UI': 'D064806', '@MajorTopicYN': 'N', '#text': 'Dysbiosis'}, 'QualifierName': {'@UI': 'Q000382', '@MajorTopicYN': 'Y', '#text': 'microbiology'}}, {'DescriptorName': {'@UI': 'D005243', '@MajorTopicYN': 'N', '#text': 'Feces'}, 'QualifierName': {'@UI': 'Q000382', '@MajorTopicYN': 'Y', '#text': 'microbiology'}}, {'DescriptorName': {'@UI': 'D005260', '@MajorTopicYN': 'N', '#text': 'Female'}}, {'DescriptorName': {'@UI': 'D000069196', '@MajorTopicYN': 'N', '#text': 'Gastrointestinal Microbiome'}}, {'DescriptorName': {'@UI': 'D041981', '@MajorTopicYN': 'N', '#text': 'Gastrointestinal Tract'}, 'QualifierName': {'@UI': 'Q000382', '@MajorTopicYN': 'N', '#text': 'microbiology'}}, {'DescriptorName': {'@UI': 'D006130', '@MajorTopicYN': 'N', '#text': 'Growth Disorders'}, 'QualifierName': {'@UI': 'Q000209', '@MajorTopicYN': 'Y', '#text': 'etiology'}}, {'DescriptorName': {'@UI': 'D006801', '@MajorTopicYN': 'N', '#text': 'Humans'}}, {'DescriptorName': {'@UI': 'D007223', '@MajorTopicYN': 'N', '#text': 'Infant'}}, {'DescriptorName': {'@UI': 'D007231', '@MajorTopicYN': 'N', '#text': 'Infant, Newborn'}}, {'DescriptorName': {'@UI': 'D007249', '@MajorTopicYN': 'N', '#text': 'Inflammation'}}, {'DescriptorName': {'@UI': 'D008137', '@MajorTopicYN': 'N', '#text': 'Longitudinal Studies'}}, {'DescriptorName': {'@UI': 'D008297', '@MajorTopicYN': 'N', '#text': 'Male'}}, {'DescriptorName': {'@UI': 'D015999', '@MajorTopicYN': 'N', '#text': 'Multivariate Analysis'}}, {'DescriptorName': {'@UI': 'D009154', '@MajorTopicYN': 'N', '#text': 'Mutation'}}, {'DescriptorName': {'@UI': 'D015997', '@MajorTopicYN': 'N', '#text': 'Neonatal Screening'}}, {'DescriptorName': {'@UI': 'D011446', '@MajorTopicYN': 'N', '#text': 'Prospective Studies'}}, {'DescriptorName': {'@UI': 'D017422', '@MajorTopicYN': 'N', '#text': 'Sequence Analysis, DNA'}}]}, 'CoiStatement': 'Competing interests. The authors declare no competing interests.'}, 'PubmedData': {'History': {'PubMedPubDate': [{'@PubStatus': 'received', 'Year': '2019', 'Month': '2', 'Day': '17'}, {'@PubStatus': 'accepted', 'Year': '2019', 'Month': '11', 'Day': '22'}, {'@PubStatus': 'pubmed', 'Year': '2020', 'Month': '1', 'Day': '22', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'medline', 'Year': '2020', 'Month': '4', 'Day': '22', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'entrez', 'Year': '2020', 'Month': '1', 'Day': '22', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'pmc-release', 'Year': '2020', 'Month': '7', 'Day': '20'}]}, 'PublicationStatus': 'ppublish', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pubmed', '#text': '31959989'}, {'@IdType': 'mid', '#text': 'NIHMS1544359'}, {'@IdType': 'pmc', '#text': 'PMC7018602'}, {'@IdType': 'doi', '#text': '10.1038/s41591-019-0714-x'}, {'@IdType': 'pii', '#text': '10.1038/s41591-019-0714-x'}]}, 'ReferenceList': [{'Reference': [{'Citation': 'Leung DH et al. 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"Error: 'list' object has no attribute 'get'\nPubMed Dictionary: {'MedlineCitation': {'@Status': 'MEDLINE', '@Owner': 'NLM', 'PMID': {'@Version': '1', '#text': '31959991'}, 'DateCompleted': {'Year': '2020', 'Month': '04', 'Day': '21'}, 'DateRevised': {'Year': '2022', 'Month': '04', 'Day': '17'}, 'Article': {'@PubModel': 'Print-Electronic', 'Journal': {'ISSN': {'@IssnType': 'Electronic', '#text': '1546-170X'}, 'JournalIssue': {'@CitedMedium': 'Internet', 'Volume': '26', 'Issue': '2', 'PubDate': {'Year': '2020', 'Month': 'Feb'}}, 'Title': 'Nature medicine', 'ISOAbbreviation': 'Nat Med'}, 'ArticleTitle': 'A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.', 'Pagination': {'StartPage': '244', 'EndPage': '251', 'MedlinePgn': '244-251'}, 'ELocationID': {'@EIdType': 'doi', '@ValidYN': 'Y', '#text': '10.1038/s41591-019-0730-x'}, 'Abstract': {'AbstractText': {'sup': ['1,2', '3', '4'], '#text': 'Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.'}}, 'AuthorList': {'@CompleteYN': 'Y', 'Author': [{'@ValidYN': 'Y', 'LastName': 'Chivukula', 'ForeName': 'Raghu R', 'Initials': 'RR', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-5264-3196'}, 'AffiliationInfo': [{'Affiliation': 'Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. raghu@wi.mit.edu.'}, {'Affiliation': 'Whitehead Institute for Biomedical Research, Cambridge, MA, USA. raghu@wi.mit.edu.'}, {'Affiliation': 'Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. raghu@wi.mit.edu.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. raghu@wi.mit.edu.'}, {'Affiliation': 'Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. raghu@wi.mit.edu.'}]}, {'@ValidYN': 'Y', 'LastName': 'Montoro', 'ForeName': 'Daniel T', 'Initials': 'DT', 'AffiliationInfo': {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Leung', 'ForeName': 'Hui Min', 'Initials': 'HM', 'AffiliationInfo': [{'Affiliation': 'Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.'}, {'Affiliation': 'Harvard Medical School, Boston, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Yang', 'ForeName': 'Jason', 'Initials': 'J', 'AffiliationInfo': [{'Affiliation': 'Whitehead Institute for Biomedical Research, Cambridge, MA, USA.'}, {'Affiliation': 'Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}, {'Affiliation': 'Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Shamseldin', 'ForeName': 'Hanan E', 'Initials': 'HE', 'AffiliationInfo': {'Affiliation': 'Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.'}}, {'@ValidYN': 'Y', 'LastName': 'Taylor', 'ForeName': 'Martin S', 'Initials': 'MS', 'AffiliationInfo': [{'Affiliation': 'Whitehead Institute for Biomedical Research, Cambridge, MA, USA.'}, {'Affiliation': 'Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}, {'Affiliation': 'Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.'}, {'Affiliation': 'Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Dougherty', 'ForeName': 'Gerard W', 'Initials': 'GW', 'AffiliationInfo': {'Affiliation': \"Department of General Pediatrics, University Children's Hospital Muenster, Münster, Germany.\"}}, {'@ValidYN': 'Y', 'LastName': 'Zariwala', 'ForeName': 'Maimoona A', 'Initials': 'MA', 'AffiliationInfo': {'Affiliation': 'Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Carson', 'ForeName': 'Johnny', 'Initials': 'J', 'AffiliationInfo': {'Affiliation': 'Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Daniels', 'ForeName': 'M Leigh Anne', 'Initials': 'MLA', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-6979-2681'}, 'AffiliationInfo': {'Affiliation': 'Division of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Sears', 'ForeName': 'Patrick R', 'Initials': 'PR', 'AffiliationInfo': {'Affiliation': 'Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Black', 'ForeName': 'Katharine E', 'Initials': 'KE', 'AffiliationInfo': {'Affiliation': 'Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Hariri', 'ForeName': 'Lida P', 'Initials': 'LP', 'AffiliationInfo': {'Affiliation': 'Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Almogarri', 'ForeName': 'Ibrahim', 'Initials': 'I', 'AffiliationInfo': {'Affiliation': 'Department of Pediatrics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.'}}, {'@ValidYN': 'Y', 'LastName': 'Frenkel', 'ForeName': 'Evgeni M', 'Initials': 'EM', 'AffiliationInfo': [{'Affiliation': 'Whitehead Institute for Biomedical Research, Cambridge, MA, USA.'}, {'Affiliation': 'Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}, {'Affiliation': 'Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Vinarsky', 'ForeName': 'Vladimir', 'Initials': 'V', 'AffiliationInfo': {'Affiliation': 'Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Omran', 'ForeName': 'Heymut', 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The authors declare the following competing interests: Provisional patent application in process: : Massachusetts General Hospital, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology; : Raghu R. Chivukula, David M. 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"Error: 'list' object has no attribute 'get'\nPubMed Dictionary: {'MedlineCitation': {'@Status': 'MEDLINE', '@Owner': 'NLM', 'PMID': {'@Version': '1', '#text': '32042191'}, 'DateCompleted': {'Year': '2020', 'Month': '04', 'Day': '21'}, 'DateRevised': {'Year': '2021', 'Month': '02', 'Day': '04'}, 'Article': {'@PubModel': 'Print-Electronic', 'Journal': {'ISSN': {'@IssnType': 'Electronic', '#text': '1546-170X'}, 'JournalIssue': {'@CitedMedium': 'Internet', 'Volume': '26', 'Issue': '2', 'PubDate': {'Year': '2020', 'Month': 'Feb'}}, 'Title': 'Nature medicine', 'ISOAbbreviation': 'Nat Med'}, 'ArticleTitle': 'Regenerative lineages and immune-mediated pruning in lung cancer metastasis.', 'Pagination': {'StartPage': '259', 'EndPage': '269', 'MedlinePgn': '259-269'}, 'ELocationID': {'@EIdType': 'doi', '@ValidYN': 'Y', '#text': '10.1038/s41591-019-0750-6'}, 'Abstract': {'AbstractText': 'Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.'}, 'AuthorList': {'@CompleteYN': 'Y', 'Author': [{'@ValidYN': 'Y', 'LastName': 'Laughney', 'ForeName': 'Ashley M', 'Initials': 'AM', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-9435-953X'}, 'AffiliationInfo': [{'Affiliation': 'Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA.'}, {'Affiliation': 'Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.'}, {'Affiliation': 'Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Hu', 'ForeName': 'Jing', 'Initials': 'J', 'AffiliationInfo': {'Affiliation': 'Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Campbell', 'ForeName': 'Nathaniel R', 'Initials': 'NR', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0002-0355-0708'}, 'AffiliationInfo': [{'Affiliation': 'Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Tri-Institutional MD-PhD Program, Weill Cornell/Rockefeller University/Sloan Kettering Institute, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Bakhoum', 'ForeName': 'Samuel F', 'Initials': 'SF', 'AffiliationInfo': [{'Affiliation': 'Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Setty', 'ForeName': 'Manu', 'Initials': 'M', 'AffiliationInfo': {'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Lavallée', 'ForeName': 'Vincent-Philippe', 'Initials': 'VP', 'AffiliationInfo': {'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Xie', 'ForeName': 'Yubin', 'Initials': 'Y', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0003-2542-2544'}, 'AffiliationInfo': [{'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Tri-Institutional Training Program in Computational Biology and Medicine, Weill Cornell/Rockefeller University/Sloan Kettering Institute, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Masilionis', 'ForeName': 'Ignas', 'Initials': 'I', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-7937-9014'}, 'AffiliationInfo': [{'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'The Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Carr', 'ForeName': 'Ambrose J', 'Initials': 'AJ', 'AffiliationInfo': {'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Kottapalli', 'ForeName': 'Sanjay', 'Initials': 'S', 'AffiliationInfo': [{'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'The Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Allaj', 'ForeName': 'Viola', 'Initials': 'V', 'AffiliationInfo': [{'Affiliation': 'Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Mattar', 'ForeName': 'Marissa', 'Initials': 'M', 'AffiliationInfo': [{'Affiliation': 'Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Rekhtman', 'ForeName': 'Natasha', 'Initials': 'N', 'AffiliationInfo': {'Affiliation': 'Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Xavier', 'ForeName': 'Joao B', 'Initials': 'JB', 'AffiliationInfo': {'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Mazutis', 'ForeName': 'Linas', 'Initials': 'L', 'AffiliationInfo': [{'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'The Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Poirier', 'ForeName': 'John T', 'Initials': 'JT', 'AffiliationInfo': {'Affiliation': 'Perlmutter Cancer Center, New York University Langone Health, New York, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Rudin', 'ForeName': 'Charles M', 'Initials': 'CM', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-5204-3465'}, 'AffiliationInfo': [{'Affiliation': 'Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}, {'Affiliation': 'Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.'}]}, {'@ValidYN': 'Y', 'LastName': \"Pe'er\", 'ForeName': 'Dana', 'Initials': 'D', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0002-9259-8817'}, 'AffiliationInfo': [{'Affiliation': 'Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. peerd@mskcc.org.'}, {'Affiliation': 'Parker Institute for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center, New York, NY, USA. peerd@mskcc.org.'}]}, {'@ValidYN': 'Y', 'LastName': 'Massagué', 'ForeName': 'Joan', 'Initials': 'J', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-9324-8408'}, 'AffiliationInfo': {'Affiliation': 'Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. j-massague@ski.mskcc.org.'}}]}, 'Language': 'eng', 'GrantList': {'@CompleteYN': 'Y', 'Grant': [{'GrantID': 'R01 CA164729', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 CA229215', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'P30 CA008748', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'DP1 HD084071', 'Acronym': 'HD', 'Agency': 'NICHD NIH HHS', 'Country': 'United States'}, {'GrantID': 'U2C CA233284', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'U54 CA209975', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'P01 CA129243', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'DP5 OD026395', 'Acronym': 'OD', 'Agency': 'NIH HHS', 'Country': 'United States'}, {'GrantID': 'F30 CA220954', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}]}, 'PublicationTypeList': {'PublicationType': [{'@UI': 'D016428', '#text': 'Journal Article'}, {'@UI': 'D052061', '#text': 'Research Support, N.I.H., Extramural'}, {'@UI': 'D013485', '#text': \"Research Support, Non-U.S. Gov't\"}]}, 'ArticleDate': {'@DateType': 'Electronic', 'Year': '2020', 'Month': '02', 'Day': '10'}}, 'MedlineJournalInfo': {'Country': 'United States', 'MedlineTA': 'Nat Med', 'NlmUniqueID': '9502015', 'ISSNLinking': '1078-8956'}, 'ChemicalList': {'Chemical': {'RegistryNumber': '0', 'NameOfSubstance': {'@UI': 'D020100', '#text': 'Hydrogels'}}}, 'CitationSubset': 'IM', 'CommentsCorrectionsList': {'CommentsCorrections': {'@RefType': 'CommentIn', 'RefSource': 'Cancer Discov. 2020 Apr;10(4):486. doi: 10.1158/2159-8290.CD-RW2020-028', 'PMID': {'@Version': '1', '#text': '32086313'}}}, 'MeshHeadingList': {'MeshHeading': [{'DescriptorName': {'@UI': 'D000230', '@MajorTopicYN': 'N', '#text': 'Adenocarcinoma'}, 'QualifierName': [{'@UI': 'Q000276', '@MajorTopicYN': 'Y', '#text': 'immunology'}, {'@UI': 'Q000473', '@MajorTopicYN': 'Y', '#text': 'pathology'}]}, {'DescriptorName': {'@UI': 'D000818', '@MajorTopicYN': 'N', '#text': 'Animals'}}, {'DescriptorName': {'@UI': 'D001980', '@MajorTopicYN': 'N', '#text': 'Bronchi'}, 'QualifierName': {'@UI': 'Q000378', '@MajorTopicYN': 'N', '#text': 'metabolism'}}, {'DescriptorName': {'@UI': 'D002454', '@MajorTopicYN': 'N', '#text': 'Cell Differentiation'}}, {'DescriptorName': {'@UI': 'D019070', '@MajorTopicYN': 'N', '#text': 'Cell Lineage'}}, {'DescriptorName': {'@UI': 'D016000', '@MajorTopicYN': 'N', '#text': 'Cluster Analysis'}}, {'DescriptorName': {'@UI': 'D030541', '@MajorTopicYN': 'N', '#text': 'Databases, Genetic'}}, {'DescriptorName': {'@UI': 'D018450', '@MajorTopicYN': 'N', '#text': 'Disease Progression'}}, 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{'DescriptorName': {'@UI': 'D008175', '@MajorTopicYN': 'N', '#text': 'Lung Neoplasms'}, 'QualifierName': [{'@UI': 'Q000276', '@MajorTopicYN': 'Y', '#text': 'immunology'}, {'@UI': 'Q000473', '@MajorTopicYN': 'Y', '#text': 'pathology'}]}, {'DescriptorName': {'@UI': 'D051379', '@MajorTopicYN': 'N', '#text': 'Mice'}}, {'DescriptorName': {'@UI': 'D009362', '@MajorTopicYN': 'Y', '#text': 'Neoplasm Metastasis'}}, {'DescriptorName': {'@UI': 'D010641', '@MajorTopicYN': 'N', '#text': 'Phenotype'}}, {'DescriptorName': {'@UI': 'D011650', '@MajorTopicYN': 'N', '#text': 'Pulmonary Alveoli'}, 'QualifierName': {'@UI': 'Q000378', '@MajorTopicYN': 'N', '#text': 'metabolism'}}, {'DescriptorName': {'@UI': 'D012038', '@MajorTopicYN': 'N', '#text': 'Regeneration'}}, {'DescriptorName': {'@UI': 'D015398', '@MajorTopicYN': 'N', '#text': 'Signal Transduction'}}]}, 'CoiStatement': 'Competing Interests. J.M. is a scientific advisor and owns company stock in Scholar Rock. C.M.R. has consulted with AbbVie, Amgen, Ascentage, Astra Zeneca, BMS, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Loxo, and Pharmar, and is on the scientific advisory boards of Elucida and Harpoon. S.F.B. owns equity in, receives compensation from, and serves as a consultant, board member, and a scientific advisory board member for Volastra Therapeutics Inc. He also has consulted for Sanofi. All other authors declare no competing conflicts.'}, 'PubmedData': {'History': {'PubMedPubDate': [{'@PubStatus': 'received', 'Year': '2019', 'Month': '5', 'Day': '8'}, {'@PubStatus': 'accepted', 'Year': '2019', 'Month': '12', 'Day': '23'}, {'@PubStatus': 'pubmed', 'Year': '2020', 'Month': '2', 'Day': '12', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'medline', 'Year': '2020', 'Month': '4', 'Day': '22', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'entrez', 'Year': '2020', 'Month': '2', 'Day': '12', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'pmc-release', 'Year': '2020', 'Month': '8', 'Day': '10'}]}, 'PublicationStatus': 'ppublish', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pubmed', '#text': '32042191'}, {'@IdType': 'mid', '#text': 'NIHMS1547395'}, {'@IdType': 'pmc', '#text': 'PMC7021003'}, {'@IdType': 'doi', '#text': '10.1038/s41591-019-0750-6'}, {'@IdType': 'pii', '#text': '10.1038/s41591-019-0750-6'}]}, 'ReferenceList': [{'Reference': [{'Citation': 'Beumer J & Clevers H Regulation and plasticity of intestinal stem cells during homeostasis and regeneration. 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"Error: 'list' object has no attribute 'get'\nPubMed Dictionary: {'MedlineCitation': {'@Status': 'MEDLINE', '@Owner': 'NLM', 'PMID': {'@Version': '1', '#text': '32025033'}, 'DateCompleted': {'Year': '2020', 'Month': '04', 'Day': '20'}, 'DateRevised': {'Year': '2022', 'Month': '05', 'Day': '03'}, 'Article': {'@PubModel': 'Print-Electronic', 'Journal': {'ISSN': {'@IssnType': 'Electronic', '#text': '1476-4687'}, 'JournalIssue': {'@CitedMedium': 'Internet', 'Volume': '578', 'Issue': '7794', 'PubDate': {'Year': '2020', 'Month': 'Feb'}}, 'Title': 'Nature', 'ISOAbbreviation': 'Nature'}, 'ArticleTitle': 'Live-animal imaging of native haematopoietic stem and progenitor cells.', 'Pagination': {'StartPage': '278', 'EndPage': '283', 'MedlinePgn': '278-283'}, 'ELocationID': {'@EIdType': 'doi', '@ValidYN': 'Y', '#text': '10.1038/s41586-020-1971-z'}, 'Abstract': {'AbstractText': {'sup': ['1,2', '3-5'], '#text': 'The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.'}}, 'AuthorList': {'@CompleteYN': 'Y', 'Author': [{'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Christodoulou', 'ForeName': 'Constantina', 'Initials': 'C', 'AffiliationInfo': [{'Affiliation': \"Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.\"}, {'Affiliation': 'Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.'}, {'Affiliation': 'Novartis Institutes for BioMedical Research, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Spencer', 'ForeName': 'Joel A', 'Initials': 'JA', 'AffiliationInfo': [{'Affiliation': 'Advanced Microscopy Program, Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA.'}, {'Affiliation': 'Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.'}, {'Affiliation': 'Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA.'}, {'Affiliation': 'Department of Bioengineering, University of California Merced, Merced, CA, USA.'}]}, {'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Yeh', 'ForeName': 'Shu-Chi A', 'Initials': 'SA', 'AffiliationInfo': [{'Affiliation': 'Advanced Microscopy Program, Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA.'}, {'Affiliation': 'Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Turcotte', 'ForeName': 'Raphaël', 'Initials': 'R', 'AffiliationInfo': [{'Affiliation': 'Advanced Microscopy Program, Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA.'}, {'Affiliation': 'Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Kokkaliaris', 'ForeName': 'Konstantinos D', 'Initials': 'KD', 'AffiliationInfo': {'Affiliation': 'Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.'}}, {'@ValidYN': 'Y', 'LastName': 'Panero', 'ForeName': 'Riccardo', 'Initials': 'R', 'AffiliationInfo': {'Affiliation': 'Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.'}}, {'@ValidYN': 'Y', 'LastName': 'Ramos', 'ForeName': 'Azucena', 'Initials': 'A', 'AffiliationInfo': [{'Affiliation': \"Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.\"}, {'Affiliation': 'Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Guo', 'ForeName': 'Guoji', 'Initials': 'G', 'AffiliationInfo': {'Affiliation': \"Dana Farber/Boston Children's Cancer and Blood Disorders Center, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.\"}}, {'@ValidYN': 'Y', 'LastName': 'Seyedhassantehrani', 'ForeName': 'Negar', 'Initials': 'N', 'AffiliationInfo': {'Affiliation': 'Department of Bioengineering, University of California Merced, Merced, CA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Esipova', 'ForeName': 'Tatiana V', 'Initials': 'TV', 'AffiliationInfo': [{'Affiliation': 'Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.'}, {'Affiliation': 'Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Vinogradov', 'ForeName': 'Sergei A', 'Initials': 'SA', 'AffiliationInfo': [{'Affiliation': 'Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.'}, {'Affiliation': 'Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Rudzinskas', 'ForeName': 'Sarah', 'Initials': 'S', 'AffiliationInfo': {'Affiliation': 'Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Zhang', 'ForeName': 'Yi', 'Initials': 'Y', 'AffiliationInfo': {'Affiliation': 'Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Perkins', 'ForeName': 'Archibald S', 'Initials': 'AS', 'AffiliationInfo': {'Affiliation': 'Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Orkin', 'ForeName': 'Stuart H', 'Initials': 'SH', 'AffiliationInfo': {'Affiliation': \"Dana Farber/Boston Children's Cancer and Blood Disorders Center, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.\"}}, {'@ValidYN': 'Y', 'LastName': 'Calogero', 'ForeName': 'Raffaele A', 'Initials': 'RA', 'AffiliationInfo': {'Affiliation': 'Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.'}}, {'@ValidYN': 'Y', 'LastName': 'Schroeder', 'ForeName': 'Timm', 'Initials': 'T', 'AffiliationInfo': {'Affiliation': 'Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.'}}, {'@ValidYN': 'Y', 'LastName': 'Lin', 'ForeName': 'Charles P', 'Initials': 'CP', 'AffiliationInfo': [{'Affiliation': 'Advanced Microscopy Program, Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA. charles_lin@hms.harvard.edu.'}, {'Affiliation': 'Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA. charles_lin@hms.harvard.edu.'}]}, {'@ValidYN': 'Y', 'LastName': 'Camargo', 'ForeName': 'Fernando D', 'Initials': 'FD', 'AffiliationInfo': [{'Affiliation': \"Stem Cell Program, Boston Children's Hospital, Boston, MA, USA. fernando.camargo@childrens.harvard.edu.\"}, {'Affiliation': 'Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA. fernando.camargo@childrens.harvard.edu.'}]}]}, 'Language': 'eng', 'GrantList': {'@CompleteYN': 'Y', 'Grant': [{'GrantID': 'R01 CA194596', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R24 NS092986', 'Acronym': 'NS', 'Agency': 'NINDS NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 DK123216', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 HL128850', 'Acronym': 'HL', 'Agency': 'NHLBI NIH HHS', 'Country': 'United States'}, {'GrantID': 'P01 HL131477', 'Acronym': 'HL', 'Agency': 'NHLBI NIH HHS', 'Country': 'United States'}, {'GrantID': 'U54 DK110805', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 EB017274', 'Acronym': 'EB', 'Agency': 'NIBIB NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 DK115577', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}, {'GrantID': 'R24 DK103074', 'Acronym': 'DK', 'Agency': 'NIDDK NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 EB018464', 'Acronym': 'EB', 'Agency': 'NIBIB NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 CA175761', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}]}, 'PublicationTypeList': {'PublicationType': {'@UI': 'D016428', '#text': 'Journal Article'}}, 'ArticleDate': {'@DateType': 'Electronic', 'Year': '2020', 'Month': '02', 'Day': '05'}}, 'MedlineJournalInfo': {'Country': 'England', 'MedlineTA': 'Nature', 'NlmUniqueID': '0410462', 'ISSNLinking': '0028-0836'}, 'ChemicalList': {'Chemical': [{'RegistryNumber': '0', 'NameOfSubstance': {'@UI': 'D000074008', '#text': 'MDS1 and EVI1 Complex Locus Protein'}}, {'RegistryNumber': '0', 'NameOfSubstance': {'@UI': 'C499305', '#text': 'Mecom protein, mouse'}}, {'RegistryNumber': 'EC 2.7.10.1', 'NameOfSubstance': {'@UI': 'C497971', '#text': 'Flt3 protein, mouse'}}, {'RegistryNumber': 'EC 2.7.10.1', 'NameOfSubstance': {'@UI': 'D051941', '#text': 'fms-Like Tyrosine Kinase 3'}}, {'RegistryNumber': 'S88TT14065', 'NameOfSubstance': {'@UI': 'D010100', '#text': 'Oxygen'}}]}, 'CitationSubset': 'IM', 'CommentsCorrectionsList': {'CommentsCorrections': {'@RefType': 'CommentIn', 'RefSource': 'Cell Stem Cell. 2020 Aug 6;27(2):189-190. doi: 10.1016/j.stem.2020.07.014', 'PMID': {'@Version': '1', '#text': '32763177'}}}, 'MeshHeadingList': {'MeshHeading': [{'DescriptorName': {'@UI': 'D000818', '@MajorTopicYN': 'N', '#text': 'Animals'}}, {'DescriptorName': {'@UI': 'D016723', '@MajorTopicYN': 'N', '#text': 'Bone Remodeling'}}, {'DescriptorName': {'@UI': 'D002465', '@MajorTopicYN': 'N', '#text': 'Cell Movement'}}, {'DescriptorName': {'@UI': 'D049109', '@MajorTopicYN': 'N', '#text': 'Cell Proliferation'}}, {'DescriptorName': {'@UI': 'D002470', '@MajorTopicYN': 'N', '#text': 'Cell Survival'}}, {'DescriptorName': {'@UI': 'D005260', '@MajorTopicYN': 'N', '#text': 'Female'}}, {'DescriptorName': {'@UI': 'D017930', '@MajorTopicYN': 'N', '#text': 'Genes, Reporter'}}, {'DescriptorName': {'@UI': 'D006412', '@MajorTopicYN': 'N', '#text': 'Hematopoietic Stem Cells'}, 'QualifierName': {'@UI': 'Q000378', '@MajorTopicYN': 'Y', '#text': 'metabolism'}}, {'DescriptorName': {'@UI': 'D000860', '@MajorTopicYN': 'N', '#text': 'Hypoxia'}, 'QualifierName': {'@UI': 'Q000378', '@MajorTopicYN': 'N', '#text': 'metabolism'}}, {'DescriptorName': {'@UI': 'D000074008', '@MajorTopicYN': 'N', '#text': 'MDS1 and EVI1 Complex Locus Protein'}, 'QualifierName': [{'@UI': 'Q000235', '@MajorTopicYN': 'N', '#text': 'genetics'}, {'@UI': 'Q000378', '@MajorTopicYN': 'N', '#text': 'metabolism'}]}, {'DescriptorName': {'@UI': 'D008297', '@MajorTopicYN': 'N', '#text': 'Male'}}, {'DescriptorName': {'@UI': 'D051379', '@MajorTopicYN': 'N', '#text': 'Mice'}}, {'DescriptorName': {'@UI': 'D057054', '@MajorTopicYN': 'Y', '#text': 'Molecular Imaging'}}, {'DescriptorName': {'@UI': 'D010100', '@MajorTopicYN': 'N', '#text': 'Oxygen'}, 'QualifierName': {'@UI': 'Q000378', '@MajorTopicYN': 'N', '#text': 'metabolism'}}, {'DescriptorName': {'@UI': 'D012886', '@MajorTopicYN': 'N', '#text': 'Skull'}, 'QualifierName': {'@UI': 'Q000166', '@MajorTopicYN': 'N', '#text': 'cytology'}}, {'DescriptorName': {'@UI': 'D051941', '@MajorTopicYN': 'N', '#text': 'fms-Like Tyrosine Kinase 3'}, 'QualifierName': [{'@UI': 'Q000235', '@MajorTopicYN': 'N', '#text': 'genetics'}, {'@UI': 'Q000378', '@MajorTopicYN': 'N', '#text': 'metabolism'}]}]}, 'CoiStatement': 'Competing interests. The authors declare no competing financial interests.'}, 'PubmedData': {'History': {'PubMedPubDate': [{'@PubStatus': 'received', 'Year': '2018', 'Month': '6', 'Day': '7'}, {'@PubStatus': 'accepted', 'Year': '2019', 'Month': '12', 'Day': '6'}, {'@PubStatus': 'pubmed', 'Year': '2020', 'Month': '2', 'Day': '7', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'medline', 'Year': '2020', 'Month': '4', 'Day': '21', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'entrez', 'Year': '2020', 'Month': '2', 'Day': '7', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'pmc-release', 'Year': '2020', 'Month': '8', 'Day': '5'}]}, 'PublicationStatus': 'ppublish', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pubmed', '#text': '32025033'}, {'@IdType': 'mid', '#text': 'NIHMS1546022'}, {'@IdType': 'pmc', '#text': 'PMC7021587'}, {'@IdType': 'doi', '#text': '10.1038/s41586-020-1971-z'}, {'@IdType': 'pii', '#text': '10.1038/s41586-020-1971-z'}]}, 'ReferenceList': [{'Reference': [{'Citation': 'Sun J et al. 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"Error: 'list' object has no attribute 'get'\nPubMed Dictionary: {'MedlineCitation': {'@Status': 'MEDLINE', '@Owner': 'NLM', 'PMID': {'@Version': '1', '#text': '32015527'}, 'DateCompleted': {'Year': '2020', 'Month': '04', 'Day': '13'}, 'DateRevised': {'Year': '2024', 'Month': '09', 'Day': '06'}, 'Article': {'@PubModel': 'Print-Electronic', 'Journal': {'ISSN': {'@IssnType': 'Electronic', '#text': '1546-1718'}, 'JournalIssue': {'@CitedMedium': 'Internet', 'Volume': '52', 'Issue': '2', 'PubDate': {'Year': '2020', 'Month': 'Feb'}}, 'Title': 'Nature genetics', 'ISOAbbreviation': 'Nat Genet'}, 'ArticleTitle': 'Identification of cancer driver genes based on nucleotide context.', 'Pagination': {'StartPage': '208', 'EndPage': '218', 'MedlinePgn': '208-218'}, 'ELocationID': {'@EIdType': 'doi', '@ValidYN': 'Y', '#text': '10.1038/s41588-019-0572-y'}, 'Abstract': {'AbstractText': 'Cancer genomes contain large numbers of somatic mutations but few of these mutations drive tumor development. Current approaches either identify driver genes on the basis of mutational recurrence or approximate the functional consequences of nonsynonymous mutations by using bioinformatic scores. Passenger mutations are enriched in characteristic nucleotide contexts, whereas driver mutations occur in functional positions, which are not necessarily surrounded by a particular nucleotide context. We observed that mutations in contexts that deviate from the characteristic contexts around passenger mutations provide a signal in favor of driver genes. We therefore developed a method that combines this feature with the signals traditionally used for driver-gene identification. We applied our method to whole-exome sequencing data from 11,873 tumor-normal pairs and identified 460 driver genes that clustered into 21 cancer-related pathways. Our study provides a resource of driver genes across 28 tumor types with additional driver genes identified according to mutations in unusual nucleotide contexts.'}, 'AuthorList': {'@CompleteYN': 'Y', 'Author': [{'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Dietlein', 'ForeName': 'Felix', 'Initials': 'F', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0002-6651-7155'}, 'AffiliationInfo': [{'Affiliation': 'Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Felix_Dietlein@dfci.harvard.edu.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Felix_Dietlein@dfci.harvard.edu.'}]}, {'@ValidYN': 'Y', '@EqualContrib': 'Y', 'LastName': 'Weghorn', 'ForeName': 'Donate', 'Initials': 'D', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-7722-8618'}, 'AffiliationInfo': [{'Affiliation': \"Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.\"}, {'Affiliation': 'Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.'}, {'Affiliation': 'Centre for Genomic Regulation, Barcelona, Spain.'}]}, {'@ValidYN': 'Y', 'LastName': 'Taylor-Weiner', 'ForeName': 'Amaro', 'Initials': 'A', 'AffiliationInfo': [{'Affiliation': 'Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Richters', 'ForeName': 'André', 'Initials': 'A', 'AffiliationInfo': [{'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}, {'Affiliation': 'Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Reardon', 'ForeName': 'Brendan', 'Initials': 'B', 'AffiliationInfo': [{'Affiliation': 'Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Liu', 'ForeName': 'David', 'Initials': 'D', 'AffiliationInfo': [{'Affiliation': 'Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}]}, {'@ValidYN': 'Y', 'LastName': 'Lander', 'ForeName': 'Eric S', 'Initials': 'ES', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0003-2662-4631'}, 'AffiliationInfo': {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.'}}, {'@ValidYN': 'Y', 'LastName': 'Van Allen', 'ForeName': 'Eliezer M', 'Initials': 'EM', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0002-0201-4444'}, 'AffiliationInfo': [{'Affiliation': 'Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. EliezerM_VanAllen@dfci.harvard.edu.'}, {'Affiliation': 'Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. EliezerM_VanAllen@dfci.harvard.edu.'}]}, {'@ValidYN': 'Y', 'LastName': 'Sunyaev', 'ForeName': 'Shamil R', 'Initials': 'SR', 'Identifier': {'@Source': 'ORCID', '#text': '0000-0001-5715-5677'}, 'AffiliationInfo': [{'Affiliation': \"Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. ssunyaev@rics.bwh.harvard.edu.\"}, {'Affiliation': 'Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA. ssunyaev@rics.bwh.harvard.edu.'}]}]}, 'Language': 'eng', 'GrantList': {'@CompleteYN': 'Y', 'Grant': [{'GrantID': 'U19 CA203654', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'K08 CA188615', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R21 CA242861', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'T32 HG002295', 'Acronym': 'HG', 'Agency': 'NHGRI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 CA227388', 'Acronym': 'CA', 'Agency': 'NCI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R35 GM127131', 'Acronym': 'GM', 'Agency': 'NIGMS NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 HG010372', 'Acronym': 'HG', 'Agency': 'NHGRI NIH HHS', 'Country': 'United States'}, {'GrantID': 'R01 MH101244', 'Acronym': 'MH', 'Agency': 'NIMH NIH HHS', 'Country': 'United States'}, {'GrantID': 'U01 HG009088', 'Acronym': 'HG', 'Agency': 'NHGRI NIH HHS', 'Country': 'United States'}]}, 'PublicationTypeList': {'PublicationType': [{'@UI': 'D016428', '#text': 'Journal Article'}, {'@UI': 'D052061', '#text': 'Research Support, N.I.H., Extramural'}, {'@UI': 'D013485', '#text': \"Research Support, Non-U.S. Gov't\"}]}, 'ArticleDate': {'@DateType': 'Electronic', 'Year': '2020', 'Month': '02', 'Day': '03'}}, 'MedlineJournalInfo': {'Country': 'United States', 'MedlineTA': 'Nat Genet', 'NlmUniqueID': '9216904', 'ISSNLinking': '1061-4036'}, 'ChemicalList': {'Chemical': [{'RegistryNumber': '0', 'NameOfSubstance': {'@UI': 'D009711', '#text': 'Nucleotides'}}, {'RegistryNumber': '0', 'NameOfSubstance': {'@UI': 'D011506', '#text': 'Proteins'}}]}, 'CitationSubset': 'IM', 'MeshHeadingList': {'MeshHeading': [{'DescriptorName': {'@UI': 'D016000', '@MajorTopicYN': 'N', '#text': 'Cluster Analysis'}}, {'DescriptorName': {'@UI': 'D019295', '@MajorTopicYN': 'N', '#text': 'Computational Biology'}, 'QualifierName': {'@UI': 'Q000379', '@MajorTopicYN': 'Y', '#text': 'methods'}}, {'DescriptorName': {'@UI': 'D006801', '@MajorTopicYN': 'N', '#text': 'Humans'}}, {'DescriptorName': {'@UI': 'D009154', '@MajorTopicYN': 'Y', '#text': 'Mutation'}}, {'DescriptorName': {'@UI': 'D009369', '@MajorTopicYN': 'N', '#text': 'Neoplasms'}, 'QualifierName': {'@UI': 'Q000235', '@MajorTopicYN': 'Y', '#text': 'genetics'}}, {'DescriptorName': {'@UI': 'D009711', '@MajorTopicYN': 'N', '#text': 'Nucleotides'}, 'QualifierName': {'@UI': 'Q000235', '@MajorTopicYN': 'Y', '#text': 'genetics'}}, {'DescriptorName': {'@UI': 'D011506', '@MajorTopicYN': 'N', '#text': 'Proteins'}, 'QualifierName': [{'@UI': 'Q000737', '@MajorTopicYN': 'N', '#text': 'chemistry'}, {'@UI': 'Q000235', '@MajorTopicYN': 'Y', '#text': 'genetics'}]}, {'DescriptorName': {'@UI': 'D000073359', '@MajorTopicYN': 'N', '#text': 'Exome Sequencing'}, 'QualifierName': {'@UI': 'Q000379', '@MajorTopicYN': 'N', '#text': 'methods'}}]}}, 'PubmedData': {'History': {'PubMedPubDate': [{'@PubStatus': 'received', 'Year': '2019', 'Month': '5', 'Day': '17'}, {'@PubStatus': 'accepted', 'Year': '2019', 'Month': '12', 'Day': '16'}, {'@PubStatus': 'pubmed', 'Year': '2020', 'Month': '2', 'Day': '6', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'medline', 'Year': '2020', 'Month': '4', 'Day': '14', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'entrez', 'Year': '2020', 'Month': '2', 'Day': '5', 'Hour': '6', 'Minute': '0'}, {'@PubStatus': 'pmc-release', 'Year': '2020', 'Month': '8', 'Day': '3'}]}, 'PublicationStatus': 'ppublish', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pubmed', '#text': '32015527'}, {'@IdType': 'mid', '#text': 'NIHMS1546846'}, {'@IdType': 'pmc', '#text': 'PMC7031046'}, {'@IdType': 'doi', '#text': '10.1038/s41588-019-0572-y'}, {'@IdType': 'pii', '#text': '10.1038/s41588-019-0572-y'}]}, 'ReferenceList': [{'Reference': [{'Citation': 'Stratton MR, Campbell PJ & Futreal PA The cancer genome. 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Nat Genet. 2020 Feb 5; 52(2):219-230
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NO-CC CODE
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KIF18A depletion impairs the mitotic fidelity of WGD+ cells(a) Relative viability of indicated cell lines 8 days after transfection with the indicated siRNAs (n = 3 independent experiments; each condition normalized to respective control; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0019, 0.0109, 0.0017, respectively). (b) Mitotic duration and fate after treatment with indicated siRNA (n = 200 cells per condition; black stars indicate p-value for two-sided Student’s t-test comparing mean mitotic duration; blue stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of mitoses that give rise to micronuclei; dotted line represents mean mitotic duration). (c) Measurement of spindle length (centrosome-to-centrosome) after transfection with indicated siRNA (n = 20 cells per condition; two-way ANOVA with interaction; graph shows mean +/− SEM; scale bar 10 μm; interaction p-values = 0.0001, 0.0011, 0.0032, respectively). (d) Image demonstrating measurement of chromosome oscillations immediately prior to anaphase by assessing the widest oscillating chromosomes in each poleward direction and the cross-sectional area of all the chromosomes (scale bar 10 μm). (e) Widest oscillating chromosome in each poleward direction immediately prior to anaphase (n = 20 cells per condition from 2 independent experiments; two-way ANOVA with interaction; interaction p-values = 0.0025, <0.0001, <0.0001, respectively). (f) Two-dimensional cross-sectional area of the entire body of chromosomes immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0012, <0.0001, 0.0525, 0.0318, <0.0001, 0.0318, 0.0432, respectively). (g) Representative confocal images showing phases of mitosis in indicated cell lines 48 hours after transfection with indicated siRNA (scale bar 10 μm).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
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nihms-1656217-f0003
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b8810094a48f8ffae948da878d429e0220afbd380b58e4745f677e1e17d56de9
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
1800,
1193
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[{'image_id': 'nihms-1656217-f0004', 'image_file_name': 'nihms-1656217-f0004.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0004.jpg', 'caption': 'WGD confers dependence on KIF18A in a panel of breast cancer cell lines(a) Relative viability of cell lines 8 days after transfection with the indicated siRNAs (n = 3 independent experiments; graph shows mean +/− SEM). (b) Mitotic duration and fate following transfection with indicated siRNA (n = 80 cells per condition across 2 independent experiments; dotted line represents mean mitotic duration; black stars indicate p-values for two-sided Student’s unpaired t-test comparing mean mitotic duration; blue stars indicate p-values for two-sided Fisher’s exact test comparing fraction of mitoses that give rise to micronuclei; red stars indicate p-values for two-sided Fisher’s exact test comparing fraction of cell that die in mitosis). (c) Depletion of KIF18A impairs WGD+ cell viability through two distinct mechanisms: A) Widely oscillating chromosomes fail to properly attach to microtubules, thus activating the spindle assembly checkpoint and leading to prolonged mitosis and death. B) Larger spindles and wider oscillations increase the distance some chromosomes must traverse in anaphase leading to lagging chromosomes, micronuclei formation, and cellular arrest.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': 'cfdd6a1177bf13e0f0ab763480e6d5e6da62d460d7780a42f7c22c2669545808'}, {'image_id': 'nihms-1656217-f0003', 'image_file_name': 'nihms-1656217-f0003.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0003.jpg', 'caption': 'KIF18A depletion impairs the mitotic fidelity of WGD+ cells(a) Relative viability of indicated cell lines 8 days after transfection with the indicated siRNAs (n = 3 independent experiments; each condition normalized to respective control; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0019, 0.0109, 0.0017, respectively). (b) Mitotic duration and fate after treatment with indicated siRNA (n = 200 cells per condition; black stars indicate p-value for two-sided Student’s t-test comparing mean mitotic duration; blue stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of mitoses that give rise to micronuclei; dotted line represents mean mitotic duration). (c) Measurement of spindle length (centrosome-to-centrosome) after transfection with indicated siRNA (n = 20 cells per condition; two-way ANOVA with interaction; graph shows mean +/− SEM; scale bar 10 μm; interaction p-values = 0.0001, 0.0011, 0.0032, respectively). (d) Image demonstrating measurement of chromosome oscillations immediately prior to anaphase by assessing the widest oscillating chromosomes in each poleward direction and the cross-sectional area of all the chromosomes (scale bar 10 μm). (e) Widest oscillating chromosome in each poleward direction immediately prior to anaphase (n = 20 cells per condition from 2 independent experiments; two-way ANOVA with interaction; interaction p-values = 0.0025, <0.0001, <0.0001, respectively). (f) Two-dimensional cross-sectional area of the entire body of chromosomes immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0012, <0.0001, 0.0525, 0.0318, <0.0001, 0.0318, 0.0432, respectively). (g) Representative confocal images showing phases of mitosis in indicated cell lines 48 hours after transfection with indicated siRNA (scale bar 10 μm).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': 'b8810094a48f8ffae948da878d429e0220afbd380b58e4745f677e1e17d56de9'}, {'image_id': 'nihms-1656217-f0012', 'image_file_name': 'nihms-1656217-f0012.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0012.jpg', 'caption': 'Effects of KIF18 depletion in aneuploid cells(a) Measurement of spindle length (centrosome-to-centrosome) after transfection with indicated siRNA (n = 20 cells per condition; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM). (b) Anaphase phenotypes following depletion of KIF18A (n = 20 cells per condition; stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of anaphases with lagging chromosomes). (c) The fraction of cells in each cell line that undergo indicated fates after completing a mitosis deficient of KIF18A that resulted in micronuclei formation (n = 25 cells per condition). (d) The fraction of cells in each cell line that experience mitotic death in their first and second mitoses following KIF18A depletion (n = 25 cells per condition). (e) KIF18A essentiality scores for WGD– and WGD+ cell lines segregated into “highly aneuploid” (AS > 10) and “non-highly aneuploid” categories based on aneuploidy score (AS) (see methods) (dotted lines show mean; Wilcoxon rank-sum test – two-sided; p-values = 0.02583, 0.3682, respectively). (f) Aneuploidy scores and WGD status for 998 cancer cell lines in the CCLE. (g) Relative viability of indicated cell lines 7 days after transfection with the indicated siRNAs (n = 3 independent experiments; each condition normalized to respective control; one-way ANOVA with Dunnett’s post hoc test; graph shows mean +/− SEM; p-values = 0.1676, > 0.9999, 0.0040, 0.2698, 0.0007, respectively).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '8106ca700a24d85e8e85340118d546ee194700c309bf9c5f8d463daaa430aff5'}, {'image_id': 'nihms-1656217-f0002', 'image_file_name': 'nihms-1656217-f0002.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0002.jpg', 'caption': 'Identification and validation of PSL genes(a) Workflow used to identify gene essentiality in WGD+ cancer cells from Project Achilles data (see methods). (b) Top hits from PSL analysis (text color indicates genes associated with indicated pathways). (c) Gene expression fold changes in WGD+ tumors relative to WGD– tumors plotted against combined FDR values across all tumor types with select PSL genes highlighted. (d) Population doublings after 8 days of AZ3146 treatment (n = 3 independent experiments; two-way ANOVA with interaction; graph shows mean +/− SEM; interaction p-values = 0.0085, 0.0020, 0.0156, respectively). (e) Relative viability of indicated cell lines 7 days after treatment with indicated siRNA (n = 3 independent experiments; graph shows mean +/− SEM). (f) Mean LC50 for 5 WGD– and 5 WGD+ breast cancer cell lines for indicated drug treatments (n = 3 independent experiments; nonlinear regression; graphs show mean LC50 +/− 95% CI).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '21e9c973c8bea6f47249be4b99ff53ea601f6077dc50235725daf6fdbdd14af9'}, {'image_id': 'nihms-1656217-f0005', 'image_file_name': 'nihms-1656217-f0005.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0005.jpg', 'caption': 'Mutational burden in WGD+ and WGD– tumors(a) Total mutational burden in indicated subtypes across 9,240 TCGA samples (dotted lines show median; Wilcoxon rank-sum test – two-sided: red stars indicate higher burden in WGD– samples and blue stars indicate higher burden in WGD+ samples). (b) Ploidy-corrected mutational burden in indicated subtypes across 9,240 TCGA samples (dotted lines show median; two-sided Wilcoxon rank-sum test: red stars indicate higher burden in WGD– samples and blue stars indicate higher burden in WGD+ samples). (c) Ploidy-corrected mutational burden in WGD+ and WGD– samples in the TCGA (n = 9,414 samples; dotted line shows mean +/− SD). (d) Ploidy-corrected mutational burden of WGD+ and WGD– samples in the TCGA with MSI/POLE mutations (n = 174 samples). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '98e576ea7fa3dbf91a7c4884456bcf800141203146cf1deb454d4e9706fefea4'}, {'image_id': 'nihms-1656217-f0010', 'image_file_name': 'nihms-1656217-f0010.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0010.jpg', 'caption': 'Ploidy-specific lethal effect of KIF18A depletion(a) Western blot showing endogenous KIF18A levels in indicated cell lines with graph showing respective protein levels normalized to GAPDH loading control (representative blot from 3 independent experiments; for gel source see Supplementary Figure 1). (b) Representative Western blot showing KIF18A levels 48 hours after transfection with indicated siRNA (n = 3 independent experiments; for gel source see Supplementary Figure 1). (c) Relative viability decrease in WGD+ and WGD– breast cancer cell lines 7 days after treatment with indicated siRNA (n = 3 independent experiments; Wilcoxon rank-sum test – two-sided; graph shows mean +/− SEM; p-value < 0.0001). (d) Relative viability 7 days after induction of Cas9 in cells with sgRNA targeting KIF18A with Western blot showing protein depletion 72 hours after induction (n = 3 independent experiments; graph shows mean +/− SEM; Student’s unpaired t-test – two-sided; p-values = 0.0007, < 0.0001, respectively; for gel source see Supplementary Figure 1). (e) Relative viability 7 days after induction of shRNA targeting KIF18A with Western blot showing protein depletion 120 hours after induction (n = 3 independent experiments; graph shows mean +/− SEM; Student’s unpaired t-test – one-sided; p-value < 0.0001; for gel source see Supplementary Figure 1). (f) Widest oscillating chromosome in each poleward direction immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; p-values = 0.0022, 0.1781, 0.1487, 0.0136, 0.0820, <0.0001, <0.0001, <0.0001, <0.0001, 0.4132, respectively). (g) Two-dimensional cross-sectional area of the entire body of chromosomes immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; p-values = 0.1178, 0.7545, 0.1440, 0.0034, 0.9989, 0.0005, 0.0033, 0.0012, 0.0110, 0.9089, respectively; graph shows mean +/− SEM).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '831b56c010840577b4e3451dbd06aa496f8b97e6ea8c6f5ed4ab924804f91b3a'}, {'image_id': 'nihms-1656217-f0006', 'image_file_name': 'nihms-1656217-f0006.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0006.jpg', 'caption': 'Characteristics of WGD+ cells(a) Correlation of stromal cell fraction and WGD (Pearson’s correlation). (b) Correlation of purity and WGD (Pearson’s correlation). (c) Illustration of our ploidy-specific lethal (PSL) analysis using gene essentiality scores for KIF18A in the Project Achilles CRISPR dataset. Starred p-values in blue represent instances where the cutoff for enrichment in WGD+ cell lines was met in either our thresholded (two-sided Fisher’s exact) or non-thresholded (two-sided Wilcoxon) analyses (see methods). (d) Fraction of responders and non-responders to PD1 blockade by WGD status (Fisher’s exact test – two-sided; p-value = 0.0351). (e) HCT116 chromosome missegregation rate (n = 3107 2N cells, 2594 4N cells; graph shows mean +/− SD). (f) DNA FACS profile of diploid and tetraploid HCT116 cells at 40 and 70 days of culture. (g) Karyotype of diploid and tetraploid HCT-116 cells with modal chromosome number and range (n = 20 karyotypes analyzed per condition). (h) Previously published data demonstrating the stability of isogenic diploid and tetraploid RPE and MCF10A cell lines.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': 'b46f7a5d6a5189b1fa80c9b6d75fad288c88644d54898fd9c78c56a5d5706fdb'}, {'image_id': 'nihms-1656217-f0001', 'image_file_name': 'nihms-1656217-f0001.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0001.jpg', 'caption': 'Genetic analysis of WGD+ tumors(a) Quantification of WGD status and total ploidy of 9,700 primary human tumor samples from the TCGA using ABSOLUTE. (b) Mean ploidy-corrected mutational burden in indicated subtypes plotted against the difference in the ploidy-corrected mutational burden between WGD+ and WGD– tumors within each subtype (Wilcoxon rank-sum test – two sided). (c) Enrichment of mutations in WGD+ tumors (log odds ratio generated by logistic regression corrected for mutation burden and tumor type). (d) Correlation of leukocyte infiltration and WGD (Pearson’s correlation). (e) Gene expression fold changes in WGD+ tumors relative to WGD– tumors plotted against combined FDR values across all tumor types with genes from the most significantly enriched gene sets highlighted.', 'hash': '97662a2cf218071d28544f33ad2561d2165b2047bef310074a129878e92859fa'}, {'image_id': 'nihms-1656217-f0008', 'image_file_name': 'nihms-1656217-f0008.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0008.jpg', 'caption': 'Validation of WGD+ vulnerabilities in breast cancer cells(a-b) Dose-response to indicated treatment after 7 days in indicated cell lines with accompanying LC50 (n = 3 independent experiments; nonlinear regression with variable slope; graphs show mean relative viability +/− SEM at each dose and mean LC50 +/− 95% CI). (c-e) Dose-response curves for 5 WGD– and 5 WGD+ breast cancer cell lines 7 days after indicated drug treatment at the indicated concentrations (n = 3 independent experiments; nonlinear regression with variable slope; graph shows mean +/− SEM at each dose). (f) Representative Western blot showing knockdown of indicated proteins in breast cancer cell lines 48 hours after treatment with indicated siRNA (n = 3 independent experiments; for gel source see Supplementary Figure 1). (g) Relative viability decrease in WGD+ and WGD– breast cancer cell lines 7 days after treatment with indicated siRNA (Wilcoxon rank-sum test – two-sided; graph shows mean +/− SEM; p-values = <0.0001, 0.0027, respectively)* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '4328f9816a3396cf9536ed7f8e16cdb15b7e1594512b039b4d5ab65423adc7ca'}, {'image_id': 'nihms-1656217-f0011', 'image_file_name': 'nihms-1656217-f0011.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0011.jpg', 'caption': 'Cell fate analysis in WGD+ cells following KIF18A depletion(a) Representative image of a 4N MCF10A cell 4 days after transfection with siKIF18A and stained for cGAS. Graph shows the fraction of micronuclei in 2N and 4N MCF10A cells with indicated treatment that stained positive for cGAS (n = 200 micronuclei per condition; Fisher’s exact test – two-sided; scale bar 10 μm; p-values = <0.0001, 0.0069, respectively). (b-c) Representative confocal images of indicated cell lines 48 hours after transfection with indicated siRNA. Arrows highlight MAD1 positive kinetochores in misaligned chromosomes (scale bar 10 μm; representative images from 2 independent experiments). (d) Representative Western blot of indicated protein levels after treatment with indicated siRNA and accompanying graphs showing relative protein levels normalized to loading control (n = 3 independent experiments; Student’s unpaired t-test – one-sided; graph shows mean +/− SEM; p-values = 0.0337, 0.0030, 0.0674, 0.0421, 0.0067, 0.0227, respectively; for gel source see Supplementary Figure 1). (e) Cell fates of indicated cell lines tracked for 3 days beginning 18 hours after transfection with indicated siRNA (n = 40 cells per condition; two-sided Fisher’s exact test comparing fraction of cells arresting/delaying in interphase relative to control group; p values = 0.0016, <0.0001, <0.0001, respectively). (f) Relative viability of indicated cell lines 4 days after transfection with the indicated siRNA (n = 3 independent experiments; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0132, 0.0310, 0.8808, 0.8615, respectively).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '70f4349df8f57442058b2228f6835f8f5e0a855ba28c8bbf49b66862e2104b44'}, {'image_id': 'nihms-1656217-f0009', 'image_file_name': 'nihms-1656217-f0009.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0009.jpg', 'caption': 'Mitotic fidelity in WGD+ cells following KIF18A depletion(a) Dose-response to MG132 treatment after 7 days in indicated cell lines with accompanying LC50 (n = 3 independent experiments; nonlinear regression with variable slope; graphs show mean relative viability +/− SEM at each dose and mean LC50 +/− 95% CI). (b) Progression free survival and overall survival in patients with upper tertile tumor expression of KIF18A in the TCGA (Cox proportional-hazards regression; graph shows hazard ratios +/− 95% CI). (c) Representative Western blot showing KIF18A levels following transfection with the indicated siRNAs in the indicated cell lines (n = 3 independent experiments; for gel source see Supplementary Figure 1). (d) Anaphase phenotypes following depletion of KIF18A (n = 20 cells per condition; stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of anaphases with lagging chromosomes; p-values = <0.0001, 0.0033, 0.0187, respectively). (e) Representative confocal images showing phases of mitosis in indicated cell lines 48 hours after transfection with indicated siRNA (representative images from 2 independent experiments; scale bar 10 μm). (f) Representative still images from 2N and 4N MCF10A cells progressing through mitosis after transfection with the indicated siRNAs. H2B-GFP labeled chromosomes are shown in white. Arrows in enlarged images show oscillating chromosomes during metaphase and the generation of a micronucleus (hrs: min; scale bar 10 μm) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': 'fb4dc4134cc6645d5bfbb5859c7ba7009bd6b30699f2c0f9eddab5f040fe5ac0'}, {'image_id': 'nihms-1656217-f0007', 'image_file_name': 'nihms-1656217-f0007.jpg', 'image_path': '../data/media_files/PMC7889737/nihms-1656217-f0007.jpg', 'caption': 'Validation of WGD+ vulnerabilities in isogenic 2N/4N cells(a) Mitotic duration of indicated cells following indicated treatments (n = 200 cells; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = <0.0001, <0.0001, 0.0265, respectively). (b) The fraction of mitoses that generate micronuclei following indicated treatments (n = 200 cells; Student’s unpaired t-test – two-sided; p-values = <0.0001, <0.0001, <0.0001, <0.0001, 0.0002, <0.0001, respectively). (c) Relative viability of 2N and 4N HCT116 cells 7 days after treatment with indicated siRNA at indicated concentrations with Western blot showing protein knockdown 48 hours after treatment with siRNA (n = 3 independent experiments; graph shows mean +/− SEM at each dose; for gel source see Supplementary Figure 1). (d) Relative viability of 2N and 4N MCF10A cells 7 days after treatment with indicated siRNA at 50 pM concentration (n =3 independent experiments; Student’s unpaired t-test – one-sided; graph shows mean +/− SEM; p-values = <0.0001, <0.0001). (e) Relative viability of 2N and 4N RPE cells 5 days after treatment with indicated siRNA at 50 pM concentration (n = 3 independent experiments; Student’s unpaired t-test – one-sided; graph shows mean +/− SEM; p-values = 0.090, 0.0007, respectively). (f) Representative Western blot showing knockdown of indicated proteins 48 hours after treatment with indicated siRNA (n = 3 independent experiments; for gel source see Supplementary Figure 1).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001', 'hash': '940bf90e6c7aedd9489df2d053ebd9a4326caa522de15a3152e8e0d43bcb9df7'}]
|
{'nihms-1656217-f0001': ['To understand the genetic differences between WGD+ and WGD– tumors, we first obtained WGD status calls made by the ABSOLUTE algorithm on ~10,000 primary tumor samples spanning 32 distinct tumor types from The Cancer Genome Atlas (TCGA). This allowed us to separate tumor samples by whether they had (WGD+) or had not (WGD–) undergone a WGD event15. Consistent with previous estimates, we found that ~36% of tumors experienced at least one WGD during their evolution8,16. We also observed a significant range in the occurrence of WGD between different tumor subtypes, implying that specific genetic, physiological, and/or microenvironmental cues can favor or repress WGD-driven tumorigenesis (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1a</xref>).).', 'We next explored the mutational landscape of WGD+ tumors, where we observed a significant enrichment of mutations in TP53 and PPPR21A (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1c</xref>), consistent with findings from advanced cancer patients), consistent with findings from advanced cancer patients8,16. The positive selection for these mutations is clear: p53 represents a major barrier to the proliferation of WGD+ cells, thus inactivating mutations in TP53 are favored in WGD+ cancers. Mutations in PPP2R1A promote centrosome clustering, an important adaptation for preventing multipolar cell division and cell death in WGD+ cells with supernumerary centrosomes3,17. We also identified mutations that are negatively enriched in WGD+ tumors, implying that these mutations are either less important for, or perhaps incompatible with, driving tumorigenesis in the context of WGD (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1c</xref>). Many of these negatively enriched genes are known to contain microsatellite indels, providing further evidence for the diverging pressures that separate the evolutionary trajectory of MSI and WGD). Many of these negatively enriched genes are known to contain microsatellite indels, providing further evidence for the diverging pressures that separate the evolutionary trajectory of MSI and WGD+ tumors18.', 'WGD is a discrete event with the capacity to fix a cell along an evolutionary path towards tumorigenesis. It gives rise to supernumerary centrosomes and a dramatically increased chromosomal burden, and the resultant chromosomal instability leads to substantial deviations away from euploidy towards highly aneuploid states. Indeed, WGD+ cells comprise the vast majority of the highly aneuploid cells observed in human cancers (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1a</xref>, , <xref rid="nihms-1656217-f0012" ref-type="fig">Extended Data Fig. 8e</xref>––<xref rid="nihms-1656217-f0012" ref-type="fig">f</xref>, , Supplementary Table 5). Our data reveal that loss of KIF18A impairs mitotic fidelity and cell viability in WGD+ cancer cells. While many factors may promote KIF18A dependency, our data show that the enhanced reliance of WGD+ cells on KIF18A is predominately due to the added chromosomal burden that inexorably follows a WGD. Two critical observations support this view: first, we observe viability defects in euploid tetraploid RPE-1 cells depleted of KIF18A despite the fact that these cells possess a normal number of centrosomes and are chromosomally stable (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3a</xref>))3,9. Second, we find that the presence of only one or two additional chromosomes in otherwise diploid cells is not sufficient to impart sensitivity to KIF18A loss (<xref rid="nihms-1656217-f0012" ref-type="fig">Extended Data Fig. 8g</xref>). This suggests that aneuploidy ). This suggests that aneuploidy per se is insufficient to drive the KIF18A dependency we observe; instead, dramatic elevations in chromosome number or the pronounced chromosomal instability and high levels of aneuploidy that arise predominately through WGD are required (<xref rid="nihms-1656217-f0012" ref-type="fig">Extended Data Fig. 8e</xref>––<xref rid="nihms-1656217-f0012" ref-type="fig">f</xref>). Our results are consistent with recent studies that have demonstrated KIF18A dependencies in highly aneuploid and chromosomally unstable cancer cell lines (Cohen-Sharir et al., Marquis et al., unpublished data). Collectively, our data highlight KIF18A as an attractive therapeutic target whose inhibition may enable the specific targeting of WGD). Our results are consistent with recent studies that have demonstrated KIF18A dependencies in highly aneuploid and chromosomally unstable cancer cell lines (Cohen-Sharir et al., Marquis et al., unpublished data). Collectively, our data highlight KIF18A as an attractive therapeutic target whose inhibition may enable the specific targeting of WGD+ tumors while sparing the normal diploid cells that comprise human tissue. Supporting this view, it has been demonstrated that KIF18A knockout mice are protected from tumorigenesis and that depletion of KIF18A from the WGD+ breast cancer cell line MDA-MB-231 impairs tumor growth in vivo40,41.'], 'nihms-1656217-f0005': ['Having differentiated WGD+ and WGD– tumors, we then assessed their mutational burdens. This analysis revealed that WGD+ tumors tend to have a higher total mutational burden8. However, we observed several tumor subtypes where the ploidy-corrected WGD– tumors had a higher mutational burden than the WGD+ tumors (<xref rid="nihms-1656217-f0005" ref-type="fig">Extended Data Fig. 1a</xref>––<xref rid="nihms-1656217-f0005" ref-type="fig">c</xref>). This tended to occur in subtypes with a high mutational load, characteristic of tumor types prone to MSI or exposure to exogenous mutagens. Conversely, in subtypes with a lower mutational burden, it was the WGD). This tended to occur in subtypes with a high mutational load, characteristic of tumor types prone to MSI or exposure to exogenous mutagens. Conversely, in subtypes with a lower mutational burden, it was the WGD+ tumors with the higher ploidy-corrected mutational burden (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1b</xref>). This supports a recent report that predicts highly mutated tumors, which experience fewer somatic copy number alterations (SCNAs), encounter selection pressures that disfavor WGD, while tumor types with a lower mutational burden and increased SCNAs will favor WGD due to its capacity to buffer against deleterious mutations in genomic regions of loss of heterozygosity). This supports a recent report that predicts highly mutated tumors, which experience fewer somatic copy number alterations (SCNAs), encounter selection pressures that disfavor WGD, while tumor types with a lower mutational burden and increased SCNAs will favor WGD due to its capacity to buffer against deleterious mutations in genomic regions of loss of heterozygosity6. We also observed that tumors with microsatellite instability (MSI) or mutations in DNA polymerase ε (POLE), which have a very high mutational burden, tend not to experience WGD events, which has been shown in other cohorts6,8 (<xref rid="nihms-1656217-f0005" ref-type="fig">Extended Data Fig. 1d</xref>).).'], 'nihms-1656217-f0006': ['To assess changes in the microenvironment of WGD tumors, we obtained ABSOLUTE purity values (i.e. the fraction of non-tumor cells) for TCGA tumor samples15. We found that WGD correlates with decreased purity and increased non-immune stromal infiltration (<xref rid="nihms-1656217-f0006" ref-type="fig">Extended Data Fig. 2a</xref>––<xref rid="nihms-1656217-f0006" ref-type="fig">b</xref>). We also assessed the correlation of WGD with TCGA estimates of tumor-infiltrating leukocytes (TILs) and found a negative correlation between WGD and TILs (). We also assessed the correlation of WGD with TCGA estimates of tumor-infiltrating leukocytes (TILs) and found a negative correlation between WGD and TILs (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1d</xref>). Gene expression analysis revealed that the most negatively enriched gene sets in WGD). Gene expression analysis revealed that the most negatively enriched gene sets in WGD+ tumors were inflammatory processes, corroborating that these tumors present with diminished host immune response similar to highly aneuploid tumors (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1e</xref>))19,20. We further identified that WGD+ tumors overexpress genes important for cellular proliferation, mitotic spindle formation, and DNA repair (<xref rid="nihms-1656217-f0001" ref-type="fig">Fig. 1e</xref>, , Supplementary Table 1). Genomic characteristics such as mutational burden, purity, and TILs interface in poorly understood ways to contribute to patient response to immunotherapy, and we hypothesized that WGD may be a contributing factor in this response. Indeed, patients with WGD+ tumors respond better to PD1 blockade across multiple tumor subtypes (<xref rid="nihms-1656217-f0006" ref-type="fig">Extended Data Fig. 2d</xref>). Collectively, our data demonstrate key genetic and phenotypic differences between WGD). Collectively, our data demonstrate key genetic and phenotypic differences between WGD+ and WGD– tumors and hint at potential adaptations and vulnerabilities that may inexorably arise following a WGD.', 'To validate these PSL genes, we first generated three isogenically matched diploid (WGD– or 2N) and tetraploid (WGD+ or 4N) cell lines as previously described3. These lines included the non-transformed epithelial cell lines RPE-1 and MCF10A, as well as the colon cancer cell line HCT116. Growth of these tetraploid cells lines over multiple passages selects for genomically stable variants that have lost their extra centrosomes (<xref rid="nihms-1656217-f0006" ref-type="fig">Extended Data Fig. 2e</xref>––<xref rid="nihms-1656217-f0006" ref-type="fig">h</xref>3,22. The development of these isogenic lines enabled us to directly compare cellular dependencies in cells differing only by WGD status.'], 'nihms-1656217-f0002': ['We examined whether WGD confers unique genetic dependencies on tumor cells by obtaining ABSOLUTE WGD calls on cancer cell lines from Project Achilles, which is a comprehensive catalog quantifying the essentiality of ~20,000 genes across ~600 cell lines following both CRISPR and RNAi-mediated gene depletion (Supplementary Table 2)21. We used Project Achilles scores to identify genes enriched for essentiality in WGD+ cell lines relative to WGD– cell lines (so-called ploidy-specific lethal (PSL) genes14) (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2a</xref>––<xref rid="nihms-1656217-f0002" ref-type="fig">b</xref>, , <xref rid="nihms-1656217-f0006" ref-type="fig">Extended Data Fig. 2c</xref>, , Supplementary Tables 3 and 4). We mapped these PSL genes against the gene expression signature of WGD+ samples in the TCGA and found several PSL genes to be significantly overexpressed, reinforcing their importance in the progression of WGD+ tumors (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2c</xref>).).', 'We also identified several PSL genes that encode for regulators of the proteasome, suggesting that WGD confers vulnerability to disruptions in protein turnover. Indeed, we found that WGD+ cells are more sensitive to the proteasome inhibitor MG132 than WGD– cells (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2f</xref>, , <xref rid="nihms-1656217-f0008" ref-type="fig">Extended Data Fig. 4e</xref>,,<xref rid="nihms-1656217-f0009" ref-type="fig">5a</xref>). This vulnerability may be due to the highly aneuploid nature of WGD). This vulnerability may be due to the highly aneuploid nature of WGD+ cells, as aneuploidy has been shown to induce proteotoxic stress28. Supporting this view, we found that tetraploid RPE-1 cells, which maintain a euploid number of chromosomes (92) (<xref rid="nihms-1656217-f0006" ref-type="fig">Extended Data Fig. 2h</xref>, were the only cell line not more sensitive to MG132 relative to diploids (, were the only cell line not more sensitive to MG132 relative to diploids (<xref rid="nihms-1656217-f0009" ref-type="fig">Extended Data Fig. 5a</xref>).).', 'Our analysis identified the gene KIF18A, which encodes for a mitotic kinesin protein, as a significant PSL hit (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2b</xref>). KIF18A functions to suppress chromosomal oscillations at the metaphase plate by regulating microtubule dynamics to facilitate proper alignment and distribution of chromosomes during mitosis). KIF18A functions to suppress chromosomal oscillations at the metaphase plate by regulating microtubule dynamics to facilitate proper alignment and distribution of chromosomes during mitosis29–32. Importantly, in contrast to the aforementioned genes that regulate essential cellular processes such as SAC function, DNA replication, and proteasome activity, KIF18A is a non-essential gene in normal diploid cells, as attested by the fact that transgenic KIF18A knockout mice survive to adulthood33. Further, KIF18A is commonly overexpressed in WGD+ tumors (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2c</xref>), and its overexpression correlates with worse patient prognosis (), and its overexpression correlates with worse patient prognosis (<xref rid="nihms-1656217-f0009" ref-type="fig">Extended Data Fig. 5b</xref>).).', 'We sought to further validate the ploidy-specific lethality of KIF18A across a panel of breast cancer cell lines. Supporting our pan-cancer gene expression analysis (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2c</xref>), we found that KIF18A protein levels are typically elevated in WGD), we found that KIF18A protein levels are typically elevated in WGD+ cells (<xref rid="nihms-1656217-f0010" ref-type="fig">Extended Data Fig. 7a</xref>). Knockdown of KIF18A from all ten breast cancer cell lines (). Knockdown of KIF18A from all ten breast cancer cell lines (<xref rid="nihms-1656217-f0010" ref-type="fig">Extended Data Fig. 7b</xref>) confirmed that WGD) confirmed that WGD+ breast cell lines experience a significantly greater reduction in viability relative to WGD– cell lines (<xref rid="nihms-1656217-f0004" ref-type="fig">Fig. 4a</xref>, , <xref rid="nihms-1656217-f0010" ref-type="fig">Extended Data Fig. 7c</xref>––<xref rid="nihms-1656217-f0010" ref-type="fig">e</xref>, , Supplementary Videos 5–6). Live-cell imaging revealed that WGD+ breast cancer cells exhibited increased spindle lengths and chromosome hyper-oscillations relative to WGD– breast cancer cells after loss of KIF18A (<xref rid="nihms-1656217-f0010" ref-type="fig">Extended Data Fig. 7f</xref>––<xref rid="nihms-1656217-f0010" ref-type="fig">g</xref>, , <xref rid="nihms-1656217-f0012" ref-type="fig">8a</xref>), thus promoting chromosome detachment, SAC activation, and prolonged mitosis (), thus promoting chromosome detachment, SAC activation, and prolonged mitosis (<xref rid="nihms-1656217-f0004" ref-type="fig">Fig. 4b</xref>, , <xref rid="nihms-1656217-f0011" ref-type="fig">Extended Fig. 6c</xref>). Notably, we observed that a large fraction of WGD). Notably, we observed that a large fraction of WGD+ cells were never able to satisfy the SAC and exhibited a dramatically prolonged mitotic arrest before ultimately undergoing mitotic cell death (<xref rid="nihms-1656217-f0004" ref-type="fig">Fig. 4b</xref>). WGD). WGD+ cells depleted of KIF18A that achieved anaphase exhibited significant increases in both anaphase lagging chromosomes and micronuclei relative to the WGD– cell lines, similar to what was observed in the isogenic tetraploid models (<xref rid="nihms-1656217-f0004" ref-type="fig">Fig. 4c</xref>, , <xref rid="nihms-1656217-f0012" ref-type="fig">Extended Data Fig. 8b</xref>). However, in contrast to the p53-proficient isogenic tetraploid cells, WGD). However, in contrast to the p53-proficient isogenic tetraploid cells, WGD+ breast cancer cell lines depleted of KIF18A were not prone to cell cycle arrest following abnormal mitosis (<xref rid="nihms-1656217-f0012" ref-type="fig">Extended Data Fig. 8c</xref>). Instead, a fraction of these cells died in interphase after experiencing catastrophic mitoses resulting in micronuclei formation, while the majority of these WGD). Instead, a fraction of these cells died in interphase after experiencing catastrophic mitoses resulting in micronuclei formation, while the majority of these WGD+ cells initiated a second round of mitosis without KIF18A, where they were just as or even more prone to mitotic cell death (<xref rid="nihms-1656217-f0012" ref-type="fig">Extended Data Fig. 8d</xref>).).', 'We employed the thresholded analysis with the Fisher’s exact test and non-thresholded analysis with the Wilcoxon rank-sum in each individual tumor type (n=12) as well as in a combined pan-cancer analysis. These analyses were also performed separately for the CRISPR and RNAi datasets. Only genes that had measurable data in 95% of total cell lines were analyzed. The final PSL score for each gene was the total number of instances a gene was found to be a hit across all analyses (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2b</xref>, , Supplementary Table 3,4). As a result, some hits may have come entirely from either the CRISPR or RNAi datasets, such as KIF18A which was only found to be enriched for essentiality in the CRISPR dataset, likely due to insufficient knockdown in the RNAi dataset.'], 'nihms-1656217-f0007': ['We first validated BUB1B and MAD2L1, the two strongest PSL gene hits from our analysis. These genes encode proteins that are essential to the function of the spindle assembly checkpoint (SAC), which delays anaphase onset until all chromosomes have attached to the mitotic spindle, promoting the faithful partitioning of genomic content during mitosis23. Increasing chromosome number prolongs the time needed to achieve full chromosome attachment and alignment24, suggesting that premature anaphase induced by disruption of the SAC should give rise to chromosome segregation errors at elevated rates in tetraploid cells. Using live-cell imaging, we found that tetraploid cells indeed require more time to attach and align chromosomes relative to diploids in all three cell lines tested (<xref rid="nihms-1656217-f0007" ref-type="fig">Extended Data Fig. 3a</xref>). Consequently, we found that inhibition of the SAC using the small molecule inhibitor AZ3146, which inhibits the MPS1 kinase and abrogates the SAC in a manner similar to MAD2 or BUBR1 depletion, leads to a significant increase in chromosome segregation defects and micronuclei formation in tetraploid cells relative to diploids (). Consequently, we found that inhibition of the SAC using the small molecule inhibitor AZ3146, which inhibits the MPS1 kinase and abrogates the SAC in a manner similar to MAD2 or BUBR1 depletion, leads to a significant increase in chromosome segregation defects and micronuclei formation in tetraploid cells relative to diploids (<xref rid="nihms-1656217-f0007" ref-type="fig">Extended Data Fig. 3b</xref>). Micronuclei and chromosome segregation errors impair cell fitness, and concordantly, population doubling assays confirmed that tetraploid cells are significantly more sensitive to SAC inhibition than diploids (). Micronuclei and chromosome segregation errors impair cell fitness, and concordantly, population doubling assays confirmed that tetraploid cells are significantly more sensitive to SAC inhibition than diploids (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2d</xref>). These data corroborate previous studies and served to validate our PSL analysis methodology). These data corroborate previous studies and served to validate our PSL analysis methodology25.', 'The identification of several genes involved in DNA replication as PSL hits suggests that WGD+ cells may also be more vulnerable to challenges to DNA replication than WGD– cells. We first validated that reductions in the levels of RRM1 and RAD51 (two PSL genes known to mitigate the DNA damage associated with replication stress) preferentially impair the viability of tetraploid cells (<xref rid="nihms-1656217-f0007" ref-type="fig">Extended Data Fig. 3c</xref>––<xref rid="nihms-1656217-f0007" ref-type="fig">f</xref>). As an orthogonal approach, we also treated isogenic diploid and tetraploid cells with hydroxyurea or gemcitabine, which inhibit ribonucleotide reductase (RRM1) activity and induce replication stress. We observed that tetraploid cell lines show an increased sensitivity to these inhibitors (). As an orthogonal approach, we also treated isogenic diploid and tetraploid cells with hydroxyurea or gemcitabine, which inhibit ribonucleotide reductase (RRM1) activity and induce replication stress. We observed that tetraploid cell lines show an increased sensitivity to these inhibitors (<xref rid="nihms-1656217-f0008" ref-type="fig">Extended Data Fig. 4a</xref>––<xref rid="nihms-1656217-f0008" ref-type="fig">b</xref>). We also confirmed this result in a panel of ten breast cancer cell lines (five WGD). We also confirmed this result in a panel of ten breast cancer cell lines (five WGD+ and five WGD–) (<xref rid="nihms-1656217-f0002" ref-type="fig">Fig. 2e</xref>––<xref rid="nihms-1656217-f0002" ref-type="fig">f</xref>, , <xref rid="nihms-1656217-f0008" ref-type="fig">Extended Data Fig. 4c</xref>––<xref rid="nihms-1656217-f0008" ref-type="fig">d</xref>,,<xref rid="nihms-1656217-f0008" ref-type="fig">f</xref>––<xref rid="nihms-1656217-f0008" ref-type="fig">g</xref>). These data reveal that WGD). These data reveal that WGD+ tumor cells are more dependent on specific DNA replication factors relative to WGD– tumor cells, perhaps as a compensation for increased replication stress induced by tetraploidy26,27.', 'Genes were assigned a binary classification (essential or non-essential) based on cutoffs established by Project Achilles. In the database, a score of −1 is assigned to a gene when its depletion in a given cell line results in a viability defect equal to the depletion of a curated list of gold standard common-essential genes49–50. Based on this scoring system, we defined any gene with a score ≤ −1 for a given cell line as essential. We then compared the fraction of cell lines in the WGD– and WGD+ groups where a gene was essential. When a gene was essential in a significantly greater fraction of WGD+ cell lines than WGD– cell lines (Fisher’s exact test, p < 0.1) in a specific tumor subtype, it was considered a “hit” in this analysis (<xref rid="nihms-1656217-f0007" ref-type="fig">Extended Data Fig. 3a</xref>).).', 'Within each tumor type, the median essentiality scores for each gene in the WGD– and WGD+ cell lines were identified. When a gene showed a statistically significant enrichment in its median essentiality score in the WGD+ compared to the WGD– cell lines (Wilcoxon test, p < 0.05), and also had an essentiality score of ≤ −0.5 in the WGD+ cell lines, it was considered at “hit” in this analysis (<xref rid="nihms-1656217-f0007" ref-type="fig">Extended Data Fig. 3a</xref>).).'], 'nihms-1656217-f0003': ['We validated KIF18A as a PSL gene by confirming that depletion of KIF18A significantly impairs the viability of tetraploid but not diploid cells (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3a</xref>, , <xref rid="nihms-1656217-f0009" ref-type="fig">Extended Data Fig. 5c</xref>). To understand the mechanism underlying this reduction in viability, we used live-cell imaging to monitor mitotic progression following KIF18A depletion. We observed that depletion of KIF18A had no effect on mitotic duration in diploid cells but led to significantly prolonged mitoses in tetraploid cells (). To understand the mechanism underlying this reduction in viability, we used live-cell imaging to monitor mitotic progression following KIF18A depletion. We observed that depletion of KIF18A had no effect on mitotic duration in diploid cells but led to significantly prolonged mitoses in tetraploid cells (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3b</xref>). We also observed that while diploid cells lacking KIF18A exhibited subtle defects in chromosome misalignment at anaphase onset, chromosome segregation proceeded relatively normally with no significant increase in micronuclei formation (). We also observed that while diploid cells lacking KIF18A exhibited subtle defects in chromosome misalignment at anaphase onset, chromosome segregation proceeded relatively normally with no significant increase in micronuclei formation (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3b</xref>,,<xref rid="nihms-1656217-f0003" ref-type="fig">g</xref>). By contrast, tetraploid cells depleted of KIF18A exhibited significant increases in chromosome misalignment, anaphase lagging chromosomes, and micronuclei formation (). By contrast, tetraploid cells depleted of KIF18A exhibited significant increases in chromosome misalignment, anaphase lagging chromosomes, and micronuclei formation (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3b</xref>,,<xref rid="nihms-1656217-f0003" ref-type="fig">g</xref>\n\n<xref rid="nihms-1656217-f0009" ref-type="fig">Extended Data Fig. 5d</xref>––<xref rid="nihms-1656217-f0009" ref-type="fig">f</xref>, , Supplementary Videos 1–4). We also observed that micronuclei in tetraploid cells depleted of KIF18A were more prone to nuclear envelope rupture than micronuclei in diploid cells, thus exposing the chromosomal contents within the micronuclei to the cytosolic environment and inducing both DNA damage and activation of the cGAS-STING pathway (<xref rid="nihms-1656217-f0011" ref-type="fig">Extended Data Fig. 6a</xref>))34,35–37', 'We speculated that the mitotic delays and aberrant chromosome segregation defects observed following KIF18A loss may be induced by changes in spindle morphology in tetraploid cells. To accommodate their doubled chromosome content, tetraploid cells assemble larger mitotic spindles (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3c</xref>))3. Depletion of KIF18A led to an additional increase in spindle length, and this effect was significantly more dramatic in tetraploid cells relative to diploids (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3c</xref>).).', 'We also measured the magnitude of chromosome oscillations immediately prior to anaphase onset in diploid and tetraploid cells by assessing the widest oscillating chromosomes in each poleward direction, as well as the overall chromosome alignment efficiency by measuring the total two-dimensional area occupied by the entire body of chromosomes (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3d</xref>). These analyses revealed that the magnitude of chromosomal oscillations is significantly greater in tetraploid cells relative to diploid cells following KIF18A depletion (). These analyses revealed that the magnitude of chromosomal oscillations is significantly greater in tetraploid cells relative to diploid cells following KIF18A depletion (<xref rid="nihms-1656217-f0003" ref-type="fig">Fig. 3e</xref>––<xref rid="nihms-1656217-f0003" ref-type="fig">f</xref>). One consequence of hyper-oscillating chromosomes in tetraploid cells depleted of KIF18A is that they have a propensity to lose their attachment to the mitotic spindle and activate the spindle assembly checkpoint, thus explaining the mitotic delays we observed (). One consequence of hyper-oscillating chromosomes in tetraploid cells depleted of KIF18A is that they have a propensity to lose their attachment to the mitotic spindle and activate the spindle assembly checkpoint, thus explaining the mitotic delays we observed (<xref rid="nihms-1656217-f0011" ref-type="fig">Extended Data Fig. 6b</xref>))38,39. A second consequence is that severely misaligned chromosomes must traverse a significantly greater distance during anaphase in tetraploid cells compared to diploid cells, thus explaining the observed increase in lagging chromosomes and micronuclei.'], 'nihms-1656217-f0011': ['We used long-term live-cell imaging to track the fates of isogenic diploid and tetraploid cells depleted of KIF18A. Our analysis revealed that while the majority of diploid cells depleted of KIF18A undergo normal cell cycle progression, isogenic tetraploid cells depleted of KIF18A are prone to interphase cell cycle arrest following abnormal mitosis, concomitant with p53 pathway activation (<xref rid="nihms-1656217-f0011" ref-type="fig">Extended Data Fig. 6d</xref>––<xref rid="nihms-1656217-f0011" ref-type="fig">e</xref>). Thus, our data reveal that loss of KIF18A in WGD). Thus, our data reveal that loss of KIF18A in WGD+ cells predisposes cells to lagging chromosomes, micronuclei formation, micronuclei rupture, and proliferative arrest. Supporting this mechanism, we found that cellular proliferation is required for the loss of KIF18A to drive our observed viability defects (<xref rid="nihms-1656217-f0011" ref-type="fig">Extended Data Fig. 6f</xref>).).']}
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Whole genome doubling confers unique genetic vulnerabilities on tumor cells
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Nature
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1614412800
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[{'@Label': 'BACKGROUD', '@NlmCategory': 'UNASSIGNED', '#text': 'Under the circumstance of school closures caused by the coronavirus outbreak, medical schools in China began implementing online teaching, including histology and embryology (HE) beginning in the middle of February 2020. The changes in HE education in responding to the pandemic in China needs to be determined, for further adaption of online teaching delivery or blended learning.'}, {'@Label': 'METHODS', '@NlmCategory': 'UNASSIGNED', '#text': 'A nationwide survey of the major medical colleges was conducted via WeChat.'}, {'@Label': 'RESULTS', '@NlmCategory': 'UNASSIGNED', '#text': 'In total, 83 medical schools (one respondent per school) were invited to survey, 78 medical schools responded which represented most medical schools across all the provinces in mainland China, as well as Hong Kong and Macao. The results revealed that 77% (n\xa0=\xa060) and 58% (n\xa0=\xa045) of the responding schools had conducted HE theoretical and practical online teaching, respectively, prior to the pandemic; however, 27% (n\xa0=\xa021) of the medical schools had temporally suspended practical sessions at the time the survey was completed. During the pandemic, 73% (n\xa0=\xa057) and 29% (n\xa0=\xa023) of the medical schools delivered HE theoretical and practical sessions by synchronous live broadcasting, respectively; 65% (n\xa0=\xa051) of the medical schools increased virtual microscopy using during practical sessions. During the pandemic, 54% (n\xa0=\xa042) of the medical schools implemented teaching activities promoting active learning; meanwhile, online assessment was implemented in 84% (n\xa0=\xa066) of the responding medical schools. With regard to the satisfaction with the effectiveness of online teaching during the pandemic, 64% (n\xa0=\xa050) of the medical schools gave positive answers and considered that it was a good opportunity to develop novel and diversified teaching methods. Despite various difficulties such as work overload and unstable online teaching environments, most medical schools are willing to continue or increase theoretical online teaching after the pandemic.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'UNASSIGNED', '#text': 'Medical institutes in China were the earliest of closing campuses and having complete online teaching experience during the pandemic. This paper presents overall HE teaching situation extracted from the survey, to assist other medical schools optimizing the transitions to quality online teaching within a short time, and to serve as reference for schools that demand essential knowledge in online teaching methods, infrastructure construction, and platform integrations.'}]
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other
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PMC7889737
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43
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[
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Nature. 2021 Feb 27; 590(7846):492-497
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KRAS membrane localization upon PDEδ disruption in KRAS-dependent cancer cells. a Intracellular localization of KRAS upon PDEδ knockdown. Cells stably expressing GFP-KRAS were transfected with PDE6D siRNA for 48 h, and KRAS localization was visualized by GFP fluorescence. b Intracellular localization of KRAS upon PDEδ inhibition. Cells were treated with 5 μM deltarasin for the indicated time. KRAS membrane association was detected as described in (a). c Quantification of (a) and (b). Cells with evident KRAS membrane association were counted and normalized to the total cell counts. At least 50 cells were counted per sample. Scale bar, 10 μm. The error bars represent the mean ± SD of three independent analyses. **P < 0.01; ***P < 0.001. NC, scrambled siRNA used as a negative control
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[{'image_id': '41401_2019_268_Fig1_HTML', 'image_file_name': '41401_2019_268_Fig1_HTML.jpg', 'image_path': '../data/media_files/PMC7471410/41401_2019_268_Fig1_HTML.jpg', 'caption': 'KRAS dependency varies in KRAS mutant cancer cell lines. a KRAS mutation status of cell lines used in this study. NSCLC, non-small cell lung cancer; PDAC, pancreatic ductal adenocarcinoma b KRAS mutant cell viability upon KRAS depletion. Cells were treated with KRAS siRNAs (siKRAS #1, #2) for 96\u2009h. Cell viability was measured by crystal violet staining assay (upper panel). siRNA interference efficiency was measured by immunoblotting analysis (lower panel). The error bars represent the mean\u2009±\u2009SD of four replicates. *P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001, n.s., not significant. c, d MAPK and AKT signaling changes upon KRAS depletion in KRAS-dependent (c) and KRAS-independent (d) cells. Cells were treated with KRAS siRNAs (siKRAS #1, #2) for 48\u2009h before being subjected to immunoblotting analysis. e Cell sensitivity to ERK inhibitors. Cells were treated with the ERK inhibitor BVD-523 for 72\u2009h, and cell viability was measured by SRB assay. The error bars represent the mean\u2009±\u2009SD of triplicates. NC, scrambled siRNA used as a negative control', 'hash': '7ea7cf8b79f43cf09da2c8cd3f79c5c6868bf5474395b02cf266bb4673e41217'}, {'image_id': '41401_2019_268_Fig2_HTML', 'image_file_name': '41401_2019_268_Fig2_HTML.jpg', 'image_path': '../data/media_files/PMC7471410/41401_2019_268_Fig2_HTML.jpg', 'caption': 'MAPK/ERK signaling alteration is associated with the response to PDEδ inhibition in KRAS-dependent cancer cells. a KRAS mutant cell viability upon PDEδ \xa0depletion. KRAS-dependent cells were treated with PDEδ siRNAs (siPDE6D #1, #2) for 96\u2009h. Cell viability was measured by crystal violet staining assay (upper panel). siRNA interference efficiency was measured by immunoblotting (lower panel). The error bars represent the mean\u2009±\u2009SD of four replicates. *P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001, n.s., not significant. b, c MAPK and AKT signaling changes upon PDEδ depletion in PDEδ-dependent (b) and PDEδ-independent cells (c). Cells were treated with siRNAs (siPDE6D #1, #2) for 48\u2009h before being subjected to immunoblotting analysis. NC, scrambled siRNA used as a negative control', 'hash': '73655a741a3964b84c72d7eb875608576711bf491489114c6a77188383568e9c'}, {'image_id': '41401_2019_268_Fig3_HTML', 'image_file_name': '41401_2019_268_Fig3_HTML.jpg', 'image_path': '../data/media_files/PMC7471410/41401_2019_268_Fig3_HTML.jpg', 'caption': 'KRAS membrane localization upon PDEδ disruption in KRAS-dependent cancer cells. a Intracellular localization of KRAS upon PDEδ knockdown. Cells stably expressing GFP-KRAS were transfected with PDE6D siRNA for 48\u2009h, and KRAS localization was visualized by GFP fluorescence. b Intracellular localization of KRAS upon PDEδ inhibition. Cells were treated with 5\u2009μM deltarasin for the indicated time. KRAS membrane association was detected as described in (a). c Quantification of (a) and (b). Cells with evident KRAS membrane association were counted and normalized to the total cell counts. At least 50 cells were counted per sample. Scale bar, 10\u2009μm. The error bars represent the mean\u2009±\u2009SD of three independent analyses. **P\u2009<\u20090.01; ***P\u2009<\u20090.001. NC, scrambled siRNA used as a negative control', 'hash': '15fc66d3a1ea763032e20fc40fce6e0f5ae1c776f72247cd04f96304effcdc7f'}, {'image_id': '41401_2019_268_Fig4_HTML', 'image_file_name': '41401_2019_268_Fig4_HTML.jpg', 'image_path': '../data/media_files/PMC7471410/41401_2019_268_Fig4_HTML.jpg', 'caption': 'Feedback activation of EPHA2 results in compensatory MAPK activation in PDEδ-depleted cells. a, b Phospho-RTK antibody and phospho-kinase antibody arrays. Cells were treated with siPDE6D (#1) for 48\u2009h, and the cell lysates were subjected to phospho-RTK antibody array (a) or phospho-kinase antibody array (b). c Immunoblotting analysis confirmed the feedback activation of EPHA2 and IGF1R in PDEδ-depleted cells. H358 cells were treated with the indicated siRNA for 48\u2009h before being subjected to immunoblotting. d MAPK signaling upon concurrent inhibition of PDEδ and EPHA2. Cells were transfected with the indicated siRNAs for 48\u2009h before exposure to the EPHA2 inhibitor ALW-II-41-27 for 4\u2009h. e MAPK signaling upon concurrent inhibition of PDEδ and IGF1R. Cells were transfected with the indicated siRNAs for 48\u2009h before exposure to the IGF1R inhibitor BMS-754807 for 5\u2009h. f, g Cell viability upon concurrent inhibition of PDEδ and MEK or EPHA2. KRAS-dependent Panc 10.05 cells were treated with PDEδ siRNA in combination with the MEK inhibitor AZD6244 or the EPHA2 inhibitor ALW-II-41-27 for 96\u2009h. Cell viability was measured by crystal violet staining assay. NC, scrambled siRNA used as a negative control. h A schematic model', 'hash': '17b9bac1c0b5abcfee7c0158d5018ae2f2d72074bd1baac45633fa895f46ecd1'}]
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{'41401_2019_268_Fig1_HTML': ['Previous studies have revealed that KRAS mutations, though they occur widely in human cancer, may not necessarily give rise to KRAS-dependent cell growth [15]. As such, we first screened the growth dependency of a small panel of human cancer cell lines bearing well-documented KRAS-activating mutations, mainly highly frequent mutations to G12 residuals (Fig.\xa0<xref rid="41401_2019_268_Fig1_HTML" ref-type="fig">1a</xref>). Indeed, knocking down KRAS expression using two independent siRNAs resulted in diverse effects on cell growth in these cells. Among the 12 tested cell lines, four cell lines, namely, Panc 02.03, H460, H1792, and H358, showed substantial growth suppression. Some cell lines, such as SW1573, H23, A549, AsPC-1, and Panc 10.05 cells, showed a moderate effect in response to KRAS depletion, while the remaining cells barely responded and were considered KRAS-independent cells (Fig.\xa0). Indeed, knocking down KRAS expression using two independent siRNAs resulted in diverse effects on cell growth in these cells. Among the 12 tested cell lines, four cell lines, namely, Panc 02.03, H460, H1792, and H358, showed substantial growth suppression. Some cell lines, such as SW1573, H23, A549, AsPC-1, and Panc 10.05 cells, showed a moderate effect in response to KRAS depletion, while the remaining cells barely responded and were considered KRAS-independent cells (Fig.\xa0<xref rid="41401_2019_268_Fig1_HTML" ref-type="fig">1b</xref>). The knockdown efficiency was comparable among the tested cells.). The knockdown efficiency was comparable among the tested cells.Fig. 1KRAS dependency varies in KRAS mutant cancer cell lines. a KRAS mutation status of cell lines used in this study. NSCLC, non-small cell lung cancer; PDAC, pancreatic ductal adenocarcinoma b KRAS mutant cell viability upon KRAS depletion. Cells were treated with KRAS siRNAs (siKRAS #1, #2) for 96\u2009h. Cell viability was measured by crystal violet staining assay (upper panel). siRNA interference efficiency was measured by immunoblotting analysis (lower panel). The error bars represent the mean\u2009±\u2009SD of four replicates. *P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001, n.s., not significant. c, d MAPK and AKT signaling changes upon KRAS depletion in KRAS-dependent (c) and KRAS-independent (d) cells. Cells were treated with KRAS siRNAs (siKRAS #1, #2) for 48\u2009h before being subjected to immunoblotting analysis. e Cell sensitivity to ERK inhibitors. Cells were treated with the ERK inhibitor BVD-523 for 72\u2009h, and cell viability was measured by SRB assay. The error bars represent the mean\u2009±\u2009SD of triplicates. NC, scrambled siRNA used as a negative control', 'KRAS activation is known to drive oncogenic malignancy, mainly via two downstream pathways, the RAF/MEK/ERK pathway, also known as the MAPK/ERK pathway, and the phosphoinositide 3-kinase (PI3K) pathway [6]. We hence examined the key signaling molecules in these two downstream pathways. In both KRAS-dependent and KRAS-independent cancer cells, KRAS depletion led to the universal downregulation of the MAPK/ERK pathway, as indicated by the decreased phosphorylation levels of c-RAF and ERK, while the impact on the PI3K/AKT pathway was variable among the cell lines (Fig.\xa0<xref rid="41401_2019_268_Fig1_HTML" ref-type="fig">1c, d</xref>; ; S1a). Consistently, pharmacological inhibition of the MAPK/ERK pathway using the clinically tested ERK kinase inhibitor BVD-523 resulted in substantial cell growth inhibition in KRAS-dependent A549 and H358 cells, which was in great contrast to KRAS-independent cell lines, such as H441 and H2030 (Fig.\xa0<xref rid="41401_2019_268_Fig1_HTML" ref-type="fig">1e;</xref>\n\nS1b). These results identified a subset of KRAS mutant cancer cells whose growth was highly dependent on KRAS activation and established a linkage between KRAS oncogenic activation and MAPK/ERK signaling activation instead of the PI3K/AKT pathway.', 'We were then interested in understanding why PDEδ disruption in some cancer cells failed to diminish MAPK/ERK signaling, as KRAS impairment in these cells was apparently associated with MAPK/ERK signaling (Fig.\xa0<xref rid="41401_2019_268_Fig1_HTML" ref-type="fig">1c</xref>). Given that PDEδ is required for KRAS plasma membrane association prior to KRAS activation, we analyzed KRAS membrane localization in these cells. To this end, GFP-conjugated KRAS was transfected into the cells to visualize KRAS subcellular localization using fluorescence microscopy. In H460 cells, which are sensitive to PDEδ deficiency, knockdown of PDE6D gene expression efficiently impaired KRAS membrane association (Fig.\xa0). Given that PDEδ is required for KRAS plasma membrane association prior to KRAS activation, we analyzed KRAS membrane localization in these cells. To this end, GFP-conjugated KRAS was transfected into the cells to visualize KRAS subcellular localization using fluorescence microscopy. In H460 cells, which are sensitive to PDEδ deficiency, knockdown of PDE6D gene expression efficiently impaired KRAS membrane association (Fig.\xa0<xref rid="41401_2019_268_Fig3_HTML" ref-type="fig">3a</xref>, left panel). Similar results were obtained using deltarasin, the first reported PDEδ inhibitor that has been shown to effectively disrupt KRAS plasma membrane localization [, left panel). Similar results were obtained using deltarasin, the first reported PDEδ inhibitor that has been shown to effectively disrupt KRAS plasma membrane localization [13] (Fig.\xa0<xref rid="41401_2019_268_Fig3_HTML" ref-type="fig">3b</xref>, left panel)., left panel).Fig. 3KRAS membrane localization upon PDEδ disruption in KRAS-dependent cancer cells. a Intracellular localization of KRAS upon PDEδ knockdown. Cells stably expressing GFP-KRAS were transfected with PDE6D siRNA for 48\u2009h, and KRAS localization was visualized by GFP fluorescence. b Intracellular localization of KRAS upon PDEδ inhibition. Cells were treated with 5\u2009μM deltarasin for the indicated time. KRAS membrane association was detected as described in (a). c Quantification of (a) and (b). Cells with evident KRAS membrane association were counted and normalized to the total cell counts. At least 50 cells were counted per sample. Scale bar, 10\u2009μm. The error bars represent the mean\u2009±\u2009SD of three independent analyses. **P\u2009<\u20090.01; ***P\u2009<\u20090.001. NC, scrambled siRNA used as a negative control'], '41401_2019_268_Fig2_HTML': ['We then attempted to assess whether the disruption of PDEδ in KRAS-dependent cells could lead to growth inhibition. As the selectivity of the reported PDEδ inhibitors remains in question [13, 16], we chose to use two independent siRNAs to specifically knockdown PDE6D, which encodes PDEδ, and cell growth was then assessed using a crystal violet staining assay. In nine KRAS-dependent or partially dependent cells, only a small fraction of the cell lines, namely, A549, H460, and SW1573 cells, responded partially to PDEδ, as indicated by partially suppressed growth inhibition (Fig.\xa0<xref rid="41401_2019_268_Fig2_HTML" ref-type="fig">2a</xref>, upper panel); however, PDEδ was similarly depleted in all the tested cells (Fig.\xa0, upper panel); however, PDEδ was similarly depleted in all the tested cells (Fig.\xa0<xref rid="41401_2019_268_Fig2_HTML" ref-type="fig">2a</xref>, lower panel). In KRAS-independent cells, as expected, none of the cells responded to PDEδ depletion (Fig.\xa0, lower panel). In KRAS-independent cells, as expected, none of the cells responded to PDEδ depletion (Fig.\xa0<xref rid="41401_2019_268_Fig2_HTML" ref-type="fig">2a</xref>).).Fig. 2MAPK/ERK signaling alteration is associated with the response to PDEδ inhibition in KRAS-dependent cancer cells. a KRAS mutant cell viability upon PDEδ \xa0depletion. KRAS-dependent cells were treated with PDEδ siRNAs (siPDE6D #1, #2) for 96\u2009h. Cell viability was measured by crystal violet staining assay (upper panel). siRNA interference efficiency was measured by immunoblotting (lower panel). The error bars represent the mean\u2009±\u2009SD of four replicates. *P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001, n.s., not significant. b, c MAPK and AKT signaling changes upon PDEδ depletion in PDEδ-dependent (b) and PDEδ-independent cells (c). Cells were treated with siRNAs (siPDE6D #1, #2) for 48\u2009h before being subjected to immunoblotting analysis. NC, scrambled siRNA used as a negative control', 'We asked whether the differential cell response upon PDEδ knockdown could possibly result from the differential impact on MAPK/ERK signaling. By simultaneously analyzing the MAPK/ERK pathway in PDEδ-responsive (red) and nonresponsive cells (blue), we discovered that siRNA depletion of PDEδ effectively diminished p-S6K, a surrogate marker for PDEδ function [12, 13, 16]. However, MAPK/ERK signaling was only decreased in PDEδ-responsive cells (Fig.\xa0<xref rid="41401_2019_268_Fig2_HTML" ref-type="fig">2b</xref>). For example, in A549, H460, and SW1573 cells, phosphorylation levels of c-RAF and ERK were effectively decreased. In contrast, in the rest of the cells in which PDEδ knockdown barely affected cell growth (Fig.\xa0). For example, in A549, H460, and SW1573 cells, phosphorylation levels of c-RAF and ERK were effectively decreased. In contrast, in the rest of the cells in which PDEδ knockdown barely affected cell growth (Fig.\xa0<xref rid="41401_2019_268_Fig2_HTML" ref-type="fig">2c</xref>), alterations to RAF/MEK/ERK signaling were not observed. These results suggested that cell growth inhibition was associated with diminished MAPK/ERK signaling.), alterations to RAF/MEK/ERK signaling were not observed. These results suggested that cell growth inhibition was associated with diminished MAPK/ERK signaling.'], '41401_2019_268_Fig3_HTML': ['The assay was also conducted in AsPC-1 cells, in which PDE6D siRNA failed to inhibit cell growth or disrupt MAPK/ERK signaling. Interestingly, in PDEδ-nonresponsive cells, KRAS membrane localization was similarly impaired (Fig.\xa0<xref rid="41401_2019_268_Fig3_HTML" ref-type="fig">3a–c</xref>). These results indicated that KRAS plasma membrane association was impaired upon PDEδ depletion in PDEδ-nonresponsive cells, suggesting that the sustained MAPK/ERK pathway activation could possibly result from compensatory upstream signaling.). These results indicated that KRAS plasma membrane association was impaired upon PDEδ depletion in PDEδ-nonresponsive cells, suggesting that the sustained MAPK/ERK pathway activation could possibly result from compensatory upstream signaling.'], '41401_2019_268_Fig4_HTML': ['To explore the possible involvement of compensatory pathways, we used a phospho-RTK antibody and phospho-kinase antibody arrays, which covered 49 phosphorylated human receptor tyrosine kinases (RTKs) and 43 intracellular kinase phosphorylation sites, respectively. This approach allowed us to screen the kinases that were activated upon PDEδ disruption. At 48\u2009h following PDEδ knockdown, we observed the feedback activation of several protein kinases, including IGF1R, EPHA2, GSK3α/β, and WNK-1, in H358 cells, which were shown to be nonresponsive to PDEδ knockdown (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4a, b</xref>). Among these altered kinases, the upregulation of IGF1R and EPHA2 induced by siPDE6D treatment was confirmed in an independent immunoblotting analysis (Fig.\xa0). Among these altered kinases, the upregulation of IGF1R and EPHA2 induced by siPDE6D treatment was confirmed in an independent immunoblotting analysis (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4c</xref>).).Fig. 4Feedback activation of EPHA2 results in compensatory MAPK activation in PDEδ-depleted cells. a, b Phospho-RTK antibody and phospho-kinase antibody arrays. Cells were treated with siPDE6D (#1) for 48\u2009h, and the cell lysates were subjected to phospho-RTK antibody array (a) or phospho-kinase antibody array (b). c Immunoblotting analysis confirmed the feedback activation of EPHA2 and IGF1R in PDEδ-depleted cells. H358 cells were treated with the indicated siRNA for 48\u2009h before being subjected to immunoblotting. d MAPK signaling upon concurrent inhibition of PDEδ and EPHA2. Cells were transfected with the indicated siRNAs for 48\u2009h before exposure to the EPHA2 inhibitor ALW-II-41-27 for 4\u2009h. e MAPK signaling upon concurrent inhibition of PDEδ and IGF1R. Cells were transfected with the indicated siRNAs for 48\u2009h before exposure to the IGF1R inhibitor BMS-754807 for 5\u2009h. f, g Cell viability upon concurrent inhibition of PDEδ and MEK or EPHA2. KRAS-dependent Panc 10.05 cells were treated with PDEδ siRNA in combination with the MEK inhibitor AZD6244 or the EPHA2 inhibitor ALW-II-41-27 for 96\u2009h. Cell viability was measured by crystal violet staining assay. NC, scrambled siRNA used as a negative control. h A schematic model', 'We next tested whether the feedback activation of IGF1R and EPHA was responsible for persistent MAPK/ERK activation upon PDE6D knockdown. To this end, cells were treated simultaneously with IGF1R\xa0or\xa0EPHA2 inhibitors [17, 18] and PDE6D siRNA. MAPK/ERK signaling was examined using an immunoblotting analysis. It was observed that the EPHA2 inhibitor ALW-II-41-27 could diminish the MAPK/ERK pathway signaling in a panel of PDEδ-nonresponsive cell lines, including H358, Panc 02.03, Panc 10.05, and AsPC-1 cells, in which PDE6D siRNA alone failed to reduce KRAS-induced MAPK/ERK signaling (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4d;</xref>\n\nS2a). In contrast, treatment with the IGF1R inhibitor BMS-754807 barely affected the phosphorylation of c-RAF and ERK upon PDEδ knockdown (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4e;</xref>\n\nS2b).', 'These results suggested that the feedback activation of EPHA2 activated the MAPK/ERK pathway upon PDEδ disruption. We further applied the EPHA2 inhibitor ALW-II-41-27 or EPHA2 siRNA to test whether the cells could be re-sensitized to PDEδ inhibition. Indeed, we discovered that while PDEδ depletion alone barely affected cell viability, combined treatment with ALW-II-41-27 (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4g</xref>) and EPHA2 siRNA (Fig.\xa0) and EPHA2 siRNA (Fig.\xa0S2c) largely inhibited the growth of Panc 10.05 cells. Similar results were obtained by combining a MEK inhibitor and PDE6D siRNA (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4f</xref>).).', 'Targeting KRAS plasma membrane association has been recently proposed for exploration as a new therapeutic opportunity for KRAS mutant cancer, which highlights PDEδ as an attractive target to impede KRAS oncogenic signaling. However, the current understanding of this new strategy is very limited, especially considering that the complex signaling network of PDEδ is known to be involved. In addition, given the heterogeneous properties of KRAS mutant cancer, it is conceivable that PDEδ inhibition may not yield therapeutic benefits in broad KRAS mutant contexts. It will be important to stratify the subset responsive to PDEδ inhibition. In this study, we discovered that only a fraction of KRAS-dependent cells responds to PDEδ inhibition, although KRAS plasma membrane association is efficiently impaired. The feedback activation of EPHA2 accounts for the limited response to PDEδ inhibition via a mechanism that involves the compensatory activation of the\xa0MAPK/ERK signaling (Fig.\xa0<xref rid="41401_2019_268_Fig4_HTML" ref-type="fig">4h</xref>). These insights help gain\xa0a better understanding of the PDEδ-targeted therapeutic strategy and suggest the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.). These insights help gain\xa0a better understanding of the PDEδ-targeted therapeutic strategy and suggest the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.']}
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EPHA2 feedback activation limits the response to PDEδ inhibition in KRAS-dependent cancer cells
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[
"KRAS",
"anticancer therapy",
"PDEδ",
"EPHA2",
"RAF/MEK/ERK signaling"
] |
Acta Pharmacol Sin
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1581926400
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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[] |
other
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PMC7471410
| null |
0
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[] |
Acta Pharmacol Sin. 2020 Feb 17; 41(2):270-277
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NO-CC CODE
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BIK1-GFP is functional in plants and undergoes endocytosis.a. BIK1-GFP is functional as confirmed by BIK1 phosphorylation in 35S::BIK1-GFP expressing Col-0 cotyledons after 1 μM flg22 treatment. MPK6 is a loading control and the black stippled line indicates discontinuous segments from the same gel. b. BIK1-GFP restored ROS production in bik1 upon flg22 treatment. Leaf discs from WT, bik1 and BIK1-GFP complementation (Line 1 and 2) were treated with 100 nM flg22 for ROS measurement using a luminometer over 50 min. Data are shown as means ± SEM (WT, bik1: n = 42; BIK1-GFP/bik1 n =45). c. Time-lapse SDCM shows that BIK1-GFP endosomal puncta are highly mobile with puncta that disappear (red circle), appear (yellow circle), and rapidly move in and out of the plane of view (white circle). Scale bar, 5 μm. d-k. BIK1-GFP localizes to endosomal puncta and PM in cross-sectional images of epidermal cells. The abaxial epidermal cells of cotyledons expressing BIK1-GFP were imaged with SDCM with a Z-step of 0.3 μm. A subset of the cross-sectional images is shown at the indicated depth (3, 6, 9, 12, 15, 18 and 21 μm) along with the maximum-intensity projection of all 67 images through epidermis. BIK1-GFP localizes to both PM and endosomal puncta (white arrows) within all sections. k-p. Quantification method for BIK1-GFP puncta within maximum-intensity projections of SDCM images. k. Maximum-intensity projections (MIP) were generated using Fiji distribution of ImageJ 1.51 (https://fiji.sc/) for each Z-series captured by SDCM imaging of BIK1-GFP cotyledons. l. Regions of MIP with non-pavement cells (e.g. stomata) are removed from the image using the Line Draw and Crop functions. The total surface area (μm2) of the image is measured using the Analyze Measure function. m. Puncta within the cropped MIP are recognized using a customized model generated and applied with the Trainable Weka Segmentation plug-in for Fiji. The same model is applied to all images to generate binary images showing the physical location of each BIK1-GFP puncta (black). n-o. Puncta within the size range of 0.1–2.5 μm2 are highlighted in green (n) and counted (o) using the Analyze Particles function in Fiji. BIK1-GFP endocytosis is quantified as the number of puncta per 1000 μm2. p. An overlay of the BIK1-GFP puncta (yellow highlight) over the cropped MIP confirms correct identification of puncta. The experiments in a-c were repeated three times with similar results.
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nihms-1565517-f0005
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2
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fb4269455917eb9f38c53e828d00e3c89a0644d01a0d63e7a760d3c369d492de
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nihms-1565517-f0005.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
1956,
1981
] |
[{'image_id': 'nihms-1565517-f0004', 'image_file_name': 'nihms-1565517-f0004.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0004.jpg', 'caption': 'RHA3A/B-mediated BIK1 monoubiquitination contributes to its function in immunity and endocytosis.a. The pBIK1::BIK19KR-HA/bik1 transgenic plants (Line 1 & 2) cannot complement bik1 for flg22-induced ROS production. Data are shown as overlay of dot plot and means of total photon count ± SEM (One-way ANOVA, WT, BIK1/bik1: n=53; bik1: n=54; BIK19KR/bik1: n=55). In all panels, data are shown as overlay of dot plot and means ± SEM, lines beneath p values indicate relevant pair-wise comparisons. b. The pBIK1::BIK19KR-HA/bik1 transgenic plants show increased bacterial growth of Pst DC3000 hrcC−. Plants were spray-inoculated and bacterial growth was measured at four days post-inoculation (dpi). One-way ANOVA, n=6. c. The amiRNA-RHA3A/B plants show reduced flg22-induced ROS production. One-way ANOVA, n=51. d. The amiRNA-RHA3A/B plants show increased bacterial growth of Pst DC3000. Plants were hand-inoculated and bacterial growth was measured at two dpi. One-way ANOVA, n=5. e. f. Flg22-induced BIK1, BIK19KR, and FLS2 endocytosis in N. benthamiana leaf epidermal cells. BIK1-TagRFP or BIK19KR-TagRFP was co-expressed with FLS2-YFP followed by 100 μM flg22 treatment and imaged at the indicated time points by a confocal microscopy (e). Scale bars, 20 μm. Quantification of BIK1-TagRFP (magenta) and FLS2-YFP (green) puncta is shown in (f). One-way ANOVA, additional images and n values are shown in Extended Data Fig. 9c. g. BIK19KR does not enable flg22-induced BIK1 dissociation from FLS2. Co-IP was performed using protoplasts expressing FLS2-HA and BIK1-FLAG, or BIK19KR-FLAG, followed by 1 μM flg22 treatment for 15 min (top). BIK1 interaction with FLS2 was quantified as intensity from IP: α-FLAG, IB: α-HA divided by intensity from IP: α-FLAG, IB: α-FLAG (bottom). Data are shown as means ± SEM of fold change (no treatment as 1.0) (One-way ANOVA, n=3). All experiments were repeated three times with similar results.', 'hash': '27d419c39e29303432add9d88849768520b8f0b522a6599e1b4f3cc377930932'}, {'image_id': 'nihms-1565517-f0003', 'image_file_name': 'nihms-1565517-f0003.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0003.jpg', 'caption': 'Identification of RHA3A-mediated BIK1 ubiquitination sites.a. BIK1 is ubiquitinated by RHA3A at multiple lysine residues. Ubiquitinated lysine residues with a diglycine remnant by LC-MS/MS analysis are marked as red with amino acid positions. b. MS/MS spectrum of the peptide containing K358 is shown. c. Structure of BIK1 labeled with six lysine identified as ubiquitination sites. Structural information was obtained from protein data bank (PDB ID: 5TOS) and analyzed by PyMOL. d. BIK19KR is compromised in flg22-induced ubiquitination. FLAG-UBQ and HA-tagged BIK1 mutants were expressed in protoplasts followed by 100 nM flg22 for 30 min. Quantification of BIK1 ubiquitination fold change is shown as overlay of dot plot and means ± SEM (middle). Different letters indicate significant difference with others (P<0.05, One-way ANOVA, n=3). Mutated lysine for BIK1 mutants are in red (bottom). e. RHA3A is unable to ubiquitinate BIK19KR. The assay was performed as in Fig. 2d. f. BIK19KR exhibits normal in vitro kinase activity. The kinase assay was performed using GST-BIK1 or GST-BIK19KR as the kinase and GST or GST-BAK1K (kinase domain) as the substrate. The experiments except MS analyses were repeated three times with similar results.', 'hash': '97118dd14afe16062d5400ec4a09fa5a394d1c6e8f8d2890256a844e66aa8b25'}, {'image_id': 'nihms-1565517-f0012', 'image_file_name': 'nihms-1565517-f0012.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0012.jpg', 'caption': 'BIK1 monoubiquitination is required for plant defense and flg22 signaling.a. BIK19KR undergoes phosphorylation similar to BIK1 upon flg22 treatment. BIK1-HA or BIK19KR-HA was expressed in WT protoplasts followed by 100 nM flg22 treatment for indicated time points. Band-shift of BIK1 was examined by IB with α-HA antibody. b. BIK19KR interacts with RHA3A in a Co-IP assay. RHA3A-HA was co-expressed with BIK1-FLAG or BIK19KR-FLAG in protoplasts followed by 100 nM flg22 treatment for 15 min. Co-IP assay was carried out with α-FLAG agarose and immunoprecipitated proteins were immunoblotted with α-HA or α-FLAG antibody (top two panels). Bottom two panels show BIK1-FLAG/BIK19KR-FLAG and RHA3A-HA proteins. c. Transgenic plants with BIK19KR overexpression in WT background show similar MAPK activation as WT. Eleven-day-old seedlings of WT or 35S::BIK19KR-HA/WT transgenic plants (Line 55 and 56) were treated with 200 nM flg22 for 15 min. MAPK activation was analyzed with α-pERK antibody (top), and protein loading is shown by CBB staining for RBC (bottom). d. Transgenic plants with BIK19KR overexpression in WT background show similar flg22-induced ROS production as WT. Leaf discs from the indicated genotypes were treated with 100 nM flg22, and ROS production was measured as relative luminescence units by a luminometer over 50 min. Data are shown as overlay of dot plot and means of total photon count ± SEM (One-way ANOVA, n=16). e. Growth phenotype of pBIK1::BIK1-HA/bik1 and pBIK1::BIK19KR-HA/bik1 transgenic plants. Five-week-old soil-grown plants are shown. Scale bar, 1 cm. f. Expression of BIK1-HA or BIK19KR-HA in transgenic plants. Total proteins from leaves of four-week-old transgenic plants were subjected to α-HA IB (top). Bottom panel shows CBB staining for RBC. g. RHA3A and RHA3B play a role in disease resistance to Pst DC3000 hrcC− infection. Plants were spray-inoculated with Pst DC3000 hrcC− and bacterial growth was measured at four days post-inoculation (dpi). Data are shown as overlay of dot plot and means ± SEM (One-way ANOVA, n=6). h. RHA3A and RHA3B play a role in disease resistance to Botrytis. Four-week-old plant leaves were deposited with 10 μL of B. cinerea BO5 at a concentration of 2.5×105 spores per mL. Disease symptom was recorded, and the lesion diameter was measured at two dpi. Data are shown as overlay of dot plot and means ± SEM (One-way ANOVA, n=34). i. ROS production is reduced in rha3a/b. Leaf discs from WT or rha3a/b were treated with 100 nM flg22 for ROS production over 50 min. Data are shown as overlay of dot plot and means of total photon count ± SEM (Two-tailed Student’s t-test, n=36 for WT and n=32 for rha3a/b). j. RHA3A and RHA3B play a role in disease resistance to Pst DC3000. Plants were spray-inoculated with Pst DC3000 and bacterial growth was measured at three dpi. Data are shown as overlay of dot plot and means ± SEM (Two-tailed Student’s t-test, n=9). The experiments were repeated three times with similar results.', 'hash': '552dcf4bedff0d0d0f8098fd7442cd899f48f64d39696fac5090c43e2efa137c'}, {'image_id': 'nihms-1565517-f0002', 'image_file_name': 'nihms-1565517-f0002.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0002.jpg', 'caption': 'E3 ligases RHA3A/B interact with and monoubiquitinate BIK1.a. Domain organization of RHA3A/B. TD: transmembrane domain; RING: E3 catalytic domain; RHA3ACD: cytoplasmic domain. Amino acid positions and the sequence of RING domain are shown. Cysteine (C) and histidine (H) residues which coordinate zinc are underlined. Isoleucine (I) involved in E2-RING interaction is labeled with *. b. BIK1 interacts with RHA3A. GST or GST-BIK1 proteins immobilized on glutathione sepharose beads were incubated with MBP or MBP-RHA3ACD-HA proteins. Washed beads were subjected to IB with α-MBP or α-GST (top two panels). Input proteins are shown by IB (middle two panels) and Coomassie Brilliant Blue (CBB) staining (bottom). c. BIK1 associates with RHA3A. Transgenic plants carrying pBIK1::BIK1-HA and pRHA3A::RHA3A-FLAG (Line 7 and 10) were used for Co-IP assay with α-FLAG agarose and immunoprecipitated proteins were immunoblotted with α-HA or α-FLAG (top two panels). Bottom two panels show BIK1-HA and RHA3A-FLAG expression. d. RHA3A ubiquitinates BIK1. GST-RHA3ACD or its I104A mutant was used in the ubiquitination reaction containing GST-BIK1-HA, E1, E2, and ATP. e. RHA3A/B are required for BIK1 ubiquitination. The rha3a/b and rha3a plants were used for protoplast isolation followed by transfection of BIK1-HA and FLAG-UBQ. The experiments were repeated three times with similar results.', 'hash': '8aa7e343febea9a39c80e161739b48b652261cc6de6fc6a363562af2abf0e9ec'}, {'image_id': 'nihms-1565517-f0005', 'image_file_name': 'nihms-1565517-f0005.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0005.jpg', 'caption': 'BIK1-GFP is functional in plants and undergoes endocytosis.a. BIK1-GFP is functional as confirmed by BIK1 phosphorylation in 35S::BIK1-GFP expressing Col-0 cotyledons after 1 μM flg22 treatment. MPK6 is a loading control and the black stippled line indicates discontinuous segments from the same gel. b. BIK1-GFP restored ROS production in bik1 upon flg22 treatment. Leaf discs from WT, bik1 and BIK1-GFP complementation (Line 1 and 2) were treated with 100 nM flg22 for ROS measurement using a luminometer over 50 min. Data are shown as means ± SEM (WT, bik1: n = 42; BIK1-GFP/bik1 n =45). c. Time-lapse SDCM shows that BIK1-GFP endosomal puncta are highly mobile with puncta that disappear (red circle), appear (yellow circle), and rapidly move in and out of the plane of view (white circle). Scale bar, 5 μm. d-k. BIK1-GFP localizes to endosomal puncta and PM in cross-sectional images of epidermal cells. The abaxial epidermal cells of cotyledons expressing BIK1-GFP were imaged with SDCM with a Z-step of 0.3 μm. A subset of the cross-sectional images is shown at the indicated depth (3, 6, 9, 12, 15, 18 and 21 μm) along with the maximum-intensity projection of all 67 images through epidermis. BIK1-GFP localizes to both PM and endosomal puncta (white arrows) within all sections. k-p. Quantification method for BIK1-GFP puncta within maximum-intensity projections of SDCM images. k. Maximum-intensity projections (MIP) were generated using Fiji distribution of ImageJ 1.51 (https://fiji.sc/) for each Z-series captured by SDCM imaging of BIK1-GFP cotyledons. l. Regions of MIP with non-pavement cells (e.g. stomata) are removed from the image using the Line Draw and Crop functions. The total surface area (μm2) of the image is measured using the Analyze Measure function. m. Puncta within the cropped MIP are recognized using a customized model generated and applied with the Trainable Weka Segmentation plug-in for Fiji. The same model is applied to all images to generate binary images showing the physical location of each BIK1-GFP puncta (black). n-o. Puncta within the size range of 0.1–2.5 μm2 are highlighted in green (n) and counted (o) using the Analyze Particles function in Fiji. BIK1-GFP endocytosis is quantified as the number of puncta per 1000 μm2. p. An overlay of the BIK1-GFP puncta (yellow highlight) over the cropped MIP confirms correct identification of puncta. The experiments in a-c were repeated three times with similar results.', 'hash': 'fb4269455917eb9f38c53e828d00e3c89a0644d01a0d63e7a760d3c369d492de'}, {'image_id': 'nihms-1565517-f0014', 'image_file_name': 'nihms-1565517-f0014.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0014.jpg', 'caption': 'Monoubiquitination mediates BIK1 release from PM upon ligand perception.a. PYR-41 impairs flg22-induced BIK1 dissociation from FLS2. FLS2-HA was co-expressed with BIK1-FLAG or Ctrl in protoplasts. After pretreatment with 50 μM PYR-41 for 30 min, protoplasts were stimulated with 100 nM flg22 for 15 min. Co-IP and IB were performed as in Fig. 4g. b. A working model of RHA3A/B-mediated BIK1 monoubiquitination in plant immunity. During non-activated, steady state condition (0 min), BIK1 remains hypo-phosphorylated and associates with FLS2 and BAK1. Upon flg22 perception, FLS2 dimerizes with BAK1, which stimulates BIK1 phosphorylation (<1 min). The phosphorylated BIK1 is monoubiquitinated by E3 ligases RHA3A and RHA3B, leading to BIK1 dissociation from FLS2-BAK1 complex, accompanied by endocytosis (10–20 min). Ligand-induced BIK1 monoubiquitination contributes to the activation of ROS and other defense responses. FLS2 is polyubiquitinated and endocytosed 40 min after flg22 perception for signaling attenuation. c. BIK19KR shows comparable protein level as BIK1 in transgenic plants. 35S::BIK1-HA or 35S::BIK19KR-HA transgenic plants in WT background were used for IB detecting BIK1 proteins by α-HA antibody. Ctrl is empty vector transgenic plants. d. BIK1 and BIK19KR protein stability after cycloheximide treatment. BIK1-HA or BIK19KR-HA was expressed in WT protoplasts for 12 hr followed by 500 μg/ml cycloheximide (CHX) treatment for the indicated time. BIK1 or BIK19KR proteins were analyzed by IB with α-HA antibody. * indicates CHX was added right after transfection, thus blocking protein synthesis. The experiments were repeated three times with similar results.', 'hash': '998ed129966bce43b8ff6ee97de8df7946d9bffedbae95b8e9d19aebf95aabee'}, {'image_id': 'nihms-1565517-f0013', 'image_file_name': 'nihms-1565517-f0013.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0013.jpg', 'caption': 'The BIK19KR mutation impairs flg22-induced BIK1 endocytosis.a. b. BIK19KR-GFP-labelled puncta colocalize less with FM4–64 than BIK1-GFP upon flg22 treatment. Five-day-old seedlings of 35S::BIK1-GFP or 35S::BIK19KR-GFP were pretreated with FM4–64 (2 μM) for 15 min and elicited with 100 nM flg22 for the indicated time points and fluorescence was detected in epidermis by a confocal microscopy (a). White arrow points to colocalized endosomes. Scale bars, 20 μm. Percentage of BIK1-GFP or BIK19KR-GFP and FM4–64 positive endosomes over time per 100% of image area is shown in (b). Data are shown as overlay of dot plot and means ± SEM (Two-tailed Student’s t-test, n = 21 images for BIK1-GFP and n= 16, 15 images for 10, 15 min respectively for BIK19KR-GFP). c. Flg22-induced BIK1, BIK19KR and FLS2 endocytosis in N. benthamiana. BIK1-TagRFP (BIK1-RFP) or BIK19KR-TagRFP (BIK19KR-RFP) was co-expressed with FLS2-YFP in N. benthamiana, infiltrated with 100 μM flg22 and imaged at the indicated time points by a confocal microscopy. The images of 30–40, 40–50 and 50–60 min after flg22 treatment from Fig. 4e are shown here. Scale bars, 20 μm. For BIK1-RFP/FLS2-YFP, n= 14, 11, 7, 10, 10, 6, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min; for BIK19KR-RFP/FLS2-YFP, n= 19, 11, 11, 9, 16, 12, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min respectively. d. Percentage of BIK1-RFP puncta colocalizing with FLS2-YFP after flg22 treatment for the indicated time points in Fig. 4e and Extended Data Fig. 9c. Data are shown as overlay of dot plot and means ± SEM (n= 14, 11, 7, 10, 10, 6, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min respectively). e-f BIK19KR-RFP shows reduced co-localization with ARA6-YFP. BIK1-RFP or BIK19KR-RFP was transiently expressed with ARA6-YFP in N. benthamiana, and the images were taken 48–72 hr after infiltration (e). Scale bars, 10 μm. Percentage of BIK1-RFP puncta colocalizing with ARA6-YFP is shown in f. Data are shown as overlay of dot plot and means ± SEM (Two-tailed Student’s t-test, n= 9 images for BIK1-RFP; n= 10 images for BIK19KR-RFP). The experiments were repeated three times with similar results.', 'hash': '72f6c5a9eaa226f2728a2a0d9891d031340792e4bb7ed034fcd3aeac90820f0c'}, {'image_id': 'nihms-1565517-f0010', 'image_file_name': 'nihms-1565517-f0010.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0010.jpg', 'caption': 'BIK1 in vitro ubiquitination sites identified by mass spectrometry.MS/MS spectrums of peptides containing ubiquitinated lysine residues of BIK1 are shown from a to i. a. K31; b. K41; c. K95; d. K106; e. K170; f. K186; g. K286; h. K337; i. K366. MS spectrums are outputs from the SEQUEST program. MS analysis was performed once.', 'hash': '034c7080212b912c39b6a74928e3246b1da2e1a9454f2006c823eaa84ba46929'}, {'image_id': 'nihms-1565517-f0006', 'image_file_name': 'nihms-1565517-f0006.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0006.jpg', 'caption': 'MAMP-triggered BIK1 family RLCK monoubiquitination.a. Flg22 induces monoubiquitination of BIK1. Protoplasts from WT plants were transfected with BIK1-HA and FLAG-UBQ or a vector (Ctrl), and followed by treatment with 100 nM flg22 for 30 min. After IP with α-FLAG, ubiquitinated BIK1 was detected by IB using α-HA antibody (top). The middle panel shows BIK1-HA proteins and the bottom shows Coomassie Brilliant Blue (CBB) staining for RuBisCO (RBC). b. Flg22 induces BIK1 monoubiquitination in pBIK1::BIK1-HA transgenic plants. Protoplasts from pBIK1::BIK1-HA/bik1 transgenic plants were transfected with FLAG-UBQ and followed by 100 nM flg22 treatment for 30 min. After IP with α-FLAG agarose, ubiquitinated BIK1 was detected by IB with α-HA antibody (top). The bottom panel shows BIK1-HA proteins. c. BAK1 is constitutively polyubiquitinated in vivo. Protoplasts from WT plants were transfected with BAK1-HA and FLAG-UBQ or control, and followed by 100 nM flg22 treatment for 30 min. IP was carried out with α-FLAG agarose (IP: α-FLAG). Ubiquitinated BAK1 (Ub-BAK1) proteins were detected as a smear with α-HA IB (IB: α-HA) (Top). The middle panel shows BAK1-HA proteins and the bottom is CBB staining for RBC. d. Flg22 induces FLS2 polyubiquitination. Protoplasts from WT plants were transfected with FLS2-HA and FLAG-UBQ and followed by 100 nM flg22 treatment for 30 min. e, f. Monoubiquitination of BIK1 with UBQK0. Protoplasts from pBIK1::BIK1-HA (e) or 35S::BIK1-HA (f) transgenic plants were transfected with FLAG-UBQK0 (all lysine residues mutated to arginine) and followed by 100 nM flg22 treatment for 30 min. The mutations of lysine-to-arginine in UBQK0 are shown on top with amino-acid positions labeled (e). g. PYR-41 blocks flg22-induced BIK1 monoubiquitination. PYR-41 (50 μM) was added 30 min prior to flg22 treatment. h. Flg22 induces BIK1 monoubiquitination in the presence of MG132. MG132 (2 μM) was added 1 hr or 2.5 hr before adding flg22. i. Flg22-induced BIK1 monoubiquitination depends on FLS2 and BAK1. Protoplasts isolated from WT, fls2 and bak1–4 plants were transfected with BIK1-HA and FLAG-UBQ, and followed by treatment with 100 nM flg22 for 30 min. j. Elf18, pep1 and chitin induce BIK1 monoubiquitination. 1 μM elf18, 200 nM pep1 or 100 μg/ml chitin was added for 30 min. k. BIK1 homolog PBL1 is monoubiquitinated upon flg22 treatment. PBL1-HA and FLAG-UBQ were expressed in protoplasts. l. Flg22 induces monoubiquitination of BIK1 family RLCK PBL10, but not BSK1. HA-tagged PBL10 or BSK1 was expressed with FLAG-UBQ in WT protoplasts. The experiments were repeated at least three times with similar results.', 'hash': '5d44926f119f72e1efe5e605d42e2071993d939369d7898bbd64b36c66f4b485'}, {'image_id': 'nihms-1565517-f0001', 'image_file_name': 'nihms-1565517-f0001.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0001.jpg', 'caption': 'MAMP-induced BIK1 endocytosis and monoubiquitination.a. BIK1-GFP localizes to cell periphery and intracellular puncta (zoom insert) in maximum intensity projections of cotyledon epidermal cells. Scale bar, 10 μm. b. BIK1-GFP colocalizes with FM4–64 in PM (star) and intracellular puncta (arrowheads). Scale bar, 5 μm. BIK1-GFP/FM4–64 Pearson’s correlation coefficient = 0.55 ± 0.14 (n=35). c-d. BIK1 and FLS2 puncta increase after 1 μM flg22 treatment. Data are shown as overlay of dot plot and means ± SEM. n=56, 48, 49, 47 images for 0, 3–17, 18–32, 33–45 min respectively for BIK1-GFP (c) and n=24, 15, 21, 36, 34, 39, 39 images for 0, 5–15, 20–30, 35–45, 50–60, 65–75, 80–90 min respectively for FLS2-GFP (d). Scale bar, 5 μm. e. Flg22 induces BIK1 monoubiquitination. Protoplasts from WT plants were transfected with BIK1-HA and FLAG-UBQ, and treated with 100 nM flg22 for 30 min. After IP with α-FLAG agarose (IP: α-FLAG), ubiquitinated BIK1 was detected by immunoblot (IB) using α-HA antibody (IB: α-HA) (Lane 1 & 2) or with GST-Usp2-cc treatment (Lane 3). Heat inactivated (HI) Usp2-cc is a control (Lane 4). Bottom panel shows BIK1-HA protein expression. Molecular weight (kDa) was labeled on the left of all immunoblots. f. Time course of flg22-induced BIK1 phosphorylation and ubiquitination. Protoplasts expressing FLAG-UBQ and BIK1-HA were treated with 100 nM flg22 for the indicated time. BIK1 band intensities were quantified with Image Lab (Bio-Rad). Quantification of BIK1 phosphorylation (underneath bottom panel) is the ratio of intensities of the upper bands (pBIK1) to the sum intensities of shifted and non-shifted bands (pBIK1+BIK1). Quantification of BIK1 ubiquitination (underneath upper panel) is the relative intensity (fold change) of Ub-BIK1 bands (no treatment as 1.0). g. BIK1 variants with impaired phosphorylation compromise flg22-induced ubiquitination. All experiments were repeated at least three times with similar results.', 'hash': 'dd5cd258ed89005aef030c5f0fa3d902de6d0b383d96bf501b738a68c80331d5'}, {'image_id': 'nihms-1565517-f0008', 'image_file_name': 'nihms-1565517-f0008.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0008.jpg', 'caption': 'RHA3A/B interacts with BIK1 in vivo.a. BIK1 interacts with RHA3A in a Co-IP assay. RHA3A-HA was co-expressed with BIK1-FLAG or Ctrl in protoplasts followed by 100 nM flg22 treatment for 15 min. The Co-IP assay was carried out with α-FLAG agarose and immunoprecipitated proteins were immunoblotted with α-HA or α-FLAG antibody (left). The right panels show BIK1-FLAG and RHA3A-HA proteins. b. RHA3A expression in pRHA3A::RHA3A-FLAG/pBIK1::BIK1-HA transgenic plants. qRT-PCR was carried out to detect RHA3A transcripts using ACTIN2 as a control. The relative gene expression from WT (set as 1), pBIK1::BIK1-HA (Ctrl) and two independent transgenic lines (line 7 and 10) is shown. Data are shown as means ± SEM (One-way ANOVA, n=3). c. BIK1 associates with RHA3B independent of flg22 treatment. RHA3B-HA was co-expressed with BIK1-FLAG or Ctrl in protoplasts followed by 100 nM flg22 treatment for 15 min. Co-IP assay was carried out with α-FLAG agarose and immunoprecipitated proteins were immunoblotted with α-HA or α-FLAG antibody (left). Right panels show BIK1-FLAG and RHA3B-HA proteins before IP. d. FLS2 interacts with RHA3A and RHA3B in a Co-IP assay. The experiments were repeated three times with similar results.', 'hash': '6c139796c10b9044f7029623274a0160d5c85baa5bb9ca552a38587200463272'}, {'image_id': 'nihms-1565517-f0011', 'image_file_name': 'nihms-1565517-f0011.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0011.jpg', 'caption': 'BIK1 in vivo ubiquitination sites identified by mass spectrometry.a. Ubiquitinated BIK1-GFP in planta was immunoprecipitated for LC-MS/MS analysis. BIK1-GFP and FLAG-UBQ were co-expressed in WT protoplasts (~4 × 106 cells) followed by 200 nM flg22 treatment for 30 min. Ubiquitinated BIK1 was immunoprecipitated by GFP-trap-agarose, separated by SDS-PAGE, digested by trypsin and subjected to LC-MS/MS analysis. Portions of cell lysates were examined for BIK1-GFP expression (left), and immunoprecipitates were analyzed by SDS-PAGE following silver staining (middle, right for longer exposure of the same gel) and SDS-PAGE following CBB staining (right). The highlighted area was cut and analyzed by MS. b. BIK1 is ubiquitinated in vivo. Ubiquitinated lysine containing a diglycine remnant by LC-MS/MS analysis are marked as red with amino acid positions. c to h. MS/MS spectrums of peptides containing ubiquitinated lysine of BIK1 are shown. c. K31; d. K41; e. K95; f. K337; g. K358; h. K366. MS spectrums are outputs from the SEQUEST program. MS analysis was performed once.', 'hash': '85ae8ac65d0466e49cd2fe0b92f93d326ed053d2c78846b178b87f75b94977e1'}, {'image_id': 'nihms-1565517-f0009', 'image_file_name': 'nihms-1565517-f0009.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0009.jpg', 'caption': 'RHA3A/B ubiquitinate BIK1 in vivo.a. GST-RHA3ACD possesses E3 ligase activity in vitro. The in vitro ubiquitination assay was performed with GST-RHA3ACD followed by deubiquitination reactions with GST-Usp2-cc. N-Ethylmaleimide (NEM) (10 mM), an inhibitor of deubiquitinases, and heat-inactivated (HI, 95°C for 5 min) Usp2-cc are controls. Samples were analyzed by SDS-PAGE and silver staining. b. GST-RHA3ACD possesses multi-monoubiquitination activity in vitro. Ubiquitination assay was done similarly as (a) except using the ubiquitin mutant with all lysine residues mutated to arginine (UBQK0). Ubiquitinated proteins were detected by IB with α-UBQ (left) or α-RHA3A (right) antibody. c. RHA3 expression in T-DNA insertion mutants. RHA3A expression in T-DNA knockout line SALK_052714 and RHA3B expression in SALK_064303 were analyzed as in Extended Data Fig. 4b. Data are shown as means ± SEM of fold change (WT as 1.0) (Two-tailed Student’s t-test, n=3). d. Screen for the optimal amiR-RHA3A and amiR-RHA3B. Protoplasts were transfected with RHA3A-HA or RHA3B-HA with Ctrl, amiR-RHA3A or amiR-RHA3B. RHA3A or RHA3B proteins were examined by IB with α-HA antibody. e. RHA3A and RHA3B are required for BIK1 ubiquitination in vivo. BIK1 ubiquitination assay was carried out by co-expressing Ctrl, artificial microRNA targeting RHA3A (amiR-RHA3A) or together with microRNA targeting RHA3B (amiR-RHA3A amiR-RHA3B). f. RHA3A and RHA3B expression in amiR-RHA3A/B transgenic plants. qRT-PCR was carried out to detect RHA3A and RHA3B transcripts with ACTIN2 as a control. Fold changes of the gene expression from two independent transgenic lines (line 1 & 2) are shown. Data are shown as means ± SEM (One-way ANOVA, n=5). g. RHA3A and RHA3B are required for BIK1 ubiquitination in transgenic plants. Protoplasts from amiR-RHA3A/B transgenic plants were transfected with BIK1-HA and FLAG-UBQ for ubiquitination assay. Quantification of BIK1 ubiquitination in amiR-RHA3A/B transgenic plants is shown on bottom. Intensity of Ub-BIK1 or BIK1 bands was quantified with Image Lab (Bio-Rad). The amount of BIK1 ubiquitination is the relative intensity of Ub-BIK1 band to BIK1 band (no treatment in WT as 1.0). Data are shown as means ± SEM. Different letters indicate significant difference with others (P < 0.05, One-way ANOVA, n=3). h. Sequencing analysis of RHA3A and RHA3B genes in the CRISPR/Cas9 rha3a/b mutant. PCR fragments corresponding to RHA3A and RHA3B in rha3a/b were amplified, sequenced, and aligned to WT coding sequences. Reverse-complement of the PAM sequence is underlined with red, and arrow indicates the theoretical Cas9 cleavage sites. The experiments were repeated three times with similar results.', 'hash': '6a12f1ec471240b56793a17bca968fbcd849d17b13c62f883d84c8de5d72c4ce'}, {'image_id': 'nihms-1565517-f0007', 'image_file_name': 'nihms-1565517-f0007.jpg', 'image_path': '../data/media_files/PMC7233372/nihms-1565517-f0007.jpg', 'caption': 'Plasma membrane localization and phosphorylation are required for BIK1 ubiquitination.a. The kinase inhibitor K252a blocks flg22-induced BIK1 ubiquitination. Protoplasts transfected with FLAG-UBQ and BIK1-HA were treated with 1 μM K252a for 30 min prior to 100 nM flg22 treatment. b. BIK1G2A no longer localizes to PM. BIK1-YFP or BIK1G2A-YFP was expressed in N. benthamiana for imaging analysis. c. BIK1G2A compromises flg22-induced monoubiquitination. BIK1-HA or BIK1G2A-HA was co-expressed with FLAG-UBQ in protoplasts. d. Single K-to-R mutations of BIK1 fail to block flg22-induced ubiquitination without altering kinase activity. HA-tagged BIK1 WT or mutants was co-expressed with FLAG-UBQ in protoplasts. e. BIK1K204R exhibits reduced autophosphorylation and phosphorylation activity on BAK1. An in vitro kinase assay was performed using GST-BIK1 or GST-BIK1K204R as a kinase and GST or GST-BAK1K (BAK1 kinase domain without detectable autophosphorylation activity) as a substrate with [γ−32P] ATP. Proteins were separated with SDS-PAGE and analyzed by autoradiography (Autorad.) (top). Protein loading is shown by CBB staining (bottom). The experiments were repeated at least twice with similar results.', 'hash': '2bc7a1129a6bc88a254edf1e100b23e984356c58d8a10df74741a2463bd510ff'}]
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{'nihms-1565517-f0001': ['We observed that BIK1-GFP localized to both the periphery of epidermal pavement cells and intracellular puncta using arabidopsis transgenic plants expressing functional 35S::BIK1-GFP by spinning disc confocal microscopy (SDCM) (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1a</xref>, , <xref rid="nihms-1565517-f0005" ref-type="fig">Extended Data Fig. 1a</xref>, , <xref rid="nihms-1565517-f0005" ref-type="fig">b</xref>). BIK1-GFP colocalized with the FM4–64-stained PM (). BIK1-GFP colocalized with the FM4–64-stained PM (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1b</xref>, star), and frequently within endosomal compartments (, star), and frequently within endosomal compartments (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1b</xref>, arrowheads). Time-lapse SDCM shows that BIK1-GFP puncta were highly mobile, disappearing (red circle), appearing (yellow circle), and moving rapidly in and out of the plane of view (white circle) (, arrowheads). Time-lapse SDCM shows that BIK1-GFP puncta were highly mobile, disappearing (red circle), appearing (yellow circle), and moving rapidly in and out of the plane of view (white circle) (<xref rid="nihms-1565517-f0005" ref-type="fig">Extended Data Fig. 1c</xref>). The abundance of BIK1-GFP puncta was increased over time (3–17 and 18–32 min) after treatment with flagellin peptide flg22 (). The abundance of BIK1-GFP puncta was increased over time (3–17 and 18–32 min) after treatment with flagellin peptide flg22 (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1c</xref>; ; <xref rid="nihms-1565517-f0005" ref-type="fig">Extended Data Fig. 1d</xref>––<xref rid="nihms-1565517-f0005" ref-type="fig">p</xref>). The timing of the ligand-induced increase in BIK1-GFP puncta differed from that of FLS2-GFP, in which puncta numbers were significantly increased 35 min after flg22 treatment (). The timing of the ligand-induced increase in BIK1-GFP puncta differed from that of FLS2-GFP, in which puncta numbers were significantly increased 35 min after flg22 treatment (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1d</xref>))11–13. Ligand-induced endocytosis of FLS2 contributes to the degradation of the activated FLS2 receptor and attenuation of the signaling11–14, whereas increased abundance of BIK1-GFP puncta precedes that of FLS2-GFP (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1c</xref>, , <xref rid="nihms-1565517-f0001" ref-type="fig">d</xref>).).', 'Ligand-induced FLS2 degradation is mediated by U-box E3 ligases PUB12 and PUB13 that polyubiquitinate FLS215–17. We tested whether BIK1 is ubiquitinated upon flg22 treatment with an in vivo ubiquitination assay in arabidopsis protoplasts co-expressing FLAG epitope-tagged ubiquitin (FLAG-UBQ) and HA epitope-tagged BIK1 (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1e</xref>; ; <xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2a</xref>). The results showed that flg22 treatment induced BIK1 ubiquitination (). The results showed that flg22 treatment induced BIK1 ubiquitination (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1e</xref>), as ubiquitinated BIK1 was detected with an α-HA immunoblot upon immunoprecipitation with an α-FLAG antibody. The flg22-induced BIK1 ubiquitination was also observed in ), as ubiquitinated BIK1 was detected with an α-HA immunoblot upon immunoprecipitation with an α-FLAG antibody. The flg22-induced BIK1 ubiquitination was also observed in pBIK1::BIK1-HA transgenic plants (<xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2b</xref>). The strong and discrete band of ubiquitinated BIK1 indicates monoubiquitination (). The strong and discrete band of ubiquitinated BIK1 indicates monoubiquitination (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1e</xref>; ; <xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2a</xref>, , <xref rid="nihms-1565517-f0006" ref-type="fig">b</xref>) compared to polyubiquitination of BAK1 and FLS2 with a ladder-like smear of protein migration () compared to polyubiquitination of BAK1 and FLS2 with a ladder-like smear of protein migration (<xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2c</xref>, , <xref rid="nihms-1565517-f0006" ref-type="fig">d</xref>). The apparent molecular mass of ubiquitinated BIK1 (~52 kDa) is ~8 kDa larger than that of unmodified BIK1 (44 kDa), supporting the attachment of a single ubiquitin to BIK1. Incubation with the catalytic domain of the deubiquitinase Usp2 (Usp2-cc), but not its heat-inactivated form, reduced the molecular mass by ~8 kDa (). The apparent molecular mass of ubiquitinated BIK1 (~52 kDa) is ~8 kDa larger than that of unmodified BIK1 (44 kDa), supporting the attachment of a single ubiquitin to BIK1. Incubation with the catalytic domain of the deubiquitinase Usp2 (Usp2-cc), but not its heat-inactivated form, reduced the molecular mass by ~8 kDa (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1e</xref>). In addition, a similar ubiquitination pattern of BIK1 was observed when we used the UBQ). In addition, a similar ubiquitination pattern of BIK1 was observed when we used the UBQK0 variant, in which all seven lysine (K) residues in UBQ were changed to arginine (R), thus preventing polyubiquitination chain formation (<xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2e</xref>, , <xref rid="nihms-1565517-f0006" ref-type="fig">f</xref>). Notably, flg22-induced BIK1 ubiquitination was blocked by treatment with the ubiquitination inhibitor PYR-41, but not by the proteasome inhibitor MG132, and was not observed in the ). Notably, flg22-induced BIK1 ubiquitination was blocked by treatment with the ubiquitination inhibitor PYR-41, but not by the proteasome inhibitor MG132, and was not observed in the fls2 or bak1–4 mutants (<xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2g</xref>––<xref rid="nihms-1565517-f0006" ref-type="fig">i</xref>). In addition to flg22, other MAMPs, including elf18, pep1, and chitin, also induced monoubiquitination of BIK1 (). In addition to flg22, other MAMPs, including elf18, pep1, and chitin, also induced monoubiquitination of BIK1 (<xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2j</xref>), in line with the notion that BIK1 is a convergent component downstream of multiple PRRs), in line with the notion that BIK1 is a convergent component downstream of multiple PRRs4. Monoubiquitination of the BIK1 family RLCKs PBL1 and PBL10, but not another RLCK BSK1, was enhanced upon flg22 treatment (<xref rid="nihms-1565517-f0006" ref-type="fig">Extended Data Fig. 2k</xref>, , <xref rid="nihms-1565517-f0006" ref-type="fig">l</xref>), suggesting that MAMP perception induces monoubiquitination of BIK1 family RLCKs.), suggesting that MAMP perception induces monoubiquitination of BIK1 family RLCKs.', 'Upon flg22 perception, BIK1 is phosphorylated, as detected by an immunoblot mobility shift within 1 min, with a plateau around 10 min (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1f</xref>))5. However, flg22-induced BIK1 ubiquitination becomes apparent only at 10 min after treatment and reaches a plateau around 30 min (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1f</xref>), suggesting that flg22-induced BIK1 ubiquitination may occur after its phosphorylation. Consistently, BIK1 phosphorylation deficient mutants, including a kinase-inactive mutant (BIK1), suggesting that flg22-induced BIK1 ubiquitination may occur after its phosphorylation. Consistently, BIK1 phosphorylation deficient mutants, including a kinase-inactive mutant (BIK1KM) and two phosphorylation site mutants (BIK1T237A and BIK1Y250A) largely compromised flg22-induced ubiquitination (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1g</xref>). In addition, the kinase inhibitor K252a blocked flg22-induced BIK1 ubiquitination (). In addition, the kinase inhibitor K252a blocked flg22-induced BIK1 ubiquitination (<xref rid="nihms-1565517-f0007" ref-type="fig">Extended Data Fig. 3a</xref>). Moreover, PM localization is required for BIK1 ubiquitination as BIK1). Moreover, PM localization is required for BIK1 ubiquitination as BIK1G2A, a mutation at the myristoylation motif essential for PM localization, was unable to be ubiquitinated upon flg22 treatment (<xref rid="nihms-1565517-f0007" ref-type="fig">Extended Data Fig. 3b</xref>, , <xref rid="nihms-1565517-f0007" ref-type="fig">c</xref>). Taken together, these data suggest that flg22-induced BIK1 phosphorylation is a prerequisite for its monoubiquitination at the PM.). Taken together, these data suggest that flg22-induced BIK1 phosphorylation is a prerequisite for its monoubiquitination at the PM.', 'Since perception of flg22 moderately increased the BIK1-GFP positive endosomal puncta (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1c</xref>), we tested whether BIK1 monoubiquitination is involved in flg22-triggered BIK1 endocytosis. Apparently, fewer FM4–64-labelled puncta were observed with BIK1), we tested whether BIK1 monoubiquitination is involved in flg22-triggered BIK1 endocytosis. Apparently, fewer FM4–64-labelled puncta were observed with BIK19KR-GFP than with BIK1-GFP after 10 or 15 min flg22 treatment (<xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9a</xref>, , <xref rid="nihms-1565517-f0013" ref-type="fig">b</xref>). In addition, we compared the flg22-triggered endocytosis of BIK1-TagRFP and BIK1). In addition, we compared the flg22-triggered endocytosis of BIK1-TagRFP and BIK19KR-TagRFP co-expressing FLS2-YFP in Nicotiana benthamiana. Similar to transgenic plants (<xref rid="nihms-1565517-f0001" ref-type="fig">Fig. 1c</xref>, , <xref rid="nihms-1565517-f0001" ref-type="fig">d</xref>), endosomal puncta of BIK1-TagRFP increased at 10–20 min, whereas FLS2-YFP puncta increased only after 60 min flg22 treatment (), endosomal puncta of BIK1-TagRFP increased at 10–20 min, whereas FLS2-YFP puncta increased only after 60 min flg22 treatment (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4e</xref>, , <xref rid="nihms-1565517-f0004" ref-type="fig">f</xref>; ; <xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9c</xref>). A large portion (~90%) of flg22-induced BIK1-TagRFP puncta did not co-localize with FLS2-YFP puncta (). A large portion (~90%) of flg22-induced BIK1-TagRFP puncta did not co-localize with FLS2-YFP puncta (<xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9d</xref>), suggesting that BIK1 and FLS2 are likely not internalized together. This is in line with different ubiquitination characteristics of BIK1 and FLS2 (mono vs. poly, 10 min vs. 1 hr). In contrast to BIK1, BIK1), suggesting that BIK1 and FLS2 are likely not internalized together. This is in line with different ubiquitination characteristics of BIK1 and FLS2 (mono vs. poly, 10 min vs. 1 hr). In contrast to BIK1, BIK19KR-TagRFP was more abundant in puncta before treatment, but the number of puncta did not increase after flg22 treatment (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4e</xref>, , <xref rid="nihms-1565517-f0004" ref-type="fig">f</xref>; ; <xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9c</xref>), indicating that BIK1), indicating that BIK19KR-TagRFP internalization does not respond to PRR activation. In addition, colocalization of BIK19KR-TagRFP with ARA6-YFP, a plant specific Rab GTPase residing on late endosomes20, was significantly reduced when compared to that of BIK1-TagRFP (<xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9e</xref>, , <xref rid="nihms-1565517-f0013" ref-type="fig">f</xref>). Notably, flg22-induced FLS2-YFP endocytosis was absent in the presence of BIK1). Notably, flg22-induced FLS2-YFP endocytosis was absent in the presence of BIK19KR-TagRFP (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4e</xref>, , <xref rid="nihms-1565517-f0004" ref-type="fig">f</xref>). Altogether, our data support the conclusion that ligand-induced BIK1 monoubiquitination contributes to its internalization from PM. Interestingly, whereas flg22 treatment induced a phosphorylation-dependent BIK1 dissociation from FLS2). Altogether, our data support the conclusion that ligand-induced BIK1 monoubiquitination contributes to its internalization from PM. Interestingly, whereas flg22 treatment induced a phosphorylation-dependent BIK1 dissociation from FLS25,6,21, flg22-triggered BIK1 dissociation from FLS2 was largely absent in the case of BIK19KR (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4g</xref>), which is consistent with the data that BIK1), which is consistent with the data that BIK19KR impaired FLS2 internalization (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4e</xref>, , <xref rid="nihms-1565517-f0004" ref-type="fig">f</xref>). In addition, we observed an increased association between BIK1). In addition, we observed an increased association between BIK19KR and FLS2 without flg22 treatment (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4g</xref>). Treatment with the ubiquitination inhibitor PYR-41 also blocked flg22-induced BIK1 dissociation from FLS2 and enhanced BIK1-FLS2 association (). Treatment with the ubiquitination inhibitor PYR-41 also blocked flg22-induced BIK1 dissociation from FLS2 and enhanced BIK1-FLS2 association (<xref rid="nihms-1565517-f0014" ref-type="fig">Extended Data Fig. 10a</xref>). Our data indicate that ligand-induced BIK1 monoubiquitination plays an important role in BIK1 dissociation from the PM-localized PRR complex, its endocytosis and immune signaling activation (). Our data indicate that ligand-induced BIK1 monoubiquitination plays an important role in BIK1 dissociation from the PM-localized PRR complex, its endocytosis and immune signaling activation (<xref rid="nihms-1565517-f0014" ref-type="fig">Extended Data Fig. 10b</xref>).).', 'The data supporting the findings of this study are available within the paper and its Supplementary Information files. Source Data (gels and graphs) for <xref rid="nihms-1565517-f0001" ref-type="fig">Figs. 1</xref>––<xref rid="nihms-1565517-f0004" ref-type="fig">4</xref> and and <xref rid="nihms-1565517-f0005" ref-type="fig">Extended Data Figs. 1</xref>––<xref rid="nihms-1565517-f0014" ref-type="fig">10</xref> are provided with the paper. are provided with the paper.'], 'nihms-1565517-f0007': ['There are 30 lysine residues in BIK1, all of which could potentially be ubiquitinated. We individually mutated 28 lysine residues to arginine except K105/K106 (located in the ATP-binding pocket and required for kinase activity), and screened for flg22-induced ubiquitination. None of the individual K-to-R mutants blocked BIK1 ubiquitination without altering its kinase activity (<xref rid="nihms-1565517-f0007" ref-type="fig">Extended Data Fig. 3d</xref>). BIK1). BIK1K204R, which compromised flg22-induced BIK1 monoubiquitination, also showed reduced phosphorylation in vivo and in vitro (<xref rid="nihms-1565517-f0007" ref-type="fig">Extended Data Fig. 3d</xref>, , <xref rid="nihms-1565517-f0007" ref-type="fig">e</xref>). To identify BIK1-associated regulators, we carried out a yeast two-hybrid screen using BIK1). To identify BIK1-associated regulators, we carried out a yeast two-hybrid screen using BIK1G2A as a bait, and identified RHA3A (AT2G17450) encoding a functionally uncharacterized E3 ubiquitin ligase with a RING-H2 finger domain and an N-terminal transmembrane domain (<xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2a</xref>). We confirmed the interaction of BIK1 with RHA3A using an ). We confirmed the interaction of BIK1 with RHA3A using an in vitro pull-down assay (<xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2b</xref>), an ), an in vivo co-immunoprecipitation (Co-IP) assay in arabidopsis protoplasts (<xref rid="nihms-1565517-f0008" ref-type="fig">Extended Data Fig. 4a</xref>), and Co-IP in transgenic plants expressing both genes under their native promoters (), and Co-IP in transgenic plants expressing both genes under their native promoters (<xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2c</xref>; ; <xref rid="nihms-1565517-f0008" ref-type="fig">Extended Data Fig. 4b</xref>). RHA3B, encoded by ). RHA3B, encoded by AT4G35480, the closest homolog of RHA3A bearing 66% amino acid identity (<xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2a</xref>), also co-immunoprecipitated with BIK1 (), also co-immunoprecipitated with BIK1 (<xref rid="nihms-1565517-f0008" ref-type="fig">Extended Data Fig. 4c</xref>). Flg22 treatment did not affect BIK1 interaction with RHA3A or RHA3B (called RHA3A/B henceforth) (). Flg22 treatment did not affect BIK1 interaction with RHA3A or RHA3B (called RHA3A/B henceforth) (<xref rid="nihms-1565517-f0008" ref-type="fig">Extended Data Fig. 4a</xref>, , <xref rid="nihms-1565517-f0008" ref-type="fig">c</xref>). Moreover, RHA3A/B co-immunoprecipitated with FLS2 (). Moreover, RHA3A/B co-immunoprecipitated with FLS2 (<xref rid="nihms-1565517-f0008" ref-type="fig">Extended Data Fig. 4d</xref>).).'], 'nihms-1565517-f0009': ['An in vitro ubiquitination assay demonstrated that RHA3A had autoubiquitination activity and monoubiquitinated itself (<xref rid="nihms-1565517-f0009" ref-type="fig">Extended Data Fig. 5a</xref>, , <xref rid="nihms-1565517-f0009" ref-type="fig">b</xref>). Importantly, glutathione-). Importantly, glutathione-S-transferase (GST)-RHA3A, but not GST-RHA3AI104A with substitution of a conserved isoleucine (I) residue, monoubiquitinated GST-BIK1-HA as evidenced by an additional discrete band that migrated ~8kD larger (<xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2d</xref>). The available ). The available rha3a and rha3b T-DNA insertion lines did not show significant reduction of the corresponding transcripts (<xref rid="nihms-1565517-f0009" ref-type="fig">Extended Data Fig. 5c</xref>). We thus generated artificial microRNAs (amiRNAs) of ). We thus generated artificial microRNAs (amiRNAs) of RHA3A/B18, and co-expression of amiR-RHA3A and amiR-RHA3B, but not amiR-RHA3A alone, suppressed flg22-induced BIK1 monoubiquitination in protoplast transient assays (<xref rid="nihms-1565517-f0009" ref-type="fig">Extended Data Fig. 5d</xref>, , <xref rid="nihms-1565517-f0009" ref-type="fig">e</xref>). Flg22-induced BIK1 monoubiquitination, but not phosphorylation, was also reduced in transgenic plants expressing ). Flg22-induced BIK1 monoubiquitination, but not phosphorylation, was also reduced in transgenic plants expressing amiR-RHA3A and amiR-RHA3B driven by the native promoters (<xref rid="nihms-1565517-f0009" ref-type="fig">Extended Data Fig. 5f</xref>, , <xref rid="nihms-1565517-f0009" ref-type="fig">g</xref>). Furthermore, we generated ). Furthermore, we generated rha3a and rha3a/b mutants using the CRISPR/Cas9 system (<xref rid="nihms-1565517-f0009" ref-type="fig">Extended Data Fig. 5h</xref>). Flg22-induced BIK1 monoubiquitination was reduced in ). Flg22-induced BIK1 monoubiquitination was reduced in rha3a/b (<xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2e</xref>). The data together indicate that RHA3A/B modulate flg22-induced BIK1 monoubiquitination.). The data together indicate that RHA3A/B modulate flg22-induced BIK1 monoubiquitination.'], 'nihms-1565517-f0003': ['To identify RHA3A-mediated BIK1 ubiquitination sites, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in vitro ubiquitinated BIK1. Among ten lysine residues identified (<xref rid="nihms-1565517-f0003" ref-type="fig">Fig. 3a</xref>, , <xref rid="nihms-1565517-f0003" ref-type="fig">b</xref>; ; <xref rid="nihms-1565517-f0010" ref-type="fig">Extended Data Fig. 6a</xref>––<xref rid="nihms-1565517-f0010" ref-type="fig">i</xref>), K), K106 residing in the ATP-binding pocket blocked BIK1 kinase activity when mutated7. Among the other nine lysine sites, all six lysine (K95, K170, K186, K286, K337, and K358) with available structural information are located on the BIK1 surface (<xref rid="nihms-1565517-f0003" ref-type="fig">Fig. 3c</xref>))19. Furthermore, six ubiquitinated lysine residues were detected by LC-MS/MS of in vivo ubiquitinated BIK1-GFP upon flg22 treatment, and they all overlap with those detected in in vitro RHA3A-BIK1 ubiquitination reactions (<xref rid="nihms-1565517-f0011" ref-type="fig">Extended Data Fig. 7a</xref>––<xref rid="nihms-1565517-f0011" ref-type="fig">h</xref>). Individual lysine mutations did not affect BIK1 ubiquitination ). Individual lysine mutations did not affect BIK1 ubiquitination in vivo (<xref rid="nihms-1565517-f0007" ref-type="fig">Extended Data Fig. 3d</xref>), whereas combined mutations of the N-terminal five lysine (BIK1), whereas combined mutations of the N-terminal five lysine (BIK1N5KR) or C-terminal four lysine (BIK1C4KR) partially compromised flg22-induced BIK1 ubiquitination, and BIK19KR with all nine lysine mutated largely blocked flg22-induced BIK1 monoubiquitination in vivo (<xref rid="nihms-1565517-f0003" ref-type="fig">Fig. 3d</xref>) and RHA3A-mediated ) and RHA3A-mediated in vitro ubiquitination (<xref rid="nihms-1565517-f0003" ref-type="fig">Fig. 3e</xref>). BIK1). BIK19KR exhibited similar activities as BIK1 with regard to its in vitro kinase activities (<xref rid="nihms-1565517-f0003" ref-type="fig">Fig. 3f</xref>), flg22-induced BIK1 phosphorylation, and association with RHA3A in protoplasts (), flg22-induced BIK1 phosphorylation, and association with RHA3A in protoplasts (<xref rid="nihms-1565517-f0012" ref-type="fig">Extended Data Fig. 8a</xref>, , <xref rid="nihms-1565517-f0012" ref-type="fig">b</xref>). Furthermore, ). Furthermore, 35S::BIK19KR-HA/WT transgenic plants had normal flg22-induced MAPK activation and ROS production (<xref rid="nihms-1565517-f0012" ref-type="fig">Extended Data Fig. 8c</xref>, , <xref rid="nihms-1565517-f0012" ref-type="fig">d</xref>). Collectively, the data indicate that RHA3A monoubiquitinates BIK1 and that BIK1 phosphorylation does not require monoubiquitination. Notably, BIK1 monoubiquitination may not be restricted to a single lysine, and multiple lysine residues could serve as monoubiquitin conjugation sites. Alternatively, monoubiquitination might be the primary form of BIK1 modification, whereas polyubiquitinated BIK1 could be short-lived.). Collectively, the data indicate that RHA3A monoubiquitinates BIK1 and that BIK1 phosphorylation does not require monoubiquitination. Notably, BIK1 monoubiquitination may not be restricted to a single lysine, and multiple lysine residues could serve as monoubiquitin conjugation sites. Alternatively, monoubiquitination might be the primary form of BIK1 modification, whereas polyubiquitinated BIK1 could be short-lived.'], 'nihms-1565517-f0012': ['BIK19KR, which eliminates BIK1 monoubiquitination but not phosphorylation, enabled us to examine the function of BIK1 monoubiquitination without compromised kinase activity. We generated BIK19KR transgenic plants driven by the BIK1 native promoter in bik1 (pBIK1::BIK19KR-HA/bik1) (<xref rid="nihms-1565517-f0012" ref-type="fig">Extended Data Fig. 8e</xref>, , <xref rid="nihms-1565517-f0012" ref-type="fig">f</xref>). Unlike ). Unlike pBIK1::BIK1-HA/bik1 transgenic plants, pBIK1::BIK19KR-HA/bik1 transgenic plants exhibited a reduced flg22-triggered ROS burst similar to the bik1 mutant (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4a</xref>). Moreover, ). Moreover, pBIK1::BIK19KR-HA/bik1 transgenic plants were more susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC− compared to wild-type (WT) or pBIK1::BIK1-HA/bik1 transgenic plants (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4b</xref>). In addition, ). In addition, amiR-RHA3A/B transgenic plants exhibited compromised flg22-triggered ROS production and enhanced susceptibility to Pst DC3000 (<xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4c</xref>, , <xref rid="nihms-1565517-f0004" ref-type="fig">d</xref>) and ) and Pst DC3000 hrcC−, and to the fungal pathogen, Botrytis cinerea (<xref rid="nihms-1565517-f0012" ref-type="fig">Extended Data Fig. 8g</xref>, , <xref rid="nihms-1565517-f0012" ref-type="fig">h</xref>). Similar results were obtained with the ). Similar results were obtained with the rha3a/b mutants (<xref rid="nihms-1565517-f0012" ref-type="fig">Extended Data Fig. 8i</xref>, , <xref rid="nihms-1565517-f0012" ref-type="fig">j</xref>). Together, the data indicate that RHA3A/B-mediated monoubiquitination of BIK1 plays a role in regulating ROS production and plant immunity.). Together, the data indicate that RHA3A/B-mediated monoubiquitination of BIK1 plays a role in regulating ROS production and plant immunity.'], 'nihms-1565517-f0014': ['The BIK1 family RLCKs are central players in plant PRR signaling with many layers of regulation4,22. BIK1 stability is critical to maintain immune homeostasis. Plant U-box proteins PUB25 and PUB26 polyubiquitinate BIK1 and regulate its stability in the steady state23. This module only regulates non-activated BIK1 homeostasis without affecting ligand-activated BIK123. Our study identified a role of RHA3A/B in monoubiquitinating BIK1 and activating PRR signaling, which is distinct from that of PUB25/26. BIK19KR protein levels in transgenic plants and protoplasts are comparable with BIK1 (<xref rid="nihms-1565517-f0014" ref-type="fig">Extended Data Fig. 10c</xref>, , <xref rid="nihms-1565517-f0014" ref-type="fig">d</xref>), suggesting that BIK1 monoubiquitination may not regulate its stability. The nature of protein ubiquitination, including monoubiquitination and polyubiquitination, dictates distinct fates of substrates, such as proteasome-mediated protein degradation, nonproteolytic functions of protein kinase activation, and membrane trafficking), suggesting that BIK1 monoubiquitination may not regulate its stability. The nature of protein ubiquitination, including monoubiquitination and polyubiquitination, dictates distinct fates of substrates, such as proteasome-mediated protein degradation, nonproteolytic functions of protein kinase activation, and membrane trafficking24. Ligand-induced polyubiquitination of FLS2 by PUB12/13 promotes FLS2 degradation, thereby attenuating immune signaling15,16, whereas ligand-induced monoubiquitination of BIK1 triggers BIK1 dissociation from PRR complexes and intracellular signaling activation. Thus, differential ubiquitination and endocytosis of distinct PRR-RLCK complex components likely serve as cues to fine-tune plant immune responses.'], 'nihms-1565517-f0008': ['a. GST-RHA3ACD possesses E3 ligase activity in vitro. The in vitro ubiquitination assay was performed with GST-RHA3ACD followed by deubiquitination reactions with GST-Usp2-cc. N-Ethylmaleimide (NEM) (10 mM), an inhibitor of deubiquitinases, and heat-inactivated (HI, 95°C for 5 min) Usp2-cc are controls. Samples were analyzed by SDS-PAGE and silver staining. b. GST-RHA3ACD possesses multi-monoubiquitination activity in vitro. Ubiquitination assay was done similarly as (a) except using the ubiquitin mutant with all lysine residues mutated to arginine (UBQK0). Ubiquitinated proteins were detected by IB with α-UBQ (left) or α-RHA3A (right) antibody. c. RHA3 expression in T-DNA insertion mutants. RHA3A expression in T-DNA knockout line SALK_052714 and RHA3B expression in SALK_064303 were analyzed as in <xref rid="nihms-1565517-f0008" ref-type="fig">Extended Data Fig. 4b</xref>. Data are shown as means ± SEM of fold change (WT as 1.0) (Two-tailed Student’s . Data are shown as means ± SEM of fold change (WT as 1.0) (Two-tailed Student’s t-test, n=3). d. Screen for the optimal amiR-RHA3A and amiR-RHA3B. Protoplasts were transfected with RHA3A-HA or RHA3B-HA with Ctrl, amiR-RHA3A or amiR-RHA3B. RHA3A or RHA3B proteins were examined by IB with α-HA antibody. e. RHA3A and RHA3B are required for BIK1 ubiquitination in vivo. BIK1 ubiquitination assay was carried out by co-expressing Ctrl, artificial microRNA targeting RHA3A (amiR-RHA3A) or together with microRNA targeting RHA3B (amiR-RHA3A amiR-RHA3B). f. RHA3A and RHA3B expression in amiR-RHA3A/B transgenic plants. qRT-PCR was carried out to detect RHA3A and RHA3B transcripts with ACTIN2 as a control. Fold changes of the gene expression from two independent transgenic lines (line 1 & 2) are shown. Data are shown as means ± SEM (One-way ANOVA, n=5). g. RHA3A and RHA3B are required for BIK1 ubiquitination in transgenic plants. Protoplasts from amiR-RHA3A/B transgenic plants were transfected with BIK1-HA and FLAG-UBQ for ubiquitination assay. Quantification of BIK1 ubiquitination in amiR-RHA3A/B transgenic plants is shown on bottom. Intensity of Ub-BIK1 or BIK1 bands was quantified with Image Lab (Bio-Rad). The amount of BIK1 ubiquitination is the relative intensity of Ub-BIK1 band to BIK1 band (no treatment in WT as 1.0). Data are shown as means ± SEM. Different letters indicate significant difference with others (P < 0.05, One-way ANOVA, n=3). h. Sequencing analysis of RHA3A and RHA3B genes in the CRISPR/Cas9 rha3a/b mutant. PCR fragments corresponding to RHA3A and RHA3B in rha3a/b were amplified, sequenced, and aligned to WT coding sequences. Reverse-complement of the PAM sequence is underlined with red, and arrow indicates the theoretical Cas9 cleavage sites. The experiments were repeated three times with similar results.'], 'nihms-1565517-f0004': ['a. b. BIK19KR-GFP-labelled puncta colocalize less with FM4–64 than BIK1-GFP upon flg22 treatment. Five-day-old seedlings of 35S::BIK1-GFP or 35S::BIK19KR-GFP were pretreated with FM4–64 (2 μM) for 15 min and elicited with 100 nM flg22 for the indicated time points and fluorescence was detected in epidermis by a confocal microscopy (a). White arrow points to colocalized endosomes. Scale bars, 20 μm. Percentage of BIK1-GFP or BIK19KR-GFP and FM4–64 positive endosomes over time per 100% of image area is shown in (b). Data are shown as overlay of dot plot and means ± SEM (Two-tailed Student’s t-test, n = 21 images for BIK1-GFP and n= 16, 15 images for 10, 15 min respectively for BIK19KR-GFP). c. Flg22-induced BIK1, BIK19KR and FLS2 endocytosis in N. benthamiana. BIK1-TagRFP (BIK1-RFP) or BIK19KR-TagRFP (BIK19KR-RFP) was co-expressed with FLS2-YFP in N. benthamiana, infiltrated with 100 μM flg22 and imaged at the indicated time points by a confocal microscopy. The images of 30–40, 40–50 and 50–60 min after flg22 treatment from <xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4e</xref> are shown here. Scale bars, 20 μm. For BIK1-RFP/FLS2-YFP, are shown here. Scale bars, 20 μm. For BIK1-RFP/FLS2-YFP, n= 14, 11, 7, 10, 10, 6, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min; for BIK19KR-RFP/FLS2-YFP, n= 19, 11, 11, 9, 16, 12, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min respectively. d. Percentage of BIK1-RFP puncta colocalizing with FLS2-YFP after flg22 treatment for the indicated time points in <xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4e</xref> and and <xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9c</xref>. Data are shown as overlay of dot plot and means ± SEM (. Data are shown as overlay of dot plot and means ± SEM (n= 14, 11, 7, 10, 10, 6, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min respectively). e-f BIK19KR-RFP shows reduced co-localization with ARA6-YFP. BIK1-RFP or BIK19KR-RFP was transiently expressed with ARA6-YFP in N. benthamiana, and the images were taken 48–72 hr after infiltration (e). Scale bars, 10 μm. Percentage of BIK1-RFP puncta colocalizing with ARA6-YFP is shown in f. Data are shown as overlay of dot plot and means ± SEM (Two-tailed Student’s t-test, n= 9 images for BIK1-RFP; n= 10 images for BIK19KR-RFP). The experiments were repeated three times with similar results.', 'a. PYR-41 impairs flg22-induced BIK1 dissociation from FLS2. FLS2-HA was co-expressed with BIK1-FLAG or Ctrl in protoplasts. After pretreatment with 50 μM PYR-41 for 30 min, protoplasts were stimulated with 100 nM flg22 for 15 min. Co-IP and IB were performed as in <xref rid="nihms-1565517-f0004" ref-type="fig">Fig. 4g</xref>. . b. A working model of RHA3A/B-mediated BIK1 monoubiquitination in plant immunity. During non-activated, steady state condition (0 min), BIK1 remains hypo-phosphorylated and associates with FLS2 and BAK1. Upon flg22 perception, FLS2 dimerizes with BAK1, which stimulates BIK1 phosphorylation (<1 min). The phosphorylated BIK1 is monoubiquitinated by E3 ligases RHA3A and RHA3B, leading to BIK1 dissociation from FLS2-BAK1 complex, accompanied by endocytosis (10–20 min). Ligand-induced BIK1 monoubiquitination contributes to the activation of ROS and other defense responses. FLS2 is polyubiquitinated and endocytosed 40 min after flg22 perception for signaling attenuation. c. BIK19KR shows comparable protein level as BIK1 in transgenic plants. 35S::BIK1-HA or 35S::BIK19KR-HA transgenic plants in WT background were used for IB detecting BIK1 proteins by α-HA antibody. Ctrl is empty vector transgenic plants. d. BIK1 and BIK19KR protein stability after cycloheximide treatment. BIK1-HA or BIK19KR-HA was expressed in WT protoplasts for 12 hr followed by 500 μg/ml cycloheximide (CHX) treatment for the indicated time. BIK1 or BIK19KR proteins were analyzed by IB with α-HA antibody. * indicates CHX was added right after transfection, thus blocking protein synthesis. The experiments were repeated three times with similar results.'], 'nihms-1565517-f0002': ['a. BIK1 is ubiquitinated by RHA3A at multiple lysine residues. Ubiquitinated lysine residues with a diglycine remnant by LC-MS/MS analysis are marked as red with amino acid positions. b. MS/MS spectrum of the peptide containing K358 is shown. c. Structure of BIK1 labeled with six lysine identified as ubiquitination sites. Structural information was obtained from protein data bank (PDB ID: 5TOS) and analyzed by PyMOL. d. BIK19KR is compromised in flg22-induced ubiquitination. FLAG-UBQ and HA-tagged BIK1 mutants were expressed in protoplasts followed by 100 nM flg22 for 30 min. Quantification of BIK1 ubiquitination fold change is shown as overlay of dot plot and means ± SEM (middle). Different letters indicate significant difference with others (P<0.05, One-way ANOVA, n=3). Mutated lysine for BIK1 mutants are in red (bottom). e. RHA3A is unable to ubiquitinate BIK19KR. The assay was performed as in <xref rid="nihms-1565517-f0002" ref-type="fig">Fig. 2d</xref>. . f. BIK19KR exhibits normal in vitro kinase activity. The kinase assay was performed using GST-BIK1 or GST-BIK19KR as the kinase and GST or GST-BAK1K (kinase domain) as the substrate. The experiments except MS analyses were repeated three times with similar results.'], 'nihms-1565517-f0013': ['a. The pBIK1::BIK19KR-HA/bik1 transgenic plants (Line 1 & 2) cannot complement bik1 for flg22-induced ROS production. Data are shown as overlay of dot plot and means of total photon count ± SEM (One-way ANOVA, WT, BIK1/bik1: n=53; bik1: n=54; BIK19KR/bik1: n=55). In all panels, data are shown as overlay of dot plot and means ± SEM, lines beneath p values indicate relevant pair-wise comparisons. b. The pBIK1::BIK19KR-HA/bik1 transgenic plants show increased bacterial growth of Pst DC3000 hrcC−. Plants were spray-inoculated and bacterial growth was measured at four days post-inoculation (dpi). One-way ANOVA, n=6. c. The amiRNA-RHA3A/B plants show reduced flg22-induced ROS production. One-way ANOVA, n=51. d. The amiRNA-RHA3A/B plants show increased bacterial growth of Pst DC3000. Plants were hand-inoculated and bacterial growth was measured at two dpi. One-way ANOVA, n=5. e. f. Flg22-induced BIK1, BIK19KR, and FLS2 endocytosis in N. benthamiana leaf epidermal cells. BIK1-TagRFP or BIK19KR-TagRFP was co-expressed with FLS2-YFP followed by 100 μM flg22 treatment and imaged at the indicated time points by a confocal microscopy (e). Scale bars, 20 μm. Quantification of BIK1-TagRFP (magenta) and FLS2-YFP (green) puncta is shown in (f). One-way ANOVA, additional images and n values are shown in <xref rid="nihms-1565517-f0013" ref-type="fig">Extended Data Fig. 9c</xref>. . g. BIK19KR does not enable flg22-induced BIK1 dissociation from FLS2. Co-IP was performed using protoplasts expressing FLS2-HA and BIK1-FLAG, or BIK19KR-FLAG, followed by 1 μM flg22 treatment for 15 min (top). BIK1 interaction with FLS2 was quantified as intensity from IP: α-FLAG, IB: α-HA divided by intensity from IP: α-FLAG, IB: α-FLAG (bottom). Data are shown as means ± SEM of fold change (no treatment as 1.0) (One-way ANOVA, n=3). All experiments were repeated three times with similar results.']}
|
Ligand-induced BIK1 monoubiquitination regulates plant immunity
| null |
Nature
|
1590130800
|
[
"Betacoronavirus",
"COVID-19",
"Coronavirus",
"Coronavirus Infections",
"Humans",
"Pandemics",
"Pneumonia, Viral",
"Reverse Transcriptase Polymerase Chain Reaction",
"SARS-CoV-2",
"Tomography, X-Ray Computed"
] |
other
|
PMC7233372
| null |
9
|
[
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"{'Citation': 'Pan F, Ye T, Sun P, et al. Time course of lung changes on chest CT during recovery from 2019 novel coronavirus (COVID-19) pneumonia. Radiology 2020 Feb 13:200370 [Epub ahead of print].', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7233367'}, {'@IdType': 'pubmed', '#text': '32053470'}]}}",
"{'Citation': 'Yang W, Cao Q, Qin L, et al. Clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (COVID-19):A multi-center study in Wenzhou city, Zhejiang, China. J Infect 2020 Feb 26 [Epub ahead of print].', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7102539'}, {'@IdType': 'pubmed', '#text': '32112884'}]}}",
"{'Citation': 'General Office of National Health Committee . Office of state administration of traditional Chinese medicine. Notice on the issuance of a program for the diagnosis and treatment of novel coronavirus (2019-nCoV) infected pneumonia (trial fifth edition). URL NEEDED. Published February 26, 2020.'}",
"{'Citation': 'General Office of National Health Committee . Office of State Administration of Traditional Chinese Medicine. Notice on the issuance of a program for the diagnosis and treatment of novel coronavirus (2019-nCoV) infected pneumonia (trial sixth edition). URL NEEDED. Published February 19, 2020.'}"
] |
Nature. 2020 May 22; 581(7807):199-203
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TRF2 over-expression leads to accumulation of telR-loops and pSer33 TIFs in HeLa cells.(a) Western blot analysis of HeLa cells infected with lentiviruses driving expression of myc-tagged TRF2 (m-TRF2) or ATRF2 (m-ATRF2) from a doxycycline inducible promoter, or with empty vector (ev) lentiviruses. Cells were treated with doxycycline for 4 days. The TRF2 antibody detects endogenous TRF2, m-TRF2 and m-ATRF2. Actin serves as a loading control. Uncropped images are shown in Supplementary Data Set 1. (b) Myc immunostaining (green) combined with telomeric DNA FISH (red) on cells as in a. Numbers are percentages of cells expressing the ectopic proteins. Scale bar, 5 μm. (c) Telomeric and Alu repeat dot-blot hybridization of S9.6 DRIPs of cells as in a. Uncropped images are shown in Supplementary Data Set 1. In, input (1%); Bd, beads only control (100%); IP, S9.6 immunoprecipitated material (100%). (d) Quantification of experiments as in c. Signals are graphed as the fraction of In DNA found in IPs after subtraction of Bd-associated signal. Values are means ± SD (n = 3 independent experiments). (e) pSer33 immunostaining (green) combined with telomeric DNA FISH (red) on cells as in a. Arrowheads point to examples of pSer33 TIFs. Scale bar, 5 μm. Percentages of cells with at least 3 pSer33 TIFs are graphed as means ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.005 (two-tailed Student’s t-test). (f) Speculative model for TRF1, TRF2 and TERRA interplay at telomeres. At a fully protected chromosome end TRF1 A domain prevents TRF2 B domain from interacting with TERRA. When TRF1 is replaced with TRF1ΔA, TERRA interaction with TRF2 B domain drives TERRA invasion into telomeric dsDNA and formation of telR-loops, which in turn promote pSer33 binding to telomeres and telomere loss. pSer33 might bind to both ss G-rich or C-rich telomeric DNA.
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emss-75371-f008
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2
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31dd6fbb05092df79bb2c1d167573cd18cd4521750b26099117693fb3c01e008
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emss-75371-f008.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
328
] |
[{'image_id': 'emss-75371-f002', 'image_file_name': 'emss-75371-f002.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f002.jpg', 'caption': 'TRF1 A domain suppresses TRF2-stimulated TERRA invasion into telomeric dsDNA.(a) Strand invasion assays with (UUAGGG)5 TERRA-like oligonucleotides (10 nM), p-Tel plasmid (0.6 nM) and increasing amounts of the indicated recombinant proteins. A control assay was performed with heat denatured (boiled) GST-TRF2. (b) Invasion assays in presence of 40 nM GST-TRF2 and increasing amounts (0, 10, 20, 40 and 80 nM) of GST-TRF1 or GST-TRF1ΔA. Invaded plasmids (inv) were quantified and graphed as the fold increase compared with samples lacking proteins. Values are means ± SD (n = 3 independent experiments). ss, single-stranded RNA oligonucleotide; RH, RNaseH treatment prior to electrophoresis; *, wells. Uncropped images are shown in Supplementary Data Set 1.', 'hash': '3962058efb78bb2209a3459f408e2b24a7f4e8d1151bb0957c846c9af4c33c63'}, {'image_id': 'emss-75371-f005', 'image_file_name': 'emss-75371-f005.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f005.jpg', 'caption': 'TRF1 A domain suppresses TRF2-mediated accumulation of telR-loops and pSer33 TIFs in HeLa cells.(a) Telomeric and Alu repeat dot-blot hybridization of S9.6 DRIPs of HeLa cells transfected with siRNAs against TRF1 (T1c) or control siRNAs (Ct). Uncropped images are shown in Supplementary Data Set 1. Nucleic acids were collected 4 days after transfection. In, input (1%); Bd, beads only control (100%); IP, S9.6 immunoprecipitated material (100%). RH, RNaseH treatment of nucleic acids prior to immunoprecipitation. Signals are graphed as the fraction of In DNA found in IPs after subtraction of Bd-associated signal. Values are means ± SD (n = 3 independent experiments). (b) Quantifications of S9.6 DRIP experiments in HeLa cells transfected with siRNAs against TRF1 (siTc or siTe) or TRF2 (siT2) and infected with retroviruses expressing siT1e-insensitive, flag tagged TRF1 (fl-TRF1) and TRFΔA (fl-ΔA), or wild type (wt) and catalytically dead (CD) forms of RNaseH1 (RH1). Nucleic acids were collected 4 days after siRNA transfections and expression of ectopic proteins. Quantifications are of fraction of telomeric In DNA found in IPs after subtraction of Bd-associated signal and normalization over empty vector (ev)-infected, siCt-transfected cells. Values are means ± SD (n = 4 independent experiments for the first two graphs from the left; n = 3 for the graph on the right). (c) Example of pSer33 immunostaining (green) combined with telomeric DNA FISH (red) on cells transfected with siT1c. Arrowheads point to pSer33 TIFs. Scale bar, 5 μm. (d) Percentages of cells with at least 3 pSer33 TIFs in cells as in b graphed as means ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.005, ***P < 0.0001 (two-tailed Student’s t-test).', 'hash': '469414392d62f3d0e4fe70ac68bd864df213f182022de4d692cebdc31c433ab6'}, {'image_id': 'emss-75371-f004', 'image_file_name': 'emss-75371-f004.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f004.jpg', 'caption': 'TRF1 A domain suppresses TERRA binding to TRF2 B domain.(a) Electromobility shift assays with (UUAGGG)5 TERRA-like oligonucleotides (0.25 nM) and increasing amounts of the indicated recombinant proteins. f, free probe; b, bound probe; *, wells. (b) Bound oligonucleotides were quantified and graphed as fraction of the total signal within each lane. Values are means ± SD (n = 3 independent experiments). (c) Strand invasion assay with (UUAGGG)5 TERRA-like oligonucleotides (10 nM), p-Tel plasmid (0.6 nM) and increasing amounts of GST-ATRF2. Invaded plasmids (inv) were quantified and graphed as the fold increase compared with samples lacking proteins. Values are means ± SD (n = 3 independent experiments). ss, single stranded oligonucleotides; *, wells. Uncropped images are shown in Supplementary Data Set 1.', 'hash': '0afd8426c15745cb26df9c628042621e161a012238df0c13185cefad78648334'}, {'image_id': 'emss-75371-f003', 'image_file_name': 'emss-75371-f003.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f003.jpg', 'caption': 'Strand invasion assays with telomeric RNA and DNA oligonucleotides and high concentrations of TRF proteins.(a) Strand invasion assays with G-rich telomeric RNA or DNA oligonucleotides (10 nM), p-Tel plasmid (0.6 nM) and increasing amounts of the indicated recombinant proteins. (b) Invasion assays in presence of 200 nM GST-TRF2 and increasing amounts (0, 25, 50, 100, 200 and 400 nM) of GST-TRF1 or GST-TRF1ΔA. Invaded plasmids (inv) were quantified and graphed as the fold increase compared with samples lacking proteins. Values are means ± SD (n = 3 independent experiments). ss, single stranded oligonucleotides; *, wells. Uncropped images are shown in Supplementary Data Set 1.', 'hash': '427a3fc1d2c7c8bdb80d190e5b40dda3fcd9949c385c0a7ec0f75a733e83fe50'}, {'image_id': 'emss-75371-f007', 'image_file_name': 'emss-75371-f007.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f007.jpg', 'caption': 'TRF1 A domain suppresses telomere loss in U2OS cells.(a) Examples of telomeric DNA FISH and CO-FISH on metaphases from U2OS cells transfected with siT1c. Cells were harvested 3 days after siRNA transfection. Filled arrowheads point to examples of TFEs, not filled arrowheads point to examples of FTs. Scale bar, 5 μm. Scatter plots are quantifications of TFEs and FTs in FISH and CO-FISH experiments. At least 2700 (for FISH) and 4300 (CO-FISH) chromosomes from 3 independent experiments were analyzed for each condition. siT1c and siT1e are two independent siRNAs against TRF1; siCt: control siRNA. (b) Telomeric DNA FISH and CO-FISH on metaphases from fl-ΔA-expressing U2OS cells. Cells were harvested after 4 days of ectopic protein expression. ev: empty vector retrovirus. Filled arrowheads point to examples of TFEs, not filled arrowheads point to examples FTs. Scale bar, 5 μm. Scatter plots are quantifications of TFEs and FTs in FISH and CO-FISH experiments. At least 4100 (FISH) and 4800 (CO-FISH) chromosomes from 3 independent experiments were analyzed for each condition. For TFEs dots are percentages per chromosome end in one metaphase, while for FTs dots are percentages per chromosome end with detectable telomeric signal in one metaphase due to the large number of TFEs accumulating in the same cells. Black bars are means. *P < 0.05, **P < 0.005, ***P < 0.0001 (Mann-Whitney U test).', 'hash': 'cd8c5a01f6ca1e337d92c37c1f41801a778cfeb426362c4ebacbdcb8f60c8c92'}, {'image_id': 'emss-75371-f006', 'image_file_name': 'emss-75371-f006.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f006.jpg', 'caption': 'TRF1 A domain suppresses telR-loop-induced telomere loss in HeLa cells.(a) Examples of telomeric DNA FISH and CO-FISH on metaphases from HeLa cells transfected with siT1c. Filled arrowheads point to TFEs, not filled arrowheads to FTs, the asterisk indicates a telomeric fragment. Scale bar, 5 μm. (b) Quantifications of TFEs and FTs in FISH and CO-FISH experiments on HeLa cells transfected with siT1c or siT1e and infected with retroviruses expressing fl-TRF1, fl-ΔA, RH1wt or RH1CD. Cells were harvested 4 days after siRNA transfection and expression of ectopic proteins. For FISH, at least 4400 chromosomes from 3 independent experiments (except for ev samples, where data are from 4 independent experiments) were analyzed for each condition. For CO-FISH, at least 3200 chromosomes from three independent experiments were analyzed for each condition. Dots are percentages of FTs or TFEs per chromosome end in one metaphase. Black bars are means. *P < 0.05, ***P < 0.0001 (Mann-Whitney U test).', 'hash': '7d9167ea267f656050eecac921bc03fd6a3deb44d140f5289e75a97f0d1f001b'}, {'image_id': 'emss-75371-f001', 'image_file_name': 'emss-75371-f001.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f001.jpg', 'caption': 'TRF2 promotes TERRA invasion into telomeric dsDNA.Strand invasion assays with the indicated telomeric RNA or DNA oligonucleotides (10 nM) in combination with p-Tel plasmid (0.6 nM) and increasing amounts of glutathione S-transferase (GST), GST-TRF1 or GST-TRF2. Invaded plasmids (inv) were quantified and graphed as the fold increase compared with samples lacking proteins (bottom). Values are means ± SD (n = 3 independent experiments). ss, single stranded oligonucleotides; *, wells. Uncropped images are shown in Supplementary Data Set 1.', 'hash': 'a37026c83b2f0661001a414aed4bf1022d6baf7006efd6fd6d5458a383fe0f8b'}, {'image_id': 'emss-75371-f008', 'image_file_name': 'emss-75371-f008.jpg', 'image_path': '../data/media_files/PMC5808845/emss-75371-f008.jpg', 'caption': 'TRF2 over-expression leads to accumulation of telR-loops and pSer33 TIFs in HeLa cells.(a) Western blot analysis of HeLa cells infected with lentiviruses driving expression of myc-tagged TRF2 (m-TRF2) or ATRF2 (m-ATRF2) from a doxycycline inducible promoter, or with empty vector (ev) lentiviruses. Cells were treated with doxycycline for 4 days. The TRF2 antibody detects endogenous TRF2, m-TRF2 and m-ATRF2. Actin serves as a loading control. Uncropped images are shown in Supplementary Data Set 1. (b) Myc immunostaining (green) combined with telomeric DNA FISH (red) on cells as in a. Numbers are percentages of cells expressing the ectopic proteins. Scale bar, 5 μm. (c) Telomeric and Alu repeat dot-blot hybridization of S9.6 DRIPs of cells as in a. Uncropped images are shown in Supplementary Data Set 1. In, input (1%); Bd, beads only control (100%); IP, S9.6 immunoprecipitated material (100%). (d) Quantification of experiments as in c. Signals are graphed as the fraction of In DNA found in IPs after subtraction of Bd-associated signal. Values are means ± SD (n = 3 independent experiments). (e) pSer33 immunostaining (green) combined with telomeric DNA FISH (red) on cells as in a. Arrowheads point to examples of pSer33 TIFs. Scale bar, 5 μm. Percentages of cells with at least 3 pSer33 TIFs are graphed as means ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.005 (two-tailed Student’s t-test). (f) Speculative model for TRF1, TRF2 and TERRA interplay at telomeres. At a fully protected chromosome end TRF1 A domain prevents TRF2 B domain from interacting with TERRA. When TRF1 is replaced with TRF1ΔA, TERRA interaction with TRF2 B domain drives TERRA invasion into telomeric dsDNA and formation of telR-loops, which in turn promote pSer33 binding to telomeres and telomere loss. pSer33 might bind to both ss G-rich or C-rich telomeric DNA.', 'hash': '31dd6fbb05092df79bb2c1d167573cd18cd4521750b26099117693fb3c01e008'}]
|
{'emss-75371-f001': ['We then carried out invasion assays with telomeric RNA and DNA oligonucleotides in the presence of glutathione S-transferase (GST)-tagged recombinant TRF proteins (Supplementary Fig. 2a,b). When incubated with radiolabeled oligonucleotides followed by electrophoresis in denaturing polyacrylamide gels, none of the recombinant proteins utilized in this study affected the radioactive signal (Supplementary Fig. 2c), thus excluding the presence of contaminating RNase, DNase or phosphatase activities. GST-TRF1, GST-TRF2 and GST alone did not alter invasion of C-rich RNA and DNA nor of G-rich DNA at concentrations up to 80 nM (<xref ref-type="fig" rid="emss-75371-f001">Fig. 1</xref>). GST-TRF2, but not GST-TRF1 or GST, stimulated invasion of G-rich TERRA-like RNA (). GST-TRF2, but not GST-TRF1 or GST, stimulated invasion of G-rich TERRA-like RNA (<xref ref-type="fig" rid="emss-75371-f001">Figs. 1</xref> and and <xref ref-type="fig" rid="emss-75371-f002">2a</xref>) and R-loop formation () and R-loop formation (<xref ref-type="fig" rid="emss-75371-f002">Fig. 2a</xref> and and Supplementary Fig. 1d) in a concentration-dependent manner. Consistent with published data16,17, GST-TRF2 stimulated G-rich DNA invasion starting at ˜100 nM concentration, where maximum G-rich RNA invasion was already achieved (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3a</xref>). High concentrations of GST-TRF1 had no effect on RNA or DNA invasion (). High concentrations of GST-TRF1 had no effect on RNA or DNA invasion (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3a</xref>). A truncated variant of TRF2 lacking the B domain (GST-TRF2ΔB) failed to promote RNA invasion at low concentrations (). A truncated variant of TRF2 lacking the B domain (GST-TRF2ΔB) failed to promote RNA invasion at low concentrations (<xref ref-type="fig" rid="emss-75371-f002">Fig. 2a</xref>; ; Supplementary Fig. 2a,b), while, at high concentrations, it stimulated RNA and DNA invasion to similar extents although less efficiently than GST-TRF2 (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3a</xref>). A TRF2 variant lacking the C-terminal dsDNA binding Myb domain). A TRF2 variant lacking the C-terminal dsDNA binding Myb domain18 (GST-TRF2ΔM), and another variant unable to induce topological changes in telomeric DNA (GST-Topless)17 did not stimulate RNA or DNA invasion at any of the tested concentrations (<xref ref-type="fig" rid="emss-75371-f002">Fig. 2a</xref>; ; Supplementary Fig. 2a,b; data not shown). Finally, heat denatured GST-TRF2 also failed to induce RNA invasion (<xref ref-type="fig" rid="emss-75371-f002">Fig. 2a</xref>).).', 'As expected, in electrophoretic mobility shift assays (EMSAs), GST-TRF2ΔM did not bind to telomeric dsDNA substrates while GST-Topless bound similarly to GST-TRF2 (Supplementary Fig. 2d and Supplementary Table 1). GST-TRF2ΔB bound to dsDNA approximately four-fold less efficiently than GST-TRF2 (Supplementary Fig. 2d,e and Table 1), which is not sufficient to explain GST-TRF2ΔB inefficiency in stimulating RNA invasion. In fact, while GST-TRF2 promoted maximum RNA invasion already at ˜40 nM concentrations (<xref ref-type="fig" rid="emss-75371-f001">Figs. 1</xref> and and <xref ref-type="fig" rid="emss-75371-f002">2a</xref>), GST-TRF2ΔB only induced a ˜1.5 fold increase in RNA invasion at ˜400 nM concentrations (), GST-TRF2ΔB only induced a ˜1.5 fold increase in RNA invasion at ˜400 nM concentrations (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3a</xref>). We conclude that TRF2 stimulates G-rich RNA invasion with higher efficiencies than it does with G-rich DNA. This novel TRF2-associated activity requires TRF2 binding to and wrapping of ds telomeric DNA, as is also the case for G-rich DNA invasion). We conclude that TRF2 stimulates G-rich RNA invasion with higher efficiencies than it does with G-rich DNA. This novel TRF2-associated activity requires TRF2 binding to and wrapping of ds telomeric DNA, as is also the case for G-rich DNA invasion16,17, and is specifically potentiated by TRF2 B domain. Further reinforcing the importance of the B domain for efficient RNA invasion, a chimeric protein where the A domain of TRF1 was swapped with the B domain of TRF2 (GST-BTRF1ΔA) readily induced RNA invasion (Supplementary Figs. 2a,b and 3a), despite binding to telomeric dsDNA ˜18 fold less efficiently than GST-TRF2 (Supplementary Fig. 2e and Supplementary Table 1).'], 'emss-75371-f002': ['We next performed G-rich RNA invasion reactions with fixed amounts of GST-TRF2 combined with increasing amounts of GST-TRF1, and observed a progressive decline in invaded plasmid species (<xref ref-type="fig" rid="emss-75371-f002">Figs. 2b</xref> and and <xref ref-type="fig" rid="emss-75371-f003">3b</xref>). In contrast, GST-TRF1 did not inhibit the G-rich DNA invasion stimulated by high concentrations of GST-TRF2 (). In contrast, GST-TRF1 did not inhibit the G-rich DNA invasion stimulated by high concentrations of GST-TRF2 (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3b</xref>). A truncated variant of TRF1 lacking the A domain (GST-TRF1ΔA) failed to neutralize GST-TRF2-stimulated RNA invasion at any of the tested concentrations (). A truncated variant of TRF1 lacking the A domain (GST-TRF1ΔA) failed to neutralize GST-TRF2-stimulated RNA invasion at any of the tested concentrations (<xref ref-type="fig" rid="emss-75371-f002">Figs. 2b</xref> and and <xref ref-type="fig" rid="emss-75371-f003">3b</xref>; ; Supplementary Fig. 2a,b). As with GST-TRF1, GST-TRF1ΔA did not promote RNA invasion (Supplementary Fig. 3a) nor did it prevent GST-TRF2 from inducing DNA invasion at high concentrations (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3b</xref>). Because the Myb domains of TRF1 and TRF2 bind the same dsDNA substrate). Because the Myb domains of TRF1 and TRF2 bind the same dsDNA substrate18, we considered the possibility that GST-TRF1 counteracted GST-TRF2 by diminishing its density on DNA and GST-TRF1ΔA simply binds dsDNA substantially less efficiently than its full-length counterpart. However, GST-TRF1ΔA bound only ˜1.1-to-1.5 fold less efficiently than GST-TRF1 to telomeric dsDNA substrates (Supplementary Fig. 2d,e and Supplementary Table 1). These minor differences are unlikely to explain why GST-TRF1ΔA did not suppress TRF2-mediated RNA invasion, as the chosen concentration ranges for TRF1 proteins covered a very wide range going from 0 to 400 nM (<xref ref-type="fig" rid="emss-75371-f002">Figs. 2b</xref> and and <xref ref-type="fig" rid="emss-75371-f003">3b</xref>).).'], 'emss-75371-f004': ['To shed some light on the mechanisms through which the A domain suppresses TRF2-stimulated RNA invasion, we set up RNA EMSAs and found that, consistent with previously published data20, GST-TRF2, GST-TRF2ΔB, GST-TRF1 and GST-TRF1ΔA bound to G-rich telomeric RNA oligonucleotides but not to C-rich or random sequence RNA oligonucleotides nor to telomeric or random sequence DNA oligonucleotides (<xref ref-type="fig" rid="emss-75371-f004">Fig. 4a,b</xref> and and Supplementary Fig. 3e). TRF2ΔB bound to G-rich RNA less efficiently than GST-TRF2, while GST-TRF1 and GST-TRF1ΔA binding kinetics were indistinguishable (<xref ref-type="fig" rid="emss-75371-f004">Fig. 4a,b</xref> and and Supplementary Table 1). Consistently, GST fused to the B domain (GST-B) bound to G-rich RNA, while GST fused to the A domain (GST-A) did not (<xref ref-type="fig" rid="emss-75371-f004">Fig. 4a,b</xref>; ; Supplementary Fig. 2a,b and Supplementary Table 1). Fusing the A domain to the N-terminus of the B domain (GST-AB) largely abolished RNA binding, while fusing the A domain to the N-terminus of TRF2 (GST-ATRF2) decreased binding to efficiencies comparable to that of GST-TRF2ΔB (<xref ref-type="fig" rid="emss-75371-f004">Fig. 4a,b</xref>; ; Supplementary Fig. 2a,b and Supplementary Table 1). As expected, GST-A, GST-B and GST-AB did not bind to telomeric dsDNA (Supplementary Fig. 2f).', 'Based on these experiments, we propose that the direct binding of G-rich RNA to the B domain drives the highly efficient TRF2-stimulated RNA invasion, and that the A domain of TRF1 counteracts this mechanism most likely by impeding the interaction between the B domain and G-rich RNA. Consistent with this hypothesis, at low concentrations, GST-ATRF2 failed to promote efficient RNA invasion although its ability to bind to telomeric dsDNA was not compromised (<xref ref-type="fig" rid="emss-75371-f004">Fig. 4c</xref>; ; Supplementary Fig. 2d and Supplementary Table 1). Similar to GST-TRF2ΔB, GST-ATRF2 mildly stimulated RNA and DNA invasion at high protein concentrations (<xref ref-type="fig" rid="emss-75371-f003">Fig. 3a</xref>).).'], 'emss-75371-f005': ['Our in vitro data suggest that TRF1 and TRF2 crosstalk to regulate TERRA interaction with telomeric dsDNA in cells. We depleted TRF1 using short interference RNAs (siRNAs) in telomerase-positive HeLa and ALT (Alternative Lengthening of Telomeres21) U2OS cancer cells, and quantified telR-loops by immunoprecipitation with the RNA:DNA hybrid-specific S9.6 antibody (S9.6 DRIPs), followed by dot-blot hybridization22,23. In both cell lines, TRF1 depletion increased telR-loops but not R-loops containing Alu repeat sequences (<xref ref-type="fig" rid="emss-75371-f005">Fig. 5a</xref>; ; Supplementary Figs. 4a,b and 5a-c). Ectopic expression of siRNA-insensitive, flag-tagged TRF1 (fl-TRF1) in TRF1-depleted HeLa cells prevented telR-loop accumulation, while an A-deleted counterpart (fl-ΔA) did not (<xref ref-type="fig" rid="emss-75371-f005">Fig. 5b</xref>; ; Supplementary Fig. 4a,b). In fact, fl-ΔA expression alone increased telR-loops (<xref ref-type="fig" rid="emss-75371-f005">Fig. 5b</xref>; ; Supplementary Figs. 4b), suggesting a dominant-negative effect likely through replacement of endogenous TRF1 at telomeres (Supplementary Fig. 6a-c). Notably, combining TRF1 depletion with fl-ΔA expression did not significantly increase telR-loops above single conditions (<xref ref-type="fig" rid="emss-75371-f005">Fig. 5b</xref>), indicating impairment of the same TRF1-associated function in suppressing telR-loops. The TRF1 A domain recruits the poly(ADP-ribose) polymerase Tankyrase 1 (TNK1) to telomeres), indicating impairment of the same TRF1-associated function in suppressing telR-loops. The TRF1 A domain recruits the poly(ADP-ribose) polymerase Tankyrase 1 (TNK1) to telomeres24. We siRNA-depleted TNK1 in HeLa and U2OS cells and observed the expected accumulation of mitotic cells25 (Supplementary Fig. 7a,b). However, TNK1 depletion did not alter telR-loop levels (Supplementary Fig. 7c), excluding that the increased telR-loops in cells depleted of TRF1 or expressing fl-ΔA results from insufficient TNK1 at telomeres.', 'In ALT cells, aberrant accumulation of telR-loops leads to telomere dysfunction-induced foci (TIFs) containing telomeric DNA and a serine 33-phosphorylated form of the ssDNA binding protein RPA32 (pSer33), and to metaphase TFEs22. pSer33 also accumulates at R-loops throughout the genome26. TRF1 depletion and fl-ΔA expression increased pSer33 TIFs and TFEs both in HeLa (<xref ref-type="fig" rid="emss-75371-f005">Figs. 5c,d</xref> and and <xref ref-type="fig" rid="emss-75371-f006">6a,b</xref>; ; Supplementary Fig. 4c,d) and U2OS (<xref ref-type="fig" rid="emss-75371-f007">Fig. 7a,b</xref> and and Supplementary Fig. 5d) cells. TFEs were detected at chromosome ends generated by both lagging and leading strand replication (<xref ref-type="fig" rid="emss-75371-f006">Figs. 6a,b</xref> and and <xref ref-type="fig" rid="emss-75371-f007">7a,b</xref>). fl-TRF1, but not fl-ΔA, prevented accumulation of pSer33 TIFs and TFEs in cells depleted of endogenous TRF1 (). fl-TRF1, but not fl-ΔA, prevented accumulation of pSer33 TIFs and TFEs in cells depleted of endogenous TRF1 (<xref ref-type="fig" rid="emss-75371-f005">Figs. 5c,d</xref> and and <xref ref-type="fig" rid="emss-75371-f006">6a,b</xref>; ; Supplementary Fig. 4c,d). As expected10, FTs also accumulated in TRF1 depleted cells (<xref ref-type="fig" rid="emss-75371-f006">Fig. 6a,b</xref> and and <xref ref-type="fig" rid="emss-75371-f007">7a</xref>; ; Supplementary Fig. 4d); both fl-TRF1 and fl-ΔA averted the increase in FTs in the same cells (<xref ref-type="fig" rid="emss-75371-f006">Fig. 6a,b</xref> and and Supplementary Fig. 4d), and suppressed spontaneous FTs in cells where endogenous TRF1 was not depleted (<xref ref-type="fig" rid="emss-75371-f006">Figs. 6b</xref> and and <xref ref-type="fig" rid="emss-75371-f007">7b</xref>).).', 'Ectopic expression of a myc-tagged version of the human RNaseH1 endoribonuclease (RH1wt) prevented accumulation of telR-loops, pSer33 TIFs and TFEs upon TRF1 depletion or fl-ΔA expression, while a catalytically dead RNaseH1 (RH1CD)19 did not (<xref ref-type="fig" rid="emss-75371-f005">Figs. 5b,d</xref> and and <xref ref-type="fig" rid="emss-75371-f006">6b</xref>; ; Supplementary Fig. 4a,b,e-l). FTs accumulated at similar frequencies in TRF1 depleted cells expressing either RH1wt or RH1CD (<xref ref-type="fig" rid="emss-75371-f006">Fig. 6b</xref>). These data establish that suppression of pSer33 TIFs and TFEs through restriction of telR-loops are previously unappreciated TRF1 functions that strictly depend on the A domain and are genetically separable from fragility suppression.). These data establish that suppression of pSer33 TIFs and TFEs through restriction of telR-loops are previously unappreciated TRF1 functions that strictly depend on the A domain and are genetically separable from fragility suppression.'], 'emss-75371-f008': ['Our biochemical data suggest that TRF1 A domain suppresses telR-loops induced by TRF2-stimulated TERRA invasion. To test this hypothesis in cells, we first over-expressed N-terminally myc-tagged TRF2 (m-TRF2) and ATRF2 (m-ATRF2) in HeLa cells and found that m-TRF2 but not m-ATRF2 increased telR-loop and pSer33 TIF levels (<xref ref-type="fig" rid="emss-75371-f008">Fig. 8 a-e</xref>). This might explain the stochastic telomere loss and consequent TFE appearance observed in human and mouse cells overexpressing TRF2). This might explain the stochastic telomere loss and consequent TFE appearance observed in human and mouse cells overexpressing TRF227,28. Furthermore, depleting endogenous TRF2 prevented accumulation of telR-loops in TRF1-depleted (Supplementary Fig. 8a,b) and in fl-ΔA-expressing (Supplementary Fig. 8c,d) cells. Consistent with the notion that uncontrolled TRF2-dependent telR-loops cause telomere instability, TRF2 depletion averted the insurgence of pSer33 TIFs and TFEs in fl-ΔA-expressing cells (<xref ref-type="fig" rid="emss-75371-f005">Fig. 5d</xref>; ; Supplementary Fig. 8e,f). As expected, TRF2 depletion did not affect FT frequencies (Supplementary Fig. 8f).', 'We have identified two novel, interdependent activities at human telomeres: i) TRF2 promotes TERRA invasion into telomeric dsDNA likely through the ability of the B domain to directly interact with TERRA possibly recruiting it to telomeres, and through the ability of the TRFH domain to wrap telomeric dsDNA and alter its topology; ii) TRF1, through its A domain, maintains constant control of this TERRA invasion activity dependent on TRF2 (<xref ref-type="fig" rid="emss-75371-f008">Fig. 8f</xref>). The molecular basis of how TRF1 discriminates RNA from DNA during TRF2-mediated strand invasion remains to be determined. Still, because TRF2 B domain is only able to interact with TERRA and not with telomeric ssDNA, this discrimination probably derives from TRF1 A domain ability to prevent TERRA from interacting with TRF2 B domain. We have also shown that in the absence of controlled crosstalk between these novel activities (a situation recapitulated by TRF1 depletion or TRF1ΔA expression in cells), aberrant telR-loops accumulate and lead to pSer33 TIFs and telomere loss (). The molecular basis of how TRF1 discriminates RNA from DNA during TRF2-mediated strand invasion remains to be determined. Still, because TRF2 B domain is only able to interact with TERRA and not with telomeric ssDNA, this discrimination probably derives from TRF1 A domain ability to prevent TERRA from interacting with TRF2 B domain. We have also shown that in the absence of controlled crosstalk between these novel activities (a situation recapitulated by TRF1 depletion or TRF1ΔA expression in cells), aberrant telR-loops accumulate and lead to pSer33 TIFs and telomere loss (<xref ref-type="fig" rid="emss-75371-f008">Fig. 8f</xref>). We therefore propose that uncontrolled TRF2 and telR-loops pose authentic threats to telomere integrity, and that TRF1 directly contributes to solving the end-protection problem by suppressing unscheduled telR-loop-induced telomere instability.). We therefore propose that uncontrolled TRF2 and telR-loops pose authentic threats to telomere integrity, and that TRF1 directly contributes to solving the end-protection problem by suppressing unscheduled telR-loop-induced telomere instability.']}
|
TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R-loops
| null |
Nat Struct Mol Biol
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1519286400
|
[{'@Label': 'BACKGROUND/OBJECTIVES', '#text': 'Sarcopenia is defined as the loss of muscle mass or function with aging and is associated with adverse outcomes. Telomere shortening is associated with mortality, yet its relationship with sarcopenia is unknown.'}, {'@Label': 'SUBJECTS/METHODS', '#text': 'Adults ≥60 years from the 1999-2002 NHANES with body composition measures were identified. Sarcopenia was defined using the two Foundation for the National Institute of Health definitions: appendicular lean mass (ALM) (men <19.75; women <15.02\u2009kg); or ALM divided by body mass index (BMI) (ALM:BMI, men <0.789; women <0.512). Telomere length was assessed using quantitative PCR. Regression models predicted telomere length with sarcopenia (referent\u2009=\u2009no sarcopenia).'}, {'@Label': 'RESULTS', '#text': 'We identified 2672 subjects. Mean age was 70.9 years (55.5% female). Prevalence of ALM and ALM:BMI sarcopenia was 29.2 and 22.1%. Deaths were higher in persons with sarcopenia as compared to those without sarcopenia (ALM: 46.4 vs. 33.4%, p\u2009<\u20090.001; ALM:BMI: 46.7 vs. 33.2%, p\u2009<\u20090.001). No adjusted differences were observed in telomere length in those with/without sarcopenia (ALM: 0.90 vs. 0.92, p\u2009=\u20090.74, ALM:BMI 0.89 vs. 0.92, p\u2009=\u20090.24). In men with ALM:BMI-defined sarcopenia, adjusted telomere length was significantly lower compared to men without sarcopenia (0.85 vs. 0.91, p\u2009=\u20090.013). With sarcopenia, we did not observe a significant association between telomere length and mortality (ALM: HR 1.11 [0.64,1.82], p\u2009=\u20090.68; ALM:BMI: HR 0.97 [0.53,1.77], p\u2009=\u20090.91), but noted significance in those without sarcopenia with mortality (ALM: HR 0.59 [0.40,0.86], p\u2009=\u20090.007; ALM:BMI: HR 0.62 [0.42,0.91]; p\u2009=\u20090.01).'}, {'@Label': 'CONCLUSIONS', '#text': 'We observed a potentially inverse relationship between telomere length and mortality in those without sarcopenia but did not observe a significant relationship between telomere length and mortality in the presence of sarcopenia.'}]
|
[
"Aged",
"Aged, 80 and over",
"Body Composition",
"Body Mass Index",
"Female",
"Humans",
"Male",
"Middle Aged",
"Nutrition Surveys",
"Sarcopenia",
"Telomere Homeostasis"
] |
other
|
PMC5808845
| null |
36
|
[
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] |
Nat Struct Mol Biol. 2018 Feb 22; 25(2):147-153
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NO-CC CODE
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(A) Fluorescence images of typical cells on various substrates
after 1 and 3 days of culture. The cells were stained for the focal
adhesion protein vinculin (green), actin cytoskeleton (red), and cellular
nuclei (blue). (B) Cytomorphometric evaluation of area, perimeter,
and Feret’s diameter. (C) Quantitative analysis of fluorescence
intensity of focal adhesions (n = 30).
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ao9b02751_0003
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2
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98c6b93e9861c21f22032642b7d7e116afc05ab939b0384f382bfbaad72f878a
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ao9b02751_0003.jpg
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multiple
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multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
729,
781
] |
[{'image_id': 'ao9b02751_0007', 'image_file_name': 'ao9b02751_0007.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0007.jpg', 'caption': 'No caption found', 'hash': 'e2837aedc2ef6691204bf5e878019be18b45eebccf271505f75348f11630491a'}, {'image_id': 'ao9b02751_0006', 'image_file_name': 'ao9b02751_0006.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0006.jpg', 'caption': '(A) Images of mineralization\ncapacity achieved by Alizarin Red\nAssay Kit after 21 days of culture. (B) Corresponding quantitative\nanalysis.', 'hash': '9c5befc14afc52c3ab79baa66e7c5342363c720e2e8efe948f1ec8b4317bbfa3'}, {'image_id': 'ao9b02751_0001', 'image_file_name': 'ao9b02751_0001.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0001.jpg', 'caption': '(A,a) SEM images of rutile nanorod films.\n(B,b) SEM images of GelMA-incorporated\nrutile nanorod films. (C) Release profile of naringin from naringin-M\nand naringin-S. (D,E) Corresponding linear fitting curves of release\nbehaviors for naringin-M and naringin-S, respectively.', 'hash': '26455a65e66f2b11b1bda9134a110a1544fc3bdbeeda7881daff28a071d72975'}, {'image_id': 'ao9b02751_0002', 'image_file_name': 'ao9b02751_0002.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0002.jpg', 'caption': 'SEM images of MSC morphology on different substrates:\n(A) Ti, (B)\nGelMA, (C) naringin-M, and (D) naringin-S. (a–d) Enlarged images\naccordingly. Filopodia were indicated by the arrow.', 'hash': 'ca8c05c50fa3f833cd2244dbceb60a30cb026c348357b4c8dc03b7ddd3b66be7'}, {'image_id': 'ao9b02751_0005', 'image_file_name': 'ao9b02751_0005.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0005.jpg', 'caption': '(A) Quantitative analysis of real-time PCR for\nrelative expression\nof osteogenesis genes after 7 and 14 days of culture. (B) Images of\nALP activity done by Alkaline Phosphatase Assay Kit after 7 days of\nculture. (C) Quantitative analysis of ALP activity.', 'hash': 'ee993bd1577c1723da2f821b9899dee3272d998b2507c45a4137cceaa08cc184'}, {'image_id': 'ao9b02751_0004', 'image_file_name': 'ao9b02751_0004.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0004.jpg', 'caption': '(A) Cell viability using staining-derived fluorescent images. The\nlive cells were stained with calcein (green), and the dead cells were\nstained with ethidium (red). (B) CCK-8 assays.', 'hash': 'e9481bcd94abd82685f62321673a84327c49cdad0ca2a249f4d8f31ad288e1bf'}, {'image_id': 'ao9b02751_0003', 'image_file_name': 'ao9b02751_0003.jpg', 'image_path': '../data/media_files/PMC6868884/ao9b02751_0003.jpg', 'caption': '(A) Fluorescence images of typical cells on various substrates\nafter 1 and 3 days of culture. The cells were stained for the focal\nadhesion protein vinculin (green), actin cytoskeleton (red), and cellular\nnuclei (blue). (B) Cytomorphometric evaluation of area, perimeter,\nand Feret’s diameter. (C) Quantitative analysis of fluorescence\nintensity of focal adhesions (n = 30).', 'hash': '98c6b93e9861c21f22032642b7d7e116afc05ab939b0384f382bfbaad72f878a'}]
|
{'ao9b02751_0001': ['The surface morphologies\nof TiO2 nanorod coatings before\nand after GelMA incorporation were observed by scanning electron microscopy\n(SEM). As shown in <xref rid="ao9b02751_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>A, the dimension and height of nanorods were nearly 100 and\n600 nm, respectively. These results were consistent with our previous\nwork.<xref rid="ao9b02751_0001" ref-type="fig">1</xref>A, the dimension and height of nanorods were nearly 100 and\n600 nm, respectively. These results were consistent with our previous\nwork.15 The distinct spacing between nanorods\nwas further beneficial for the incorporation of GelMA (<xref rid="ao9b02751_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>B). As shown in <xref rid="ao9b02751_0001" ref-type="fig">1</xref>B). As shown in <xref rid="ao9b02751_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>C, the release profile of naringin\nwas sustained after an apparent burst release. We also analyzed the\nrelease kinetics (<xref rid="ao9b02751_0001" ref-type="fig">1</xref>C, the release profile of naringin\nwas sustained after an apparent burst release. We also analyzed the\nrelease kinetics (<xref rid="ao9b02751_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>D,E). During the first stage of burst release, the intercept\nrepresented the initial percentage of released naringin. The parameter\nwas 2.70 for naringin-M and 14.99 for naringin-S, which suggested\nthat more drug might be reserved in naringin-M. During the second\nstage of sustained release, the slope represented the rate of released\nbehavior. The parameter was 3.76 for naringin-M and 3.01 for naringin-S,\nwhich revealed higher concentration of naringin released from naringin-M\nin unit time. According to the release kinetics, we inferred that\nthese two typical coatings with distinct release kinetics of naringin\ncould provide an ideal model to further explore their activities for\nosteogenic differentiation of MSCs.<xref rid="ao9b02751_0001" ref-type="fig">1</xref>D,E). During the first stage of burst release, the intercept\nrepresented the initial percentage of released naringin. The parameter\nwas 2.70 for naringin-M and 14.99 for naringin-S, which suggested\nthat more drug might be reserved in naringin-M. During the second\nstage of sustained release, the slope represented the rate of released\nbehavior. The parameter was 3.76 for naringin-M and 3.01 for naringin-S,\nwhich revealed higher concentration of naringin released from naringin-M\nin unit time. According to the release kinetics, we inferred that\nthese two typical coatings with distinct release kinetics of naringin\ncould provide an ideal model to further explore their activities for\nosteogenic differentiation of MSCs.', 'Recently, GelMA has been\nwidely used to control the drug delivery.\nGelMA, acting as carriers, can interact with drug by physisorption\nand covalent linking. In general, drug delivery from GelMA is mediated\nby diffusion and degradation.16 At first,\ndiffusion dominances the release profile because matrix degradation\nis slow.17 Drug is immobilized by macro/nano-entrapment.\nOnce GelMA is dissolved in the solvent, the diffusion of drug from\nthe porous structure occurs. The molecular weight of drugs and the\npore size of GelMA play important roles in the release process.18−20 The degradation of GelMA can be divided into bulk and surface erosion.16 Bulk erosion is homogenous when GelMA swelling\nis faster than the polymer disintegration. In contrast, surface erosion\nis heterogeneous when the polymer disintegration is predominant. A\nnumber of parameters are related in the process such as the chemical\nstructure of GelMA, exposure time to UV light, the concentration of\nthe GelMA hydrogel, and others.21,22 In this work, we designed\ntwo coatings to achieve degradation-type release (naringin-M) and\ndiffusion-type release (naringin-S). Naringin delivery was constant\nand sustained after a burst release from two coatings (<xref rid="ao9b02751_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>C). However, the release kinetics\nof two coating was different (<xref rid="ao9b02751_0001" ref-type="fig">1</xref>C). However, the release kinetics\nof two coating was different (<xref rid="ao9b02751_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>D,E). Because the molecular weight of naringin was\nlow, the entrapped naringin could be released from the porous structure\nof GelMA easily. Therefore, the initial percentage of released naringin\nfrom naringin-S was higher than that of naringin-M.<xref rid="ao9b02751_0001" ref-type="fig">1</xref>D,E). Because the molecular weight of naringin was\nlow, the entrapped naringin could be released from the porous structure\nof GelMA easily. Therefore, the initial percentage of released naringin\nfrom naringin-S was higher than that of naringin-M.'], 'ao9b02751_0002': ['We first observed that the cellular morphology on various\nsubstrates.\nAs shown in <xref rid="ao9b02751_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>, the morphology of MSCs cultured on naringin-M and naringin-S remarkably\ndisplayed more filopodia compared to that of the other two substrates.\nThese results suggested that the coatings loaded with naringin promoted\nthe spreading of MSCs.<xref rid="ao9b02751_0002" ref-type="fig">2</xref>, the morphology of MSCs cultured on naringin-M and naringin-S remarkably\ndisplayed more filopodia compared to that of the other two substrates.\nThese results suggested that the coatings loaded with naringin promoted\nthe spreading of MSCs.'], 'ao9b02751_0003': ['Immunofluorescence was further employed to observe the adhesion\nbehaviors of MSCs on various substrates. As shown in <xref rid="ao9b02751_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>A, the cellular area was larger\non naringin-M and naringin-S compared to on that of cells on Ti and\nGelMA. Moreover, quantitative analysis also confirmed the obvious\nattachment and spreading of MSCs on the coatings loaded with naringin\nespecially on day 3 (<xref rid="ao9b02751_0003" ref-type="fig">3</xref>A, the cellular area was larger\non naringin-M and naringin-S compared to on that of cells on Ti and\nGelMA. Moreover, quantitative analysis also confirmed the obvious\nattachment and spreading of MSCs on the coatings loaded with naringin\nespecially on day 3 (<xref rid="ao9b02751_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>B). It was worth to note that MSCs on naringin-M displayed\nmore vinculin with higher fluorescence intensity than the others on\nday 3.<xref rid="ao9b02751_0003" ref-type="fig">3</xref>B). It was worth to note that MSCs on naringin-M displayed\nmore vinculin with higher fluorescence intensity than the others on\nday 3.', 'Moreover,\nwe demonstrated that the release of naringin was beneficial\nto the attachment (<xref rid="ao9b02751_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>), osteogenesis (<xref rid="ao9b02751_0003" ref-type="fig">3</xref>), osteogenesis (<xref rid="ao9b02751_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>), and mineralization (<xref rid="ao9b02751_0005" ref-type="fig">5</xref>), and mineralization (<xref rid="ao9b02751_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>) of MSCs. Though the biological activities\nof naringin have been confirmed,<xref rid="ao9b02751_0006" ref-type="fig">6</xref>) of MSCs. Though the biological activities\nof naringin have been confirmed,23−25 the mechanism of its\nosteo-conductivity is complicated and yet to be illuminated. Several\nstudies manifested that extracellular regulated protein kinases (ERK)\n1/2 were found to be activated by naringin, and osteogenic differentiation\nwas repressed when the inhibitor of ERK 1/2 was used.26,27 The activation of ERK 1/2 is downstream of the Ras family.28 Lin et al. demonstrated that the Ras family\nwas remarkably activated by naringin.29 Furthermore, the ERK 1/2 pathway can regulate osteogenic differentiation\nthrough microRNA.30 Meanwhile, GelMA hydrogels\nand collagen have also been demonstrated to regulate the osteogenic\ndifferentiation of MSCs via ERK signaling pathways.31,32 In this study, the osteogenic differentiation potential of MSCs\non naringin-M was more remarkable compared to that of naringin-S.\nFor the naringin-S, the naringin was entrapped in the pore structure\nof GelMA and released quickly. When it comes to the naringin-M, the\nnaringin not only physically absorbed on GelMA but also covalently\nbonded with GelMA during the curing process. Therefore, the detected\nconcentration of naringin released from naringin-M was lower than\nthat from naringin-S at the initial stage. Nonetheless, the synergistic\nrelease of naringin with degraded GelMA from naringin-M enhanced the\nosteogenic differentiation of MSCs more effectively. These results\nsuggested that there might be a synergistic effect of naringin and\nGelMA to regulate the osteogenic differentiation of MSCs because of\nthe similar signaling pathway potentially involved, which needs further\ninvestigation.'], 'ao9b02751_0004': ['The cellular viability and proliferation were evaluated by\nusing\nlive/dead assay and CCK-8 assay. As shown in <xref rid="ao9b02751_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>A, the density of MSCs increased obviously\non naringin-M and naringin-S. Especially the number of attached cells\nwas significantly upregulated on naringin-M and naringin-S even after\n5 days of culture. These results were further confirmed by the quantitative\nanalysis of CCK-8 results (<xref rid="ao9b02751_0004" ref-type="fig">4</xref>A, the density of MSCs increased obviously\non naringin-M and naringin-S. Especially the number of attached cells\nwas significantly upregulated on naringin-M and naringin-S even after\n5 days of culture. These results were further confirmed by the quantitative\nanalysis of CCK-8 results (<xref rid="ao9b02751_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>B), which could be attributed to the bioactivity of\nnaringin.<xref rid="ao9b02751_0004" ref-type="fig">4</xref>B), which could be attributed to the bioactivity of\nnaringin.'], 'ao9b02751_0005': ['Assessment of osteogenesis genes was achieved by real-time polymerase\nchain reaction (PCR). The results are shown in <xref rid="ao9b02751_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>A. After 7 days of culture, all expressions\nof osteogenic-related genes were upregulated on naringin-M compared\nto the others. After 14 days of culture, there was no obvious difference\nbetween the coatings loaded with naringin, but expression of osteogenesis\ngenes was notably upregulated when compared to Ti and GelMA. What\nis more, the larger area of ALP-positive with higher intensity displayed\non naringin-M and naringin-S than on the two others after 7 days of\nculture as shown in <xref rid="ao9b02751_0005" ref-type="fig">5</xref>A. After 7 days of culture, all expressions\nof osteogenic-related genes were upregulated on naringin-M compared\nto the others. After 14 days of culture, there was no obvious difference\nbetween the coatings loaded with naringin, but expression of osteogenesis\ngenes was notably upregulated when compared to Ti and GelMA. What\nis more, the larger area of ALP-positive with higher intensity displayed\non naringin-M and naringin-S than on the two others after 7 days of\nculture as shown in <xref rid="ao9b02751_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>B. Moreover, the quantitative analysis revealed remarkably\nupregulated ALP activity on naringin-M (<xref rid="ao9b02751_0005" ref-type="fig">5</xref>B. Moreover, the quantitative analysis revealed remarkably\nupregulated ALP activity on naringin-M (<xref rid="ao9b02751_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>C).<xref rid="ao9b02751_0005" ref-type="fig">5</xref>C).'], 'ao9b02751_0006': ['The ability of mineralization was evaluated by Alizarin Red\nAssay\nkit after long-term culture. The results are shown in <xref rid="ao9b02751_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>. The more obvious area of\nAlizarin-positive on naringin-M and naringin-S compared to on the\ntwo others. Furthermore, the quantitative analysis confirmed the significantly\nupregulated osteogenesis on naringin-M.<xref rid="ao9b02751_0006" ref-type="fig">6</xref>. The more obvious area of\nAlizarin-positive on naringin-M and naringin-S compared to on the\ntwo others. Furthermore, the quantitative analysis confirmed the significantly\nupregulated osteogenesis on naringin-M.']}
|
Controlled Release of Naringin in GelMA-Incorporated
Rutile Nanorod Films to Regulate Osteogenic Differentiation of Mesenchymal
Stem Cells
| null |
ACS Omega
|
1573027200
|
Naringin, a Chinese herbal medicine, has been demonstrated to concentration-dependently promote osteogenic differentiation of mesenchymal stem cells (MSCs). However, it remains a challenge to load naringin on coatings for osteogenesis and further control the release kinetics. Here, we demonstrated that the release behavior of naringin on rutile nanorod films could be controlled by either mixing naringin with gelatin methacryloyl (GelMA) before spinning onto the films or soaking the obtained GelMA-incorporated films with the naringin solution to achieve the distinct degradation-type release and diffusion-type release, respectively. We further revealed that the naringin-loaded coatings facilitated adhesion, proliferation and late differentiation, and mineralization of MSCs. Our findings provided a novel strategy to engineer the coatings with controlled release of naringin and emphasized the bioactivity of naringin for the osteogenic differentiation of MSCs.
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other
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PMC6868884
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32
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[
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] |
ACS Omega. 2019 Nov 6; 4(21):19350-19357
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NO-CC CODE
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Adhesion to nascent proteins controls hMSC mechanosensing in degradable hydrogels.a Representative images (scale bar 200 μm, inset 20 μm) of nascent protein deposition in MMP-degradable NorHA hydrogels after 6 days treatment without (control, note same image as in Fig. 2b), with monoclonal antibodies against integrin alpha 2 (anti α2, 20 μg/mL) or human fibronectin (HFN7.1, 5 μg/mL) or with soluble RGD (sol RGD, 0.5 mM). b Quantification of cell aspect ratio and accumulated nascent protein thickness deposited by hMSCs encapsulated in degradable NorHA hydrogels (n = 133 cells (control), n = 155 cells (anti α2), and n = 152 cells (HFN7.1), n = 124 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in a). c Representative images and d quantification of nuclear/cytoplasmic (nuc/cyto) YAP/TAZ ratios of hMSCs encapsulated in degradable NorHA hydrogels, cultured for 6 days in adipogenic-osteogenic media (scale bar 50 μm), quantifications: n = 60 cells (control), n = 51 cells (anti α2), and n = 40 cells (HFN7.1), n = 39 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in c). e Immunostaining for fatty-acid binding protein (FABP, adipogenic marker) and osteocalcin (Oc, osteogenic marker) after 14 days in adipogenic-osteogenic media (scale bar 50 μm). f Quantification of positively stained cells (percentage,%) towards osteogenesis (Oc positive) and adipogenesis (FABP positive) after 14 days, n = 6 samples from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, two-way ANOVA with Bonferroni post hoc).
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multiple panels: images & plots
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[
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[{'image_id': 'nihms-1520816-f0004', 'image_file_name': 'nihms-1520816-f0004.jpg', 'image_path': '../data/media_files/PMC6650309/nihms-1520816-f0004.jpg', 'caption': 'Dynamic hydrogel composition modulates viscoelastic properties and cell spreading.a Schematic of guest-host double-network (DN) hydrogel formation, from the combination of (i) covalently crosslinked HA hydrogel (MeHA) without RGD peptide and crosslinked with 1,4-dithiothreitol (DTT) and (ii) guest-host (GH) hydrogel assembled through mixing of HA modified with cyclodextrin (host) or adamantane (guest). Rheological measurements (1 Hz, 0.5% strain) of storage modulus (G’, elastic component) and loss modulus (G”, viscous component) for DN hydrogels with b increasing GH polymer concentration at a given covalent crosslink ratio (0.3) and c increasing covalent crosslinking (ratio of thiols to methacrylates) at a given GH polymer concentration (2.50%, n = 3 independent measurements per group, mean ± SD). d Representative images of F-Actin immunostaining and quantification of aspect ratio of hMSCs encapsulated in DN hydrogels (G’ 3.5 ± 0.45 kPa, n = 3 independent measurements per group, mean ± SD) with different GH concentration but same covalent crosslinking ratio (0.3) (scale bar 20 μm), quantifications: n = 34 cells (0.00%), n = 58 cells (1.25%, 2.50%) and n = 56 cells (3.50%) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for images on the left. e Representative images of F-Actin immunostaining and quantification of aspect ratio of hMSCs encapsulated in DN hydrogels with different covalent crosslinking ratios but same GH polymer concentration (2.50%). Note that image of 0.3 corresponds to 2.50% in d) (scale bar 20 μm), quantifications: n = 46 cells (0.2) and n = 49 (0.4) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for images on the left, # GH only hydrogels (i.e. no covalent crosslinks) were not stable over 6 days in culture).', 'hash': 'd568fd4cf58bd8c37aaa9ceb22f256a0ce6e6280b07891fbc563b7cc3693daaa'}, {'image_id': 'nihms-1520816-f0003', 'image_file_name': 'nihms-1520816-f0003.jpg', 'image_path': '../data/media_files/PMC6650309/nihms-1520816-f0003.jpg', 'caption': 'Adhesion to nascent proteins controls hMSC mechanosensing in degradable hydrogels.a Representative images (scale bar 200 μm, inset 20 μm) of nascent protein deposition in MMP-degradable NorHA hydrogels after 6 days treatment without (control, note same image as in Fig. 2b), with monoclonal antibodies against integrin alpha 2 (anti α2, 20 μg/mL) or human fibronectin (HFN7.1, 5 μg/mL) or with soluble RGD (sol RGD, 0.5 mM). b Quantification of cell aspect ratio and accumulated nascent protein thickness deposited by hMSCs encapsulated in degradable NorHA hydrogels (n = 133 cells (control), n = 155 cells (anti α2), and n = 152 cells (HFN7.1), n = 124 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in a). c Representative images and d quantification of nuclear/cytoplasmic (nuc/cyto) YAP/TAZ ratios of hMSCs encapsulated in degradable NorHA hydrogels, cultured for 6 days in adipogenic-osteogenic media (scale bar 50 μm), quantifications: n = 60 cells (control), n = 51 cells (anti α2), and n = 40 cells (HFN7.1), n = 39 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in c). e Immunostaining for fatty-acid binding protein (FABP, adipogenic marker) and osteocalcin (Oc, osteogenic marker) after 14 days in adipogenic-osteogenic media (scale bar 50 μm). f Quantification of positively stained cells (percentage,%) towards osteogenesis (Oc positive) and adipogenesis (FABP positive) after 14 days, n = 6 samples from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, two-way ANOVA with Bonferroni post hoc).', 'hash': 'f171cad4da83dd3783c6406008b2d31d47f44da912d12974853d4ffa6b39ea3a'}, {'image_id': 'nihms-1520816-f0002', 'image_file_name': 'nihms-1520816-f0002.jpg', 'image_path': '../data/media_files/PMC6650309/nihms-1520816-f0002.jpg', 'caption': 'Nascent ECM proteins create an adhesive layer at the cell-hydrogel interface.a Schematic illustrating norbornene-modified hyaluronic acid (NorHA) hydrogels crosslinked via a thiol-ene reaction with MMP-degradable dithiol peptide crosslinkers and incorporating RGD for adhesion. b Representative images of nascent proteins (white, visualized via fluorescent DBCO labeling) deposited by hMSCs encapsulated in degradable NorHA hydrogels (9.0 ± 0.7 kPa, mean ± SD, n = 3 independent measurements) and cultured in growth media for up to 6 days (see Supplementary Figure 5 for daily changes up to 14 days, scale bar 200 μm, inset 20 μm). c Quantification of the accumulated nascent protein thickness deposited by hMSCs encapsulated in degradable NorHA hydrogels (n = 40 cells (4 hours), 68 cells (day 1, day 2, day 4) and 72 cells (day 6) from 3 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified representative images in b). d Representative transmission electron microscopy (TEM, * hydrogel) images of encapsulated hMSC after 6 days (scale bar 0.5 μm). Orange box in left image indicates magnification in right image (arrow indicates cell membrane and arrowheads show randomly aligned collagen fibrils). e Representative images (magnifications on right) of nascent proteins and fibronectin, laminin α5 and collagen type 1 and type 4 at 6 days (scale bars 20 μm). f Representative image (magnifications of single channels on right) of accumulated nascent proteins and focal adhesions stained for paxillin (scale bars 20 μm). g Schematic (left) illustrating the region used to generate intensity profiles (right) emanating from single FAs (representative image in f), n = 50 adhesions from 10 individual cells (2 biologically independent experiments), lines show median intensity profile, shaded areas demonstrate 95% confidence interval).', 'hash': '7d2607429d2f3b1bdc8f1c3a1856bf41512bb1b965f1bb31179e78190db69d6a'}, {'image_id': 'nihms-1520816-f0005', 'image_file_name': 'nihms-1520816-f0005.jpg', 'image_path': '../data/media_files/PMC6650309/nihms-1520816-f0005.jpg', 'caption': 'Nascent protein remodeling is required for cell spreading and osteogenesis in dynamic hydrogels.a Representative images of nascent protein deposition of hMSCs encapsulated in DN hydrogels (G’ 3.6 ± 0.1 kPa, G” 0.7 ± 0.05 kPa, n = 3 independent measurements, mean ± SD) treated with an inhibitor of exocytosis and vesicular trafficking Exo-1 (120 nM) and a recombinant tissue inhibitor of metalloproteinases-3 (TIMP-3, 5 nM encapsulated) during 6 days in growth media (scale bar 200 μm, insets 20 μm). b Quantification of cell aspect ratio and accumulated nascent protein thickness deposited by hMSCs encapsulated in dynamic hydrogels (aspect ratio: n = 161 cells (control), n = 150 cells (TIMP-3), and n = 122 cells (Exo-1), protein thickness: n = 76 cells (control), n = 61 cells (TIMP-3), and n = 55 cells (Exo-1), from 3 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in a). c Representative images and d quantification of nuclear/cytoplasmic (nuc/cyto) YAP/TAZ ratios of hMSCs encapsulated in dynamic hydrogels and cultured for 6 days in adipogenic-osteogenic media (scale bar 50 μm, n = 59 cells (Control) and n = 39 cells (TIMP-3, Exo-1), from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, ** p ≤ 0.01, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for images in c). e Immunostaining for fatty-acid binding protein (FABP, adipogenic marker) and osteocalcin (Oc, osteogenic marker) after 14 days in adipogenic-osteogenic media (scale bar 50 μm). f Quantification of positively stained cells (percentage,%) towards osteogenesis (Oc positive) and adipogenesis (FABP positive) after 14 days, n = 6 samples from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, two-way ANOVA with Bonferroni post hoc).', 'hash': '61877755dac88b0ff7885d72a459d38fe9f81fc2c1296e14b320e031bbba3122'}, {'image_id': 'nihms-1520816-f0006', 'image_file_name': 'nihms-1520816-f0006.jpg', 'image_path': '../data/media_files/PMC6650309/nihms-1520816-f0006.jpg', 'caption': 'Nascent protein adhesion and remodeling enhance cell spreading in degradable/dynamic hydrogels.Cells interact with a 3D hydrogel and presented ligands for a short time period before depositing nascent proteins to form a pericellular matrix. The hydrogel properties determine if encapsulated cells can spread, but adhesion and active remodeling of the nascent proteins are required for spreading. For example, in hydrogels that cells can locally degrade (e.g., protease-sensitive crosslinkers), nascent proteins guide cell behavior as an assembled interfacial layer that cells adhere to. Perturbation of cell-ECM adhesion through inhibiting specific cell-nascent protein interactions (e.g., sol RGD, anti α2, HFN7.1) inhibits spreading and decreases its downstream cellular outcomes (YAP/TAZ nuclear translocation, osteogenic differentiation). Similarly, in dynamic microenvironments (e.g., viscoelastic hydrogels) where spreading is protease-independent, nascent protein deposition and remodeling are needed for mechanosensing (YAP/TAZ nuclear translocation, osteogenic differentiation) and are blocked by inhibiting nascent protein secretion (Exo-1) and remodeling (TIMP-3).', 'hash': '558348ec429a39deee4746fe3f1db44611445b800848362f7e90ac9bdbd60880'}, {'image_id': 'nihms-1520816-f0001', 'image_file_name': 'nihms-1520816-f0001.jpg', 'image_path': '../data/media_files/PMC6650309/nihms-1520816-f0001.jpg', 'caption': 'Nascent protein deposition by encapsulated hMSCs occurs early, independent of hydrogel type.a Schematic of nascent extracellular protein labeling. The methionine analog azidohomoalanine (AHA) is added to the culture media and incorporated into nascent proteins (e.g., fibronectin, collagens, laminins). The bio-orthogonal Cu(I)-free strain-promoted cyclo-addition between the azide and DBCO-modified fluorophore (DBCO-488) enables visualization of the nascent proteins. b Representative images of nascent proteins (white) deposited by hMSCs encapsulated in various hydrogels (alginate, agarose, maleimide modified poly(ethylene glycol) (PEG-MAL), methacrylated hyaluronic acid (MeHA), norbornene modified hyaluronic acid (NorHA)), E = ~9 kPa, scale bar 200 μm, inset 20 μm). c Representative transmission electron microscopy (TEM, * hydrogel, # nucleus; scale bar 5 μm left, 1 μm right) image of encapsulated hMSC after 1 day (24 h in culture). Orange box in top image indicates magnification in bottom image (arrow indicates cell membrane and arrowheads show collagen fibrils). d Representative images of nascent proteins (white) deposited by hMSCs encapsulated in non-degradable NorHA hydrogels (9.0 ± 0.7 kPa, mean ± SD, n = 3 independent measurements) and cultured in growth media (supplemented with AHA) up to 6 days (see Supplementary Figure 3 for daily changes up to 14 days, scale bar 200 μm, inset 20 μm). e Quantification of the accumulated nascent protein thickness deposited by hMSCs encapsulated in non-degradable NorHA hydrogels (n = 40 cells (4 hours), 55 cells (Day 2, Day 4) and 70 cells (Day 6) from 3 biologically independent experiments, mean ± SD, **** p ≤ 0.0001, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified representative images in d).', 'hash': '9939eea04eb2142905a6ecc558715fbb5c0f573e93e2f1396f4a211de18f91d4'}]
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{'nihms-1520816-f0001': ['To visualize nascent protein deposition by hMSCs within 3D hydrogels, we adapted a labeling technique where methionine analogs containing azide groups (azidohomoalanine, AHA) are incorporated into proteins as they are synthesized26 and a bio-orthogonal strain-promoted cyclo-addition is then performed with a fluorophore conjugated cyclooctyne (DBCO-488) for visualization (<xref rid="nihms-1520816-f0001" ref-type="fig">Fig. 1a</xref>). The cyclo-addition is performed prior to cell fixation to reduce labeling of intracellular proteins (). The cyclo-addition is performed prior to cell fixation to reduce labeling of intracellular proteins (Supplementary Fig. 1) while maintaining high cell viability (97 ± 2% viability). Thus, this approach allows spatiotemporal visualization of methionine-containing proteins around individual cells15.', 'To illustrate early nascent protein deposition across a range of hydrogel environments, hMSCs were encapsulated within both physically (i.e., agarose, ionically-crosslinked alginate) and covalently (i.e., maleimide poly(ethylene glycol) (PEG-MAL) and methacrylated HA (MeHA) crosslinked via Michael-addition, norbornene modified HA (NorHA) crosslinked via thiol-ene reaction) crosslinked hydrogels, all with ~9 kPa elastic modulus (<xref rid="nihms-1520816-f0001" ref-type="fig">Fig. 1b</xref>). DBCO cyclo-addition labeling after 1 day culture in AHA-supplemented growth media revealed nascent protein deposition across all hydrogels (). DBCO cyclo-addition labeling after 1 day culture in AHA-supplemented growth media revealed nascent protein deposition across all hydrogels (<xref rid="nihms-1520816-f0001" ref-type="fig">Fig. 1b</xref>). Transmission electron microscopy (TEM) images of hMSCS in NorHA hydrogels confirmed the presence of loosely organized fibrils with a morphology consistent with collagen at the cell-hydrogel interface after 1 day in culture (). Transmission electron microscopy (TEM) images of hMSCS in NorHA hydrogels confirmed the presence of loosely organized fibrils with a morphology consistent with collagen at the cell-hydrogel interface after 1 day in culture (<xref rid="nihms-1520816-f0001" ref-type="fig">Fig. 1c</xref>) that were not present directly after encapsulation () that were not present directly after encapsulation (Supplementary Fig. 2). These findings indicate that nascent protein deposition by hMSCs within the pericellular space occurs rapidly across a range of hydrogel environments.', 'To further assess the spatio-temporal distribution of nascent proteins using this bio-orthogonal labeling technique, hMSCs were encapsulated within RGD-modified (1 mM) NorHA hydrogels with elastic moduli of 9.0 ± 0.7 kPa and cultured (continuous presence of AHA during entire culture) up to 14 days, consistent with previous studies8,27 (Supplementary Fig. 3). An extensive mesh-like protein structure surrounded the cell body and increased with culture time (<xref rid="nihms-1520816-f0001" ref-type="fig">Fig. 1d</xref>). Additional labeling of the cell membrane confirmed that the proteins were extracellular and permitted quantification of the thickness of cell-produced proteins (). Additional labeling of the cell membrane confirmed that the proteins were extracellular and permitted quantification of the thickness of cell-produced proteins (Supplementary Fig. 4). The accumulated nascent protein thickness increased over 6 days (<xref rid="nihms-1520816-f0001" ref-type="fig">Fig. 1e</xref>) and was dependent on the initial hydrogel modulus () and was dependent on the initial hydrogel modulus (Supplementary Fig. 3), suggesting that cells accumulate proteins in the pericellular space within environments that restrict cell spreading6. These observations highlight that, even in the absence of differentiation factors, nascent protein deposition occurs early and likely contributes to how cells experience their physical and chemical environment.'], 'nihms-1520816-f0002': ['Previous studies with covalently crosslinked hydrogels indicated that cell spreading in 3D is dependent on the local degradability of the network6,7,27. Thus, we next encapsulated hMSCs in proteolytically degradable HA hydrogels to determine whether nascent protein deposition modulates cell spreading (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2a</xref>). Extracellular nascent proteins were observed during 6 days in culture within degradable NorHA hydrogels (). Extracellular nascent proteins were observed during 6 days in culture within degradable NorHA hydrogels (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2b</xref>) and nascent protein thickness and cell spreading aspect ratios increased with culture () and nascent protein thickness and cell spreading aspect ratios increased with culture (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2c</xref>, , Supplementary Fig. 5). TEM imaging after 6 days further confirmed that ECM fibrils were still present in close association with the cells and along the cell membrane (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2d</xref>, , Supplementary Fig. 6). Encapsulation of hMSCs into degradable NorHA hydrogels of lower (~3 kPa) and higher (~20 kPa) elastic moduli resulted in similar cell spreading and nascent protein deposition; however, the protein thickness was lower for stiffer hydrogels (Supplementary Fig. 5), likely indicating that the distribution and assembly of nascent proteins is mediated through hydrogel crosslinking.', 'We then asked how the spatial pattern of AHA labeled proteins colocalized with specific ECM proteins such as cellular fibronectin, laminin α5, and collagen types 1 and 4. High degrees of structural similarity were observed (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2e</xref>), suggesting that AHA incorporates into nascent proteins of the pericellular matrix. No staining of ECM proteins was observed directly after encapsulation (), suggesting that AHA incorporates into nascent proteins of the pericellular matrix. No staining of ECM proteins was observed directly after encapsulation (Supplementary Fig. 7) and we confirmed that serum proteins did not alter cell behavior and adhesion to the hydrogels, as hMSCs cultured in defined serum-free media exhibited similar ECM protein deposition and spreading relative to hMSCs in serum-containing growth media (Supplementary Fig. 8) and there was minimal adhesion of hMSCs on 2D hydrogels (without RGD presentation) cultured in serum-containing media (Supplementary Fig. 9). To investigate whether these changes in the pericellular matrix alter how hMSCs interact and sense the hydrogel environment, we next examined adhesive contacts between cells and extracellular ligands28. Paxillin was used to visualize focal adhesions (FAs) in hMSCs (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2f</xref>). We observed paxillin complexes directly coupled to the actin cytoskeleton (). We observed paxillin complexes directly coupled to the actin cytoskeleton (Supplementary Fig. 10), indicating formation of FAs within degradable NorHA hydrogels. Intensity profile quantification across single FAs (paxillin) relative to the location of nascent protein revealed that the localization of single FAs is consistent with adhesion to the deposited ECM (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2g</xref>), with an average protein thickness of 1.2 ± 0.8 μm, consistent with the accumulated protein thickness at day 6 (), with an average protein thickness of 1.2 ± 0.8 μm, consistent with the accumulated protein thickness at day 6 (<xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2c</xref>). This indicates that hMSCs directly interact with their nascent ECM during adhesion and spreading in degradable hydrogels, and do not rely solely on epitopes presented by the hydrogel.). This indicates that hMSCs directly interact with their nascent ECM during adhesion and spreading in degradable hydrogels, and do not rely solely on epitopes presented by the hydrogel.', 'a Representative images (scale bar 200 μm, inset 20 μm) of nascent protein deposition in MMP-degradable NorHA hydrogels after 6 days treatment without (control, note same image as in <xref rid="nihms-1520816-f0002" ref-type="fig">Fig. 2b</xref>), with monoclonal antibodies against integrin alpha 2 (anti α2, 20 μg/mL) or human fibronectin (HFN7.1, 5 μg/mL) or with soluble RGD (sol RGD, 0.5 mM). ), with monoclonal antibodies against integrin alpha 2 (anti α2, 20 μg/mL) or human fibronectin (HFN7.1, 5 μg/mL) or with soluble RGD (sol RGD, 0.5 mM). b Quantification of cell aspect ratio and accumulated nascent protein thickness deposited by hMSCs encapsulated in degradable NorHA hydrogels (n = 133 cells (control), n = 155 cells (anti α2), and n = 152 cells (HFN7.1), n = 124 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in a). c Representative images and d quantification of nuclear/cytoplasmic (nuc/cyto) YAP/TAZ ratios of hMSCs encapsulated in degradable NorHA hydrogels, cultured for 6 days in adipogenic-osteogenic media (scale bar 50 μm), quantifications: n = 60 cells (control), n = 51 cells (anti α2), and n = 40 cells (HFN7.1), n = 39 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in c). e Immunostaining for fatty-acid binding protein (FABP, adipogenic marker) and osteocalcin (Oc, osteogenic marker) after 14 days in adipogenic-osteogenic media (scale bar 50 μm). f Quantification of positively stained cells (percentage,%) towards osteogenesis (Oc positive) and adipogenesis (FABP positive) after 14 days, n = 6 samples from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, two-way ANOVA with Bonferroni post hoc).'], 'nihms-1520816-f0003': ['Having visualized the local accumulation of and cellular adhesion to nascent proteins, we sought to understand whether this adhesion alters cellular behavior within degradable hydrogels. The binding of integrins to specific matrix ligands may actively induce conformational changes in ECM proteins, triggering downstream cell signaling cascades29, such as with the binding of the integrin α2 domain with collagen (GFOGER)30,31 and fibronectin (RGD)22,32. To investigate how this binding regulates nascent protein assembly and cell behavior, we added function-perturbing monoclonal antibodies that selectively block interactions with either secreted collagen (anti Integrin alpha2 (anti-α2)) or human fibronectin (HFN7.1). Soluble RGD peptides (sol RGD) were also used as a competitive inhibitor to integrin-mediated binding33. All inhibitors were administered daily during the 6-day culture period. After 1 day, there were minimal changes in protein thickness with HFN7.1 (5 ug/mL) and sol RGD (0.5 mM) administration and a small increase with anti-α2 (20 ug/mL) treatment (Supplementary Fig. 11a,b). When treatment with the antibodies/peptides was continued for 6 days, hMSC spreading was reduced across all groups when compared to untreated and IgG isotype controls, with minimal changes in nascent protein thickness observed (<xref rid="nihms-1520816-f0003" ref-type="fig">Fig. 3a</xref>, , Supplementary Fig. 11c). hMSC spreading depended on the dose of antibodies/peptides at day 6, and concentrations were selected that had minimal impact on cell viability (Supplementary Fig. 11d,e). Although significantly reduced, blocking of collagen or fibronectin interactions did not completely abrogate cell spreading during the 6-day culture period, suggesting that multiple protein binding interactions are involved for cell spreading in 3D hydrogels.', 'To assess whether binding to these adhesive domains of collagen and fibronectin alters downstream behaviors of hMSCs, mechanosensitive signaling pathways were analyzed. Yes-associated protein/Transcriptional co-activator (YAP/TAZ) is critical in cellular sensing and transduction of mechanical signals, with enhanced nuclear translocation in response to increased traction/tension27,34,35. After 6 days (culture in bipotential adipogenic-osteogenic media6,8), YAP/TAZ was primarily nuclear for hMSCs. Conversely, YAP/TAZ was more cytoplasmic (reduced nuclear to cytoplasmic intensity (nuc/cyto) ratio) when adhesion to nascent proteins was blocked, particularly with HFN7.1 treatment (<xref rid="nihms-1520816-f0003" ref-type="fig">Fig. 3c,d</xref>). As such, the cell-adhesive domain of secreted fibronectin seems to mediate YAP/TAZ mechanosensitive signaling, resulting from cell-nascent protein sensing.). As such, the cell-adhesive domain of secreted fibronectin seems to mediate YAP/TAZ mechanosensitive signaling, resulting from cell-nascent protein sensing.', 'Given that cell contractility and YAP/TAZ nuclear localization are functionally important in directing MSC fate6,34, changes in downstream osteogenic and adipogenic differentiation were also investigated (<xref rid="nihms-1520816-f0003" ref-type="fig">Fig. 3e</xref>). Trends in nascent protein deposition and spreading were similar for hMSCs cultured in bipotential adipogenic-osteogenic differentiation media to those observed for hMSCs that were cultured in growth media (). Trends in nascent protein deposition and spreading were similar for hMSCs cultured in bipotential adipogenic-osteogenic differentiation media to those observed for hMSCs that were cultured in growth media (Supplementary Fig. 12). At 14 days, hMSCs showed primarily osteogenic differentiation as indicated with most cells being positive for osteocalcin (Oc), whereas adipogenesis (positive for fatty-acid binding protein (FABP)) was favored in groups treated with sol RGD, anti-α2 or HFN7.1 (<xref rid="nihms-1520816-f0003" ref-type="fig">Fig. 3f</xref>). Interestingly, anti-α2 treatment reduced osteogenesis to a lesser extent than HFN7.1. Not only does HFN7.1 abrogate cell adhesion to fibronectin, but perturbation of α5β1 and αvβ3 integrin recognition also alters fibronectin conformation and fibril formation). Interestingly, anti-α2 treatment reduced osteogenesis to a lesser extent than HFN7.1. Not only does HFN7.1 abrogate cell adhesion to fibronectin, but perturbation of α5β1 and αvβ3 integrin recognition also alters fibronectin conformation and fibril formation36,37, further enhancing its effects. This is also consistent with the observation that sol RGD peptides compete against the cell-adhesive domain of fibronectin for interactions with integrins, and thus similarly reduces hMSC osteogenic differentiation (<xref rid="nihms-1520816-f0003" ref-type="fig">Fig. 3e,f</xref>). These results highlight that, in hydrogels permissive to cell spreading, nascent protein adhesion guides cell behavior and fate by complementing signals supplied by the material itself.). These results highlight that, in hydrogels permissive to cell spreading, nascent protein adhesion guides cell behavior and fate by complementing signals supplied by the material itself.'], 'nihms-1520816-f0004': ['Recent studies have shown that viscoelastic hydrogels with dynamic crosslinks can significantly impact cell behavior through mechanisms that include local crosslink remodeling and ligand clustering8,38,39, in the absence of proteases. To assess whether nascent proteins mediate these behaviors, we designed a dynamic double network (DN) HA hydrogel system based on both covalent and supramolecular guest-host (GH) crosslinking40. Here, the first network is formed through covalent crosslinking of methacrylated HA (MeHA) and dithiols (DTT) via Michael addition (<xref rid="nihms-1520816-f0004" ref-type="fig">Fig. 4a</xref>). The second network consists of a HA hydrogel that forms through non-covalent guest-host interactions of β-cyclodextrin (CD, host) with adamantane (Ad, guest)). The second network consists of a HA hydrogel that forms through non-covalent guest-host interactions of β-cyclodextrin (CD, host) with adamantane (Ad, guest)41,42. Dynamic hydrogels were formed upon mixing and simultaneous crosslinking with interpenetration of the two networks. Fluorescent labeling and confocal imaging of the MeHA and GH networks demonstrated uniformity and microstructural homogeneity of the networks (Supplementary Fig. 13). Because the properties of each network can be controlled independently, we tailored the viscous and elastic properties of the dynamic hydrogels through alterations of the concentration of either network. At a given MeHA crosslink ratio of 0.3 (ratio of thiols to methacrylates), higher concentrations of the GH network (up to 3 wt%) increased the oscillatory viscous moduli (G”), but did not significantly alter the oscillatory elastic moduli (G’) (<xref rid="nihms-1520816-f0004" ref-type="fig">Fig. 4b</xref>). Conversely, G’ of dynamic hydrogels was altered by varying the covalent crosslink ratio of the MeHA network (0.0–0.4), with only small changes in G” (). Conversely, G’ of dynamic hydrogels was altered by varying the covalent crosslink ratio of the MeHA network (0.0–0.4), with only small changes in G” (<xref rid="nihms-1520816-f0004" ref-type="fig">Fig. 4c</xref>). Modulation of viscoelasticity was confirmed by the frequency-response of DN hydrogels with an increase in both G’ and G” at higher frequencies, whereas the MeHA network without the GH network behaved as an elastic hydrogel, with little frequency-dependent response (). Modulation of viscoelasticity was confirmed by the frequency-response of DN hydrogels with an increase in both G’ and G” at higher frequencies, whereas the MeHA network without the GH network behaved as an elastic hydrogel, with little frequency-dependent response (Supplementary Fig. 14). This is consistent with previous studies that varied hydrogel physical and covalent crosslinking to tune viscoelasticity43.', 'Using this material, we showed that encapsulated hMSC behavior was altered by the hydrogel composition and viscoelasticity, as measured through morphological changes at 6 days. When encapsulated within hydrogels of the same G’ (3.6 ± 0.4 kPa, without RGD), but altered viscosity, cell spreading was suppressed within MeHA only (0.00% GH) and DN hydrogels with low GH concentration (1.25% GH) (<xref rid="nihms-1520816-f0004" ref-type="fig">Fig. 4d</xref>); however, cell spreading greatly increased at higher GH concentrations, reaching aspect ratios greater than those observed in the covalent degradable hydrogels. Furthermore, when comparing dynamic hydrogels with increasing G’ (ranging from 1.9 ± 0.2 to 5.7 ± 0.3 kPa), cell spreading decreased as a function of covalent crosslink ratio (); however, cell spreading greatly increased at higher GH concentrations, reaching aspect ratios greater than those observed in the covalent degradable hydrogels. Furthermore, when comparing dynamic hydrogels with increasing G’ (ranging from 1.9 ± 0.2 to 5.7 ± 0.3 kPa), cell spreading decreased as a function of covalent crosslink ratio (<xref rid="nihms-1520816-f0004" ref-type="fig">Fig. 4e</xref>). Note that single GH hydrogels (no covalent crosslinks) were observed to disassemble during the 6-day culture period, which prevented any analysis of cell spreading). Note that single GH hydrogels (no covalent crosslinks) were observed to disassemble during the 6-day culture period, which prevented any analysis of cell spreading42. Generally, these findings suggest that increasing the viscous component of dynamic hydrogels influences the ability of hMSCs to spread by physically remodeling their pericellular environment through protease-independent mechanisms, consistent with previous reports8,44.'], 'nihms-1520816-f0005': ['Given that hMSCs were able to spread in dynamic hydrogels without local hydrogel degradation, we used one formulation (GH 2.50%, crosslink ratio 0.3) to investigate how nascent protein deposition and remodeling regulates cell behavior within these viscoelastic environments. At 3 days, encapsulated hMSCs spread in the DN hydrogels with a nascent ECM layer that was evident by 4 hours at the cell-hydrogel interface; RGD did not alter cell spreading (Supplementary Fig. 15). This indicates that the mechanism driving spreading in dynamic hydrogels is not greatly influenced by tethered adhesive ligands; thus, subsequent studies were performed without RGD modification. Cell spreading was then investigated in response to alterations in the nascent proteins. Specifically, 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo-1) was added to perturb the transport and secretion of extracellular proteins45–47 and TIMP-3 (endogenous tissue inhibitor of metalloproteinase 3) was encapsulated48 with hMSCs to locally limit nascent protein remodeling over the 6 day culture period49 (<xref rid="nihms-1520816-f0005" ref-type="fig">Fig. 5a</xref>). Blocking exocytosis with Exo-1 (120 nM) reduced the nascent protein thickness and cell spreading compared to controls (). Blocking exocytosis with Exo-1 (120 nM) reduced the nascent protein thickness and cell spreading compared to controls (<xref rid="nihms-1520816-f0005" ref-type="fig">Fig. 5b</xref>), whereas blocking protein remodeling with TIMP-3 (5 nM encapsulated and added daily to media) increased the average nascent protein thickness and reduced hMSC spreading (), whereas blocking protein remodeling with TIMP-3 (5 nM encapsulated and added daily to media) increased the average nascent protein thickness and reduced hMSC spreading (<xref rid="nihms-1520816-f0005" ref-type="fig">Fig. 5b</xref>). Inhibition of exocytosis may be associated with limitations, including its influence on the secretion of extracellular vesicles and growth factors; though these treatments did not significantly alter 2D YAP/TAZ nuclear localization and contractility relative to controls (). Inhibition of exocytosis may be associated with limitations, including its influence on the secretion of extracellular vesicles and growth factors; though these treatments did not significantly alter 2D YAP/TAZ nuclear localization and contractility relative to controls (Supplementary Fig. 16).', 'Noting that cell spreading and matrix remodeling are important for the functional behavior of these cells, we next evaluated if nascent protein deposition and remodeling alters hMSC fate. Upon changing the media to bipotential adipogenic-osteogenic media for 6 days, the same trends in nascent protein deposition and spreading as observed in growth media were maintained (Supplementary Fig. 17). After 6 days of culture, YAP/TAZ nuc/cyto ratios were significantly lower with Exo-1 or TIMP-3 treatment compared to the control group, indicating that these treatments resulted in YAP/TAZ retention in the cytoplasm (<xref rid="nihms-1520816-f0005" ref-type="fig">Fig. 5c,d</xref>). Following 14 days of culture in bipotential media, adipogenesis was significantly increased in both Exo-1 and TIMP-3 treated groups relative to the increased osteogenesis observed in the control group (). Following 14 days of culture in bipotential media, adipogenesis was significantly increased in both Exo-1 and TIMP-3 treated groups relative to the increased osteogenesis observed in the control group (<xref rid="nihms-1520816-f0005" ref-type="fig">Fig. 5e,f</xref>). This is consistent with the results in degradable hydrogels and previous findings of enhanced spreading and osteogenic commitment as functional outcomes of increased YAP/TAZ nuclear localization). This is consistent with the results in degradable hydrogels and previous findings of enhanced spreading and osteogenic commitment as functional outcomes of increased YAP/TAZ nuclear localization8,27, and highlights the essential role of nascent protein adhesion and remodeling in enabling MSC behavior and fate. Similar observations occurred in the absence of differentiation factors, albeit to a lesser extent (Supplementary Fig. 18) and similar trends in cell behavior and nascent protein accumulation were observed across multiple donors (Supplementary Fig. 19). These findings suggest that cell behavior when encapsulated within engineered materials is influenced by nascent protein deposition and subsequent pericellular remodeling at the cell-hydrogel interface.'], 'nihms-1520816-f0006': ['Numerous synthetic hydrogels have been used to investigate how biophysical cues regulate cell behavior in 3D. Often, direct interactions of cells with the engineered microenvironment are implicated in explaining observed phenomena. However, our results indicate that cellular outcomes are not only influenced by the initial engineered interface presented to the cell, but also by adhesion to and remodeling of nascent proteins deposited locally by cells very soon after cultures are initiated. This is often overlooked in the assessment of results, potentially due to the difficulty in analyzing this interface. By adapting a nascent extracellular protein labeling approach, we elucidated insight into the dynamic nature of the cell-hydrogel interface, which plays synergistic effects in directing cell behavior. Specifically, we found that secreted proteins increasingly mask the presentation of signals from the engineered hydrogel, that this process has meaningful consequences as early as day 1, and that the contributions of these nascent proteins persist through differentiation events occurring over several weeks (<xref rid="nihms-1520816-f0006" ref-type="fig">Fig. 6</xref>). Cellular adhesion naturally emerged from focal adhesions interacting with nascent proteins and local matrix remodeling within hydrogels that underwent either proteolytic degradation or dynamic polymer reorganization to permit cell spreading. When specific nascent protein adhesion or remodeling was blocked, the ability of the cell to spread and respond to the material environment was diminished, and this had downstream consequences on YAP/TAZ signaling and differentiation. This work demonstrates that, in many hydrogel systems, nascent proteins shape and define the pericellular microenvironment, supplementing cues from engineered hydrogels.). Cellular adhesion naturally emerged from focal adhesions interacting with nascent proteins and local matrix remodeling within hydrogels that underwent either proteolytic degradation or dynamic polymer reorganization to permit cell spreading. When specific nascent protein adhesion or remodeling was blocked, the ability of the cell to spread and respond to the material environment was diminished, and this had downstream consequences on YAP/TAZ signaling and differentiation. This work demonstrates that, in many hydrogel systems, nascent proteins shape and define the pericellular microenvironment, supplementing cues from engineered hydrogels.']}
|
Local nascent protein deposition and remodeling guide mesenchymal stromal cell mechanosensing and fate in three-dimensional hydrogels
| null |
Nat Mater
|
1566111600
|
Hydrogels serve as valuable tools for studying cell-extracellular matrix interactions in three-dimensional environments that recapitulate aspects of native extracellular matrix. However, the impact of early protein deposition on cell behaviour within hydrogels has largely been overlooked. Using a bio-orthogonal labelling technique, we visualized nascent proteins within a day of culture across a range of hydrogels. In two engineered hydrogels of interest in three-dimensional mechanobiology studies-proteolytically degradable covalently crosslinked hyaluronic acid and dynamic viscoelastic hyaluronic acid hydrogels-mesenchymal stromal cell spreading, YAP/TAZ nuclear translocation and osteogenic differentiation were observed with culture. However, inhibition of cellular adhesion to nascent proteins or reduction in nascent protein remodelling reduced mesenchymal stromal cell spreading and nuclear translocation of YAP/TAZ, resulting in a shift towards adipogenic differentiation. Our findings emphasize the role of nascent proteins in the cellular perception of engineered materials and have implications for in vitro cell signalling studies and application to tissue repair.
|
[
"Cell Adhesion",
"Cell Lineage",
"Humans",
"Hydrogels",
"Mechanotransduction, Cellular",
"Mesenchymal Stem Cells",
"Proteins",
"Signal Transduction"
] |
other
|
PMC6650309
| null |
64
|
[
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Nat Mater. 2019 Aug 18; 18(8):883-891
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VP56 locates in the cytoplasm and interacts with the RLRs. (A) HEK 293T cells seeded into 10-cm2 dishes were transfected with empty vector or Flag-MAVS/TBK1/MITA/IRF3/IRF7 and Myc-VP56 (5 μg each). After 24 h, cell lysates were IP with anti-Flag affinity gel. The immunoprecipitates and WCLs were then analyzed by IB with anti-Flag and anti-Myc Abs, respectively. (B) EPC cells seeded on microscopy cover glass in 6-well plates were transfected with 2 μg of VP56-EGFP and 2 μg of empty vector or DsRed-MAVS/TBK1/MITA/IRF3/IRF7. After 24 h, the cells were fixed and subjected to confocal microscopy analysis. Green signals represent overexpressed VP56 protein, red signals represent overexpressed MAVS, TBK1, MITA, IRF3, or IRF7, and blue staining indicates the nucleus region (a × 63 oil immersion objective). Scale bar, 10 μm. All experiments were repeated at least three times with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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[{'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC7111710/gr1_lrg.jpg', 'caption': 'VP56 is stimulated by virus infection. . (A) qPCR detection of the transcriptional levels of s7 on stimulation. GCO cells were seeded on 6-well plates overnight and infected with GCRV (100\xa0μl of the filtered virus-containing supernatant of frozen and thawed GCO cells, which was diluted 100 times with PBS) At the time points 0, 1, 2, 3, 5 and 7 day, total RNA was extracted for further qPCR assays.(B and C) VP56 inhibits poly I:C-induced IFN expression. GCO cells seeded into 6-well plates overnight were transfected with 2\xa0μg Myc-VP56 or the empty vector and transfected with poly I:C (2\xa0μg/ml) at 24\xa0h post-transfection. At 24\xa0h post transfection, total RNAs were extracted to examine the transcriptional levels of cellular ifn and isg15 by qPCR. The relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1. Data were expressed as mean\xa0±\xa0SEM, n\xa0=\xa03. Asterisks indicate significant differences from control (*, p\xa0<\xa00.05). All experiments were repeated for at least three times with similar result.', 'hash': '87e2b168e1cce9f50efb4563afd57b209f987be3a3bd98f015c683c975120f48'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC7111710/gr5_lrg.jpg', 'caption': 'VP56 promotes K48-linked ubiquitination and degradation of IRF7. (A) HEK 293T cells were seeded in 6-well plates overnight and transfected with 1\xa0μg HA-IRF7 and 1\xa0μg empty vector, or Myc-VP56 and 1\xa0μg Flag-TBK1. At 18\xa0h post-transfection, the cells were treated with the indicated inhibitors for 6\xa0h prior to being harvested for IB analysis of WCLs with the anti-HA, anti-Myc, anti-Flag, and anti-β-actin Abs. (B) EPC cells were seeded in 10-cm2 dishes and transfected with 4\xa0μg Flag-TBK1, 4\xa0μg Myc-IRF7, 4\xa0μg HA-VP56 or empty vector, and 2\xa0μg HA-Ub. At 18\xa0h post transfection, the cells were treated with DMSO or MG132 for 6\xa0h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCLs were analyzed by IB with the Abs indicated. (C) EPC cells were seeded in 10-cm2 dishes and transfected with 4\xa0μg Flag-TBK1, 4\xa0μg Myc-IRF7, 4\xa0μg HA-VP56 or empty vector, and 2\xa0μg HA-Ub-K48O or HA-Ub-K63O. At 18\xa0h post-transfection, the cells were treated with MG132 for 6\xa0h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. All experiments were repeated for at least three times with similar results.', 'hash': 'c0bea94e5f3d73706fdb4035c136465f89757ad968f034eba33ec39511603073'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC7111710/gr4_lrg.jpg', 'caption': 'VP56 decreases the phosphorylation of IRF7 mediated by TBK1. (A) VP56 has no significant effect on RLR protein expression. HEK 293T cells were seeded in 6-well plates overnight and transfected with 2\xa0μg of HA-RLRs and 2\xa0μg of empty vector or Myc-VP56 for 24\xa0h. The WCLs were subjected to IB with the anti-HA, anti-Myc, and anti-β-actin Abs. (B and C) TBK1 mediates the phosphorylation of IRF3 and IRF7. HEK 293T cells were seeded into 6-well plates overnight and transfected with Flag-TBK1 and HA-IRF3/IRF7 (2\xa0μg for each) for 24\xa0h. The cell lysates (100\xa0μg) were treated with or without CIP (10 U) for 40\xa0min\xa0at 37\xa0°C. The lysates were then detected by IB with anti-HA, anti-Flag and anti-β-actin Abs. (D and F) HEK 293T cells were seeded into 6-well plates overnight and co-transfected with 1\xa0μg Flag-TBK1 or Flag-DrTBK1 plus 1\xa0μg empty vector or HA-VP56, together with 1\xa0μg Myc-IRF7 or Myc-DrIRF7 for 24\xa0h. The lysates were then subjected to IB with anti-Myc, anti-Flag, anti-HA, and anti-β-actin Abs. (E and G)HEK 293T cells were seeded into 6-well plates overnight and co-transfected with 1\xa0μg Flag-TBK1 or Flag-DrTBK1 plus various concentration of HA-VP56 (0.5\xa0μg, or 1\xa0μg, or 2\xa0μg, empty vector was used to make up the rest), together with 1\xa0μg Myc-IRF7 or Myc-DrIRF7 for 24\xa0h. The lysates were then subjected to IB with anti-Myc, anti-Flag, anti-HA and anti-β-actin Abs. All experiments were repeated at least three times with similar results.', 'hash': '0c9bc7a49ad94fae88a4c4898dc6a3fc166721505230770d1bb5b4f5b17545be'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC7111710/gr2_lrg.jpg', 'caption': 'VP56 inhibits MAVS-mediated IFN1 activation. (A and B) EPC cells were seeded on 24-well plates overnight and co-transfected with MAVS, IRF7, and pcDNA3.1-VP56 or pcDNA3.1 (+) plus IFN1pro (A) or ISRE-Luc (B) at the ratio of 1:1:1 (0.5\xa0μg for each). pRL-TK was used as a control. At 24\xa0h post transfection, cells were collected for detection of luciferase activities. The promoter activity is presented as relative light units normalized to Renilla luciferase activity. Data were expressed as mean\xa0±\xa0SEM, n\xa0=\xa03. Asterisks indicate significant differences from control (*, p\xa0<\xa00.05). All experiments were repeated for at least three times with similar result.', 'hash': '6cbccca56a964ee95ce455b9503b14803341f0cdddad342681e7ac24526baa11'}, {'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC7111710/gr6_lrg.jpg', 'caption': 'VP56 dampens the cellular IFN response and facilitates SVCV proliferation. (A and B) EPC cells seeded into 6-well plates overnight were infected with SVCV (MOI\xa0=\xa01). After 24\xa0h, total RNAs were extracted to examine the transcriptional levels of cellular epcifn and epcvig1 by qPCR. The relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1. Data were expressed as mean\xa0±\xa0SEM, n\xa0=\xa03. Asterisks indicate significant differences from control (*, p\xa0<\xa00.05). (C and D) EPC cells seeded in 24-well plates overnight were transfected with 0.5\xa0μg pcDNA3.1 (+)-VP56 or pcDNA3.1 (+) vector. At 24\xa0h post-transfection, cells were infected with SVCV (MOI\xa0=\xa00.001) for 48\xa0h. (C) Then, cells were fixed with 4% PFA and stained with 1% crystal violet. (D) Culture supernatants from the cells infected with SVCV were collected, and the viral titer was measured by standard TCID50 method. All experiments were repeated for at least three times with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)', 'hash': '6bfd061a58f2e7f13d405659f12170eaa10546324f5bb8a6161f739f8d138391'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC7111710/gr3_lrg.jpg', 'caption': 'VP56 locates in the cytoplasm and interacts with the RLRs. (A) HEK 293T cells seeded into 10-cm2 dishes were transfected with empty vector or Flag-MAVS/TBK1/MITA/IRF3/IRF7 and Myc-VP56 (5\xa0μg each). After 24\xa0h, cell lysates were IP with anti-Flag affinity gel. The immunoprecipitates and WCLs were then analyzed by IB with anti-Flag and anti-Myc Abs, respectively. (B) EPC cells seeded on microscopy cover glass in 6-well plates were transfected with 2\xa0μg of VP56-EGFP and 2\xa0μg of empty vector or DsRed-MAVS/TBK1/MITA/IRF3/IRF7. After 24\xa0h, the cells were fixed and subjected to confocal microscopy analysis. Green signals represent overexpressed VP56 protein, red signals represent overexpressed MAVS, TBK1, MITA, IRF3, or IRF7, and blue staining indicates the nucleus region (a\xa0×\xa063 oil immersion objective). Scale bar, 10\xa0μm. All experiments were repeated at least three times with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)', 'hash': '7d9043797d7d971937f02ccccbe3f3c872598cb6a3eeba48452b0ff63661856a'}]
|
{'gr1_lrg': ['Previously, our study has demonstrated that GCRV VP41 reduces MITA phosphorylation and blocks IFN production, thus escaping the host immune response. Given that one virus should possess multiple strategies to elude host defense mechanisms, other immune escape mechanisms of GCRV should be identified. Here, to further investigate the other strategies used by GCRV to combat the host, other constructs of GCRV segments were employed for luciferase experiments in vitro, and the s7-encoded protein (VP56) exhibited the potential to inhibit host IFN activation. Upon infection with GCRV, the viral s7 gene was increased significantly in GCO cells, which indicated that the cells were successfully infected with GCRV (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>\nA). Four type I IFNs (IFN1-IFN4) have been identified in grass carp, but only IFN1 can be significantly activated by poly I:C, a mimic of viral RNA [\nA). Four type I IFNs (IFN1-IFN4) have been identified in grass carp, but only IFN1 can be significantly activated by poly I:C, a mimic of viral RNA [35] (data not shown). Thus, grass carp IFN1 was used in this study. As shown in <xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B, poly I:C stimulation induced the upregulation of IFN1 transcripts; however, such induction was significantly impeded by the overexpression of VP56. Moreover, VP56 also blocked the poly I:C-activated expression of B, poly I:C stimulation induced the upregulation of IFN1 transcripts; however, such induction was significantly impeded by the overexpression of VP56. Moreover, VP56 also blocked the poly I:C-activated expression of isg15 (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>C). These data indicate that GCRV VP56 serves as a negative regulator to interfere with host IFN production.C). These data indicate that GCRV VP56 serves as a negative regulator to interfere with host IFN production.Fig. 1VP56 is stimulated by virus infection. . (A) qPCR detection of the transcriptional levels of s7 on stimulation. GCO cells were seeded on 6-well plates overnight and infected with GCRV (100\xa0μl of the filtered virus-containing supernatant of frozen and thawed GCO cells, which was diluted 100 times with PBS) At the time points 0, 1, 2, 3, 5 and 7 day, total RNA was extracted for further qPCR assays.(B and C) VP56 inhibits poly I:C-induced IFN expression. GCO cells seeded into 6-well plates overnight were transfected with 2\xa0μg Myc-VP56 or the empty vector and transfected with poly I:C (2\xa0μg/ml) at 24\xa0h post-transfection. At 24\xa0h post transfection, total RNAs were extracted to examine the transcriptional levels of cellular ifn and isg15 by qPCR. The relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1. Data were expressed as mean\xa0±\xa0SEM, n\xa0=\xa03. Asterisks indicate significant differences from control (*, p\xa0<\xa00.05). All experiments were repeated for at least three times with similar result.Fig. 1'], 'gr2_lrg': ['Fish RLR factors are efficient for triggering IFN production [14]. Consequently, grass carp RLR constructs and IFN1 promoter (IFN1pro) were employed in the following studies. As shown in <xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\nA, the overexpression of MAVS and IRF7 upregulated the activation of IFN1pro, and the activation of IFN1pro induced by MAVS was inhibited by co-transfection with VP56. However, the ectopic expression of VP56 did not affect the IRF7-stimulated IFN1pro activity. In the host IFN response, the ISRE motif is considered the binding site of ISGs that responds to transcriptional factors. After co-transfection with VP56 and ISRE-Luc and RLR factors, the upregulation of ISRE activity activated by MAVS but not IRF7 was reduced by VP56 (\nA, the overexpression of MAVS and IRF7 upregulated the activation of IFN1pro, and the activation of IFN1pro induced by MAVS was inhibited by co-transfection with VP56. However, the ectopic expression of VP56 did not affect the IRF7-stimulated IFN1pro activity. In the host IFN response, the ISRE motif is considered the binding site of ISGs that responds to transcriptional factors. After co-transfection with VP56 and ISRE-Luc and RLR factors, the upregulation of ISRE activity activated by MAVS but not IRF7 was reduced by VP56 (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B). Collectively, these results suggest that VP56 decreases IFN production via negatively regulating MAVS.B). Collectively, these results suggest that VP56 decreases IFN production via negatively regulating MAVS.Fig. 2VP56 inhibits MAVS-mediated IFN1 activation. (A and B) EPC cells were seeded on 24-well plates overnight and co-transfected with MAVS, IRF7, and pcDNA3.1-VP56 or pcDNA3.1 (+) plus IFN1pro (A) or ISRE-Luc (B) at the ratio of 1:1:1 (0.5\xa0μg for each). pRL-TK was used as a control. At 24\xa0h post transfection, cells were collected for detection of luciferase activities. The promoter activity is presented as relative light units normalized to Renilla luciferase activity. Data were expressed as mean\xa0±\xa0SEM, n\xa0=\xa03. Asterisks indicate significant differences from control (*, p\xa0<\xa00.05). All experiments were repeated for at least three times with similar result.Fig. 2'], 'gr3_lrg': ['To further explore the function of VP56, whether VP56 interacts with RLRs at the protein level was investigated. HEK 293T cells were co-transfected with Myc-VP56 and Flag-tagged RLR factors, including MAVS, TBK1, MITA, IRF3, and IRF7. The results showed that most of the anti-Flag Ab-immunoprecipitated protein complexes were recognized by the anti-Myc Ab, which suggests that VP56 associates with TBK1, MITA, IRF3, and IRF7 but not MAVS (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\nA). Next, the subcellular location of VP56 was monitored in EPC cells. Confocal microscopy revealed that the VP56-EGFP signal was mainly distributed in the cytoplasm (\nA). Next, the subcellular location of VP56 was monitored in EPC cells. Confocal microscopy revealed that the VP56-EGFP signal was mainly distributed in the cytoplasm (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>B). We co-transfected DsRed-MAVS, DsRed-MITA, DsRed-TBK1, DsRed-IRF3, or DsRed- IRF7 with VP56-EGFP. A red signal from TBK1, IRF3, and IRF7 was observed in the cytosol and almost overlapped with the green signal from VP56 (B). We co-transfected DsRed-MAVS, DsRed-MITA, DsRed-TBK1, DsRed-IRF3, or DsRed- IRF7 with VP56-EGFP. A red signal from TBK1, IRF3, and IRF7 was observed in the cytosol and almost overlapped with the green signal from VP56 (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C–G). Taken together, these data suggest that VP56 is located in the cytosol and associates with RLR factors.C–G). Taken together, these data suggest that VP56 is located in the cytosol and associates with RLR factors.Fig. 3VP56 locates in the cytoplasm and interacts with the RLRs. (A) HEK 293T cells seeded into 10-cm2 dishes were transfected with empty vector or Flag-MAVS/TBK1/MITA/IRF3/IRF7 and Myc-VP56 (5\xa0μg each). After 24\xa0h, cell lysates were IP with anti-Flag affinity gel. The immunoprecipitates and WCLs were then analyzed by IB with anti-Flag and anti-Myc Abs, respectively. (B) EPC cells seeded on microscopy cover glass in 6-well plates were transfected with 2\xa0μg of VP56-EGFP and 2\xa0μg of empty vector or DsRed-MAVS/TBK1/MITA/IRF3/IRF7. After 24\xa0h, the cells were fixed and subjected to confocal microscopy analysis. Green signals represent overexpressed VP56 protein, red signals represent overexpressed MAVS, TBK1, MITA, IRF3, or IRF7, and blue staining indicates the nucleus region (a\xa0×\xa063 oil immersion objective). Scale bar, 10\xa0μm. All experiments were repeated at least three times with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 3'], 'gr4_lrg': ['To investigate the regulatory mechanism of VP56 on the RLR axis, we examined the effect of VP56 on RLR molecules at the protein level. MAVS-, TBK1-, MITA-, IRF3-, and IRF7-HA expression vectors were co-transfected with Myc-VP56 or an empty vector. As shown in <xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\nA, overexpressed VP56 did not cause any obvious change in RLR molecules at the protein level. Given that IRF3/7 phosphorylation is indispensable for mediating the IFN response, whether the phosphorylation of IRF3/7 is influenced by VP56 needs to be clarified. First, the function of grass carp TBK1 was investigated. As shown in \nA, overexpressed VP56 did not cause any obvious change in RLR molecules at the protein level. Given that IRF3/7 phosphorylation is indispensable for mediating the IFN response, whether the phosphorylation of IRF3/7 is influenced by VP56 needs to be clarified. First, the function of grass carp TBK1 was investigated. As shown in <xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>B and C, both IRF3 and IRF7 caused a band shift and exhibited higher mobilities when they were co-transfected with TBK1-Flag in 293T cells. Subsequently, the cell lysates were incubated with CIP. As expected, the band shifts disappeared, demonstrating that IRF3 and IRF7 are also phosphorylated by TBK1 in grass carp. Then, we evaluated whether IRF3/7 phosphorylation would be impaired by the overexpression of VP56. As shown in B and C, both IRF3 and IRF7 caused a band shift and exhibited higher mobilities when they were co-transfected with TBK1-Flag in 293T cells. Subsequently, the cell lysates were incubated with CIP. As expected, the band shifts disappeared, demonstrating that IRF3 and IRF7 are also phosphorylated by TBK1 in grass carp. Then, we evaluated whether IRF3/7 phosphorylation would be impaired by the overexpression of VP56. As shown in <xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>D and E, the amount of IRF7 was dramatically reduced by the overexpression of VP56 in a dose-dependent manner; in contrast, VP56 had minimal effects on the IRF3 level. Similar results occur in zebrafish (D and E, the amount of IRF7 was dramatically reduced by the overexpression of VP56 in a dose-dependent manner; in contrast, VP56 had minimal effects on the IRF3 level. Similar results occur in zebrafish (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>F and G). These results suggest that VP56 specifically promotes the degradation of IRF7.F and G). These results suggest that VP56 specifically promotes the degradation of IRF7.Fig. 4VP56 decreases the phosphorylation of IRF7 mediated by TBK1. (A) VP56 has no significant effect on RLR protein expression. HEK 293T cells were seeded in 6-well plates overnight and transfected with 2\xa0μg of HA-RLRs and 2\xa0μg of empty vector or Myc-VP56 for 24\xa0h. The WCLs were subjected to IB with the anti-HA, anti-Myc, and anti-β-actin Abs. (B and C) TBK1 mediates the phosphorylation of IRF3 and IRF7. HEK 293T cells were seeded into 6-well plates overnight and transfected with Flag-TBK1 and HA-IRF3/IRF7 (2\xa0μg for each) for 24\xa0h. The cell lysates (100\xa0μg) were treated with or without CIP (10 U) for 40\xa0min\xa0at 37\xa0°C. The lysates were then detected by IB with anti-HA, anti-Flag and anti-β-actin Abs. (D and F) HEK 293T cells were seeded into 6-well plates overnight and co-transfected with 1\xa0μg Flag-TBK1 or Flag-DrTBK1 plus 1\xa0μg empty vector or HA-VP56, together with 1\xa0μg Myc-IRF7 or Myc-DrIRF7 for 24\xa0h. The lysates were then subjected to IB with anti-Myc, anti-Flag, anti-HA, and anti-β-actin Abs. (E and G)HEK 293T cells were seeded into 6-well plates overnight and co-transfected with 1\xa0μg Flag-TBK1 or Flag-DrTBK1 plus various concentration of HA-VP56 (0.5\xa0μg, or 1\xa0μg, or 2\xa0μg, empty vector was used to make up the rest), together with 1\xa0μg Myc-IRF7 or Myc-DrIRF7 for 24\xa0h. The lysates were then subjected to IB with anti-Myc, anti-Flag, anti-HA and anti-β-actin Abs. All experiments were repeated at least three times with similar results.Fig. 4'], 'gr5_lrg': ['Protein degradation is one of the main strategies involved in modulating protein functions in biological processes and there are two main systems for protein degradation: ubiquitin proteasome and autophagosome pathways. To identify the degradation pathway of IRF7, the cells were treated with indicated inhibitors. The VP56-mediated degradation of IRF7 was completely inhibited by the proteasome inhibitor MG132 but not 3-MA, which is an autophagosome pathway inhibitor (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>\nA). Since ubiquitination is an important process during proteasome-dependent degradation, we further determined whether the degradation of IRF7 was due to ubiquitination. HEK 293T cells were transfected with Flag-TBK1, Myc-IRF7, HA-VP56, and HA-Ub in the presence or absence of MG132. Following the immunoprecipitation of Myc-IRF7, IB revealed that VP56 potentiated the ubiquitination of IRF7 (\nA). Since ubiquitination is an important process during proteasome-dependent degradation, we further determined whether the degradation of IRF7 was due to ubiquitination. HEK 293T cells were transfected with Flag-TBK1, Myc-IRF7, HA-VP56, and HA-Ub in the presence or absence of MG132. Following the immunoprecipitation of Myc-IRF7, IB revealed that VP56 potentiated the ubiquitination of IRF7 (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>B). K48 and K63, the lysines at positions 48 and 63 of ubiquitin linked with polyubiquitin chains, are two canonical polyubiquitin chain linkages. Given that K48-linked polyubiquitin chain modification leads to the targeting of proteins for proteasome recognition and degradation, whereas K63-linked polyubiquitin chain modification enhances the stability of target proteins [B). K48 and K63, the lysines at positions 48 and 63 of ubiquitin linked with polyubiquitin chains, are two canonical polyubiquitin chain linkages. Given that K48-linked polyubiquitin chain modification leads to the targeting of proteins for proteasome recognition and degradation, whereas K63-linked polyubiquitin chain modification enhances the stability of target proteins [[36], [37], [38]], we chose to investigate whether VP56 promoted the K48- or K63-linked ubiquitination of IRF7. To achieve this goal, plasmids expressing ubiquitin mutants and retaining only a single lysine residue either K48 (ubiquitin-K48) or K63 (ubiquitin-K63) were used. As shown in <xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>C, immunoprecipitation and IB indicated that VP56 promoted IRF7 ubiquitination with wild-type ubiquitin and ubiquitin-K48 but not with ubiquitin-K63. The above results indicate that VP56 induces the K48-linked ubiquitination of IRF7, which is recognized and subsequently degraded by the proteasome pathway.C, immunoprecipitation and IB indicated that VP56 promoted IRF7 ubiquitination with wild-type ubiquitin and ubiquitin-K48 but not with ubiquitin-K63. The above results indicate that VP56 induces the K48-linked ubiquitination of IRF7, which is recognized and subsequently degraded by the proteasome pathway.Fig. 5VP56 promotes K48-linked ubiquitination and degradation of IRF7. (A) HEK 293T cells were seeded in 6-well plates overnight and transfected with 1\xa0μg HA-IRF7 and 1\xa0μg empty vector, or Myc-VP56 and 1\xa0μg Flag-TBK1. At 18\xa0h post-transfection, the cells were treated with the indicated inhibitors for 6\xa0h prior to being harvested for IB analysis of WCLs with the anti-HA, anti-Myc, anti-Flag, and anti-β-actin Abs. (B) EPC cells were seeded in 10-cm2 dishes and transfected with 4\xa0μg Flag-TBK1, 4\xa0μg Myc-IRF7, 4\xa0μg HA-VP56 or empty vector, and 2\xa0μg HA-Ub. At 18\xa0h post transfection, the cells were treated with DMSO or MG132 for 6\xa0h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCLs were analyzed by IB with the Abs indicated. (C) EPC cells were seeded in 10-cm2 dishes and transfected with 4\xa0μg Flag-TBK1, 4\xa0μg Myc-IRF7, 4\xa0μg HA-VP56 or empty vector, and 2\xa0μg HA-Ub-K48O or HA-Ub-K63O. At 18\xa0h post-transfection, the cells were treated with MG132 for 6\xa0h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. All experiments were repeated for at least three times with similar results.Fig. 5'], 'gr6_lrg': ['To determine whether VP56 interferes with the cellular IFN response to facilitate virus proliferation, EPC cells were transfected with VP56 or the empty vector and infected with SVCV. Total RNAs were extracted and monitored by qPCR. As shown in <xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>\nA and B, the expression of the \nA and B, the expression of the ifn transcript in the cells that overexpress VP56 was reduced compared to their levels in the control cells and the reduced expression of host vig1 was also observed. Moreover, more CPE was observed in the VP56 group at 2\xa0d post-infection (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>C). This was confirmed by the titer of SVCV, which had significantly increased (5,800-fold) in the VP56-overexpressing cells compared to the control cells (C). This was confirmed by the titer of SVCV, which had significantly increased (5,800-fold) in the VP56-overexpressing cells compared to the control cells (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>D). These data demonstrate that VP56 suppresses the cellular IFN response and enhances the capacity of SVCV to replicate.D). These data demonstrate that VP56 suppresses the cellular IFN response and enhances the capacity of SVCV to replicate.Fig. 6VP56 dampens the cellular IFN response and facilitates SVCV proliferation. (A and B) EPC cells seeded into 6-well plates overnight were infected with SVCV (MOI\xa0=\xa01). After 24\xa0h, total RNAs were extracted to examine the transcriptional levels of cellular epcifn and epcvig1 by qPCR. The relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1. Data were expressed as mean\xa0±\xa0SEM, n\xa0=\xa03. Asterisks indicate significant differences from control (*, p\xa0<\xa00.05). (C and D) EPC cells seeded in 24-well plates overnight were transfected with 0.5\xa0μg pcDNA3.1 (+)-VP56 or pcDNA3.1 (+) vector. At 24\xa0h post-transfection, cells were infected with SVCV (MOI\xa0=\xa00.001) for 48\xa0h. (C) Then, cells were fixed with 4% PFA and stained with 1% crystal violet. (D) Culture supernatants from the cells infected with SVCV were collected, and the viral titer was measured by standard TCID50 method. All experiments were repeated for at least three times with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 6']}
|
Grass carp reovirus VP56 represses interferon production by degrading phosphorylated IRF7
|
[
"VP56",
"GCRV",
"Immune evasion",
"IRF7",
"Interferon"
] |
Fish Shellfish Immunol
|
1585983600
|
Grass carp reovirus (GCRV) is an efficient pathogen causing high mortality in grass carp, meanwhile, fish interferon (IFN) is a powerful cytokine enabling host cells to establish an antiviral state; therefore, the strategies used by GCRV to escape the cellular IFN response need to be investigated. Here, we report that GCRV VP56 inhibits host IFN production by degrading the transcription factor IFN regulatory factor 7 (IRF7). First, overexpression of VP56 inhibited the IFN production induced by the polyinosinic-polycytidylic acid (poly I:C) and mitochondrial antiviral signaling protein (MAVS), while the capacity of IRF7 on IFN induction was unaffected. Second, VP56 interacted with RLRs but did not affect the stabilization of the proteins in the normal state, while the phosphorylated IRF7 activated by TBK1 was degraded by VP56 through K48-linked ubiquitination. Finally, overexpression of VP56 remarkably reduced the host cellular ifn transcription and facilitated viral proliferation. Taken together, our results demonstrate that GCRV VP56 suppresses the host IFN response by targeting phosphorylated IRF7 for ubiquitination and degradation.
|
[
"Animals",
"Carps",
"Female",
"HEK293 Cells",
"Humans",
"Immunity, Innate",
"Interferon Regulatory Factor-7",
"Interferons",
"Ovary",
"Phosphorylation",
"Poly I-C",
"Reoviridae",
"Reoviridae Infections",
"Ubiquitination",
"Viral Proteins"
] |
other
|
PMC7111710
| null |
55
|
[
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Fish Shellfish Immunol. 2020 Apr 4; 99:99-106
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Subcellular localization and membrane association of wild type and mutant SARS-CoV E protein. (a) HeLa cells expressing the Flag-tagged E protein (A–C), and BHK cells expressing the Flag-tagged (D–F) and untagged (G–I) SARS-CoV E were stained with either anti-Flag (A–F) or anti-E (G–I) antibodies at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. The same HeLa cells were also stained with anti-calnexin antibody (B), and the same BHK cells were also stained with anti-p230 trans Golgi antibodies (panels E and H). Panels C, F, and I show the overlapping images. (b) BHK cells expressing the Flag-tagged wild type (E) and mutant E protein (Em1, Em2, Em3, Em4, Em5, and Em6) were stained with anti-Flag antibody at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. (c) HeLa cells expressing the Flag-tagged wild type and mutant E protein were harvested at 12 h posttransfection, broken by 20 stokes with a Dounce cell homogenizer, and fractionated into cytosol (C) and membrane (M) fractions after removal of cell debris and nuclei. Polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. The percentages of E protein detected in the membrane fraction were determined by densitometry and indicated on the right. Numbers on the left indicate molecular masses in kilodaltons.
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multiple panels: images & plots
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[
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"Immuno Assays"
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"fluorescence microscopy",
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[{'image_id': 'gr3', 'image_file_name': 'gr3.jpg', 'image_path': '../data/media_files/PMC7111751/gr3.jpg', 'caption': 'Mutational analysis of the three cysteine residues of SARS-CoV E protein. (a) HeLa cells were transfected with the Flag-tagged wild type and seven mutant E constructs containing mutations of either a single (C40-A, C43-A, and C44-A), combination of two (C40/43-A, C40/44-A, and C43/44-A) or all three (C40/43/44-A) cysteine residues. Cell lysates were prepared at 24 h posttransfection, polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. Numbers on the left indicate molecular masses in kilodaltons. (b) Entry of hygromycin B into HeLa cells expressing wild type and mutant E proteins. HeLa cells expressing the Flag-tagged wild type E (lanes 1, 2, and 3) and seven cysteine to alanine mutation constructs (lanes 4–24) were treated with 0, 0.5, and 1 mM of hygromycin B for 30 min at 12 h posttransfection, and radiolabeled with [35S] methionine–cysteine for 3 h. Cell lysates were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. SARS-CoV N protein was coexpressed with wild type and mutant E protein, and the expression of N protein was detected by immunoprecipitation with polyclonal anti-N antibodies. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. The percentages of E and N proteins detected in the presence of hygromycin B were determined by densitometry and indicated at the bottom. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '68d14c5eb8125d4af1a52e2c8e1737a58b2b9723048e0430a8ed1cdbdc025186'}, {'image_id': 'gr4', 'image_file_name': 'gr4.jpg', 'image_path': '../data/media_files/PMC7111751/gr4.jpg', 'caption': 'Mutational analysis of the transmembrane domain of SARS-CoV E protein. (a) HeLa cells were transfected with the Flag-tagged wild type and six mutant constructs containing mutations in the transmembrane domain of the E protein. Cell lysates were prepared 24 h posttransfection, polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. Numbers on the left indicate molecular masses in kilodaltons. (b) Entry of hygromycin B into HeLa cells expressing wild type and mutant E proteins. HeLa cells expressing the Flag-tagged wild type E (lanes 1, 2, and 3) and six mutant E constructs (lanes 4–21), respectively, were treated with 0, 0.5, and 1 mM of hygromycin B for 30 min at 12 h posttransfection, and radiolabeled with [35S] methionine–cysteine for 3 h. Cell lysates were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. SARS-CoV N protein was coexpressed with wild type and mutant E protein, and the expression of N protein was detected by immunoprecipitation with polyclonal anti-N antibodies. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. The percentages of E and N proteins detected in the presence of hygromycin B were determined by densitometry and indicated at the bottom. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '8b0aaa29a5e3f73efcef68a68017216f4672d5bd58c0fa9b02303dd9a7ec89ad'}, {'image_id': 'gr8a', 'image_file_name': 'gr8a.jpg', 'image_path': '../data/media_files/PMC7111751/gr8a.jpg', 'caption': 'Subcellular localization and membrane association of wild type and mutant SARS-CoV E protein. (a) HeLa cells expressing the Flag-tagged E protein (A–C), and BHK cells expressing the Flag-tagged (D–F) and untagged (G–I) SARS-CoV E were stained with either anti-Flag (A–F) or anti-E (G–I) antibodies at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. The same HeLa cells were also stained with anti-calnexin antibody (B), and the same BHK cells were also stained with anti-p230 trans Golgi antibodies (panels E and H). Panels C, F, and I show the overlapping images. (b) BHK cells expressing the Flag-tagged wild type (E) and mutant E protein (Em1, Em2, Em3, Em4, Em5, and Em6) were stained with anti-Flag antibody at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. (c) HeLa cells expressing the Flag-tagged wild type and mutant E protein were harvested at 12 h posttransfection, broken by 20 stokes with a Dounce cell homogenizer, and fractionated into cytosol (C) and membrane (M) fractions after removal of cell debris and nuclei. Polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. The percentages of E protein detected in the membrane fraction were determined by densitometry and indicated on the right. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': 'f2a03a97879d4d148c2ff2152ad1dd9c828aab88c8a514242a422c3c2965b2ef'}, {'image_id': 'gr5', 'image_file_name': 'gr5.jpg', 'image_path': '../data/media_files/PMC7111751/gr5.jpg', 'caption': 'Determination of SARS-CoV E protein as an integral membrane protein. HeLa cells expressing the Flag-tagged SARS-CoV and IBV E proteins, respectively, were harvested at 12 h posttransfection, broken by 20 stokes with a Dounce cell homogenizer, and fractionated into cytosol (C) and membrane (M) fractions after removal of cell debris and nuclei. The membrane fraction was treated with 1% Triton X-100, 100 mM Na2CO3 (pH 11), and 1 M KCl, respectively, and further fractionated into soluble (S) and pellet (P) fractions. Polypeptides were separated by SDS-PAGE and analyzed by Western blot using either anti-Flag antibody or anti-GM130 antibody (Abcam). Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '2c63dd2c6a8da4f2a55571a1471a9449df0b351a4b956dc36ac49e9641cc3bb9'}, {'image_id': 'gr2', 'image_file_name': 'gr2.jpg', 'image_path': '../data/media_files/PMC7111751/gr2.jpg', 'caption': 'Amino acid sequences of wild type and mutant SARS-CoV E protein. The putative transmembrane domain is underlined, and the three cysteine residues are in bold. Also indicated are the amino acid substitutions in each mutant construct.', 'hash': '2a2ad6ab90da7df453e93f896dcb4d3f83f203fcc8d80e765967bfd2ef2741fd'}, {'image_id': 'gr1', 'image_file_name': 'gr1.jpg', 'image_path': '../data/media_files/PMC7111751/gr1.jpg', 'caption': 'Modification of the membrane permeability of mammalian cells by SARS-CoV E protein. HeLa cells expressing the Flag-tagged E protein were treated with 0, 1, and 2 mM of hygromycin B for 30 min at 12 h posttransfection (lanes 1, 2, and 3), and radiolabeled with [35S] methionine–cysteine for 3 h. Cell lysates were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. Cells expressing SARS-CoV N proteins were included as negative control (lanes 4, 5, and 6). The expression of N protein was detected by immunoprecipitation with polyclonal anti-N antibodies. The percentages of E and N proteins detected in the presence of hygromycin B were determined by densitometry and indicated at the bottom. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': 'c2815ecd37aeff5c358c81c6ce80946fb9cf6d25f0ed19a67a2ea87419aded40'}, {'image_id': 'gr8bc', 'image_file_name': 'gr8bc.jpg', 'image_path': '../data/media_files/PMC7111751/gr8bc.jpg', 'caption': 'Subcellular localization and membrane association of wild type and mutant SARS-CoV E protein. (a) HeLa cells expressing the Flag-tagged E protein (A–C), and BHK cells expressing the Flag-tagged (D–F) and untagged (G–I) SARS-CoV E were stained with either anti-Flag (A–F) or anti-E (G–I) antibodies at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. The same HeLa cells were also stained with anti-calnexin antibody (B), and the same BHK cells were also stained with anti-p230 trans Golgi antibodies (panels E and H). Panels C, F, and I show the overlapping images. (b) BHK cells expressing the Flag-tagged wild type (E) and mutant E protein (Em1, Em2, Em3, Em4, Em5, and Em6) were stained with anti-Flag antibody at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. (c) HeLa cells expressing the Flag-tagged wild type and mutant E protein were harvested at 12 h posttransfection, broken by 20 stokes with a Dounce cell homogenizer, and fractionated into cytosol (C) and membrane (M) fractions after removal of cell debris and nuclei. Polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. The percentages of E protein detected in the membrane fraction were determined by densitometry and indicated on the right. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '0f1435d73d957bba23428daff691cbde712f384b9e1b67ecba75cd2a13836a20'}, {'image_id': 'gr6', 'image_file_name': 'gr6.jpg', 'image_path': '../data/media_files/PMC7111751/gr6.jpg', 'caption': 'Oligomerization of SARS-CoV E protein. The His-tagged E protein expressed in Sf9 insect cells was purified using Ni-NTA purification system (Qiagen), and incubated with three different concentrations of glutaraldehyde (0.1, 0.25, and 0.5 mM) for 1 h at room temperature. The reaction was quenched by adding 100 mM glycine. Polypeptides were separated on SDS-15% polyacrylamide gel in the presence or absence of 1% β-mercaptoethanol, and analyzed by Western blot with anti-His antibody. Different oligomers of the E protein are indicated on the right. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '4a7f42ce5f60891398f4cdbd3eeca6b178131f5313c7d5b8faf94c0af656b31f'}, {'image_id': 'gr7', 'image_file_name': 'gr7.jpg', 'image_path': '../data/media_files/PMC7111751/gr7.jpg', 'caption': 'Palmitoylation of SARS-CoV E protein. (a) Total cell lysates prepared from HeLa cells expressing the Flag-tagged SARS-CoV E protein (lanes 1 and 2) and IBV E protein (lanes 3 and 4) were treated either with 1 M Tris–HCl (lanes 1 and 3) or 1M hydroxylamine (lanes 2 and 4). Polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. Numbers on the left indicate molecular masses in kilodaltons. (b) HeLa cells expressing the Flag-tagged wild type SARS-CoV E protein (lane 1), mutant C40/44-A (lane 2), C40/44-A (lane 3), C43/44-A (lane4), C40/43/44-A (lane 5), and IBV E protein (lane 6) were radiolabeled with [35S] methionine–cysteine (upper panel) and [3H] palmitic acid (lower panel). Cell lysates were prepared and subjected to immunoprecipitation with anti-Flag antibody. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '5a84948608bec6b64b7f860ec1d345c4491bd3f1ec9669bf25d6d2261a0a0431'}, {'image_id': 'gr9', 'image_file_name': 'gr9.jpg', 'image_path': '../data/media_files/PMC7111751/gr9.jpg', 'caption': 'Association of SARS-CoV E protein with lipid rafts. HeLa cells expressing the Flag-tagged SARS E protein were lysed with 1% Triton, and centrifuged to remove insoluble material and nuclei. The supernatants were fractionated by ultracentrifugation with a sucrose gradient, and 11 fractions were collected. The presence of the SARS-CoV E protein in each fraction was analyzed by Western blot using anti-Flag antibody, and the presence of GM1 was determined by dot blot. Numbers on the left indicate molecular masses in kilodaltons.', 'hash': '415dbd5bb33717624439112890a1c1cf6a48957d95790cac318a0b0f3707174f'}]
|
{'gr1': ['To test if the SARS-CoV E protein could affect the membrane permeability of mammalian cells, the Flag-tagged E protein was expressed in HeLa cells. At 12 h posttransfection, cells were treated with two different concentrations of hygromycin B for 30 min, and then radiolabeled with [35S] methionine–cysteine for 3 h. Cell extracts were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. As shown in <xref rid="gr1" ref-type="fig">Fig. 1</xref>\n, extracts prepared from cells without treatment with hygromycin B showed the detection of the E protein and some other cellular proteins. In cells treated with 1 and 2 mM of hygromycin B, the expression of the E protein was reduced to 6 and 2%, respectively (\n, extracts prepared from cells without treatment with hygromycin B showed the detection of the E protein and some other cellular proteins. In cells treated with 1 and 2 mM of hygromycin B, the expression of the E protein was reduced to 6 and 2%, respectively (<xref rid="gr1" ref-type="fig">Fig. 1</xref>). However, in cells transfected with the SARS-CoV N protein, a similar amount of the N protein was detected in cells both treated and untreated with hygromycin B (). However, in cells transfected with the SARS-CoV N protein, a similar amount of the N protein was detected in cells both treated and untreated with hygromycin B (<xref rid="gr1" ref-type="fig">Fig. 1</xref>). The expression of the N protein was marginally reduced to 90 and 85%, respectively (). The expression of the N protein was marginally reduced to 90 and 85%, respectively (<xref rid="gr1" ref-type="fig">Fig. 1</xref>). These results confirm that expression of E protein in mammalian cells alters the membrane permeability of these cells to hygromycin B.). These results confirm that expression of E protein in mammalian cells alters the membrane permeability of these cells to hygromycin B.Fig. 1Modification of the membrane permeability of mammalian cells by SARS-CoV E protein. HeLa cells expressing the Flag-tagged E protein were treated with 0, 1, and 2 mM of hygromycin B for 30 min at 12 h posttransfection (lanes 1, 2, and 3), and radiolabeled with [35S] methionine–cysteine for 3 h. Cell lysates were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. Cells expressing SARS-CoV N proteins were included as negative control (lanes 4, 5, and 6). The expression of N protein was detected by immunoprecipitation with polyclonal anti-N antibodies. The percentages of E and N proteins detected in the presence of hygromycin B were determined by densitometry and indicated at the bottom. Numbers on the left indicate molecular masses in kilodaltons.'], 'gr2': ['SARS-CoV E protein contains three cysteine residues at amino acid positions 40, 43, and 44, respectively. These residues are located 3–7 amino acids downstream of the C-terminal residue of the transmembrane domain (<xref rid="gr2" ref-type="fig">Fig. 2</xref>\n). The first and third cysteine residues, at amino acid positions 40 and 44, respectively, were previously shown to play certain roles in oligomerization of the E protein (\n). The first and third cysteine residues, at amino acid positions 40 and 44, respectively, were previously shown to play certain roles in oligomerization of the E protein (Liao et al., 2004). They may also be involved in the E protein-induced alteration of membrane permeability in bacterial cells (Liao et al., 2004). To systematically test the effects of these residues on the expression, posttranslational modification, folding, oligomerization, and the membrane-permeabilizing activities of E protein, seven mutants, C40-A, C43-A, C44-A, C40/44-A, C40/43-A, C43/44-A, and C40/43/44-A, with mutations of the three cysteine residues to alanine either individually or in combination of two or three, were made by site-directed mutagenesis (<xref rid="gr2" ref-type="fig">Fig. 2</xref>). Western blotting analysis of cells expressing wild type and most mutant constructs showed specific detection of three species migrating at the range of molecular masses from 14 to 18 kDa under reducing conditions and representing three isoforms of the E protein (). Western blotting analysis of cells expressing wild type and most mutant constructs showed specific detection of three species migrating at the range of molecular masses from 14 to 18 kDa under reducing conditions and representing three isoforms of the E protein (<xref rid="gr3" ref-type="fig">Fig. 3</xref>a). These isoforms may be derived from posttranslational modification of the protein. The apparent molecular masses of these isoforms on SDS-PAGE are significantly larger than the calculated molecular mass of approximately 10 kDa for the Flag-tagged E protein.a). These isoforms may be derived from posttranslational modification of the protein. The apparent molecular masses of these isoforms on SDS-PAGE are significantly larger than the calculated molecular mass of approximately 10 kDa for the Flag-tagged E protein.Fig. 2Amino acid sequences of wild type and mutant SARS-CoV E protein. The putative transmembrane domain is underlined, and the three cysteine residues are in bold. Also indicated are the amino acid substitutions in each mutant construct.Fig. 3Mutational analysis of the three cysteine residues of SARS-CoV E protein. (a) HeLa cells were transfected with the Flag-tagged wild type and seven mutant E constructs containing mutations of either a single (C40-A, C43-A, and C44-A), combination of two (C40/43-A, C40/44-A, and C43/44-A) or all three (C40/43/44-A) cysteine residues. Cell lysates were prepared at 24 h posttransfection, polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. Numbers on the left indicate molecular masses in kilodaltons. (b) Entry of hygromycin B into HeLa cells expressing wild type and mutant E proteins. HeLa cells expressing the Flag-tagged wild type E (lanes 1, 2, and 3) and seven cysteine to alanine mutation constructs (lanes 4–24) were treated with 0, 0.5, and 1 mM of hygromycin B for 30 min at 12 h posttransfection, and radiolabeled with [35S] methionine–cysteine for 3 h. Cell lysates were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. SARS-CoV N protein was coexpressed with wild type and mutant E protein, and the expression of N protein was detected by immunoprecipitation with polyclonal anti-N antibodies. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. The percentages of E and N proteins detected in the presence of hygromycin B were determined by densitometry and indicated at the bottom. Numbers on the left indicate molecular masses in kilodaltons.', 'SARS-CoV E protein contains an unusually long putative transmembrane domain of 29 amino acid residues with a high leucine/isoleucine/valine content (55.17%) (Arbely et al., 2004). Recent molecular simulation and biochemical evidence showed that this domain may be involved in the formation of ion channel by oligomerization (Torres et al., 2005). Mutations of the putative transmembrane domain were therefore carried out to study its functions in membrane association and permeabilizing activity of the E protein. As shown in <xref rid="gr2" ref-type="fig">Fig. 2</xref>, four mutants, Em1, Em2, Em3, and Em4, were initially made by mutation of 3–7 leucine/valine residues to charged amino acid residues in the transmembrane domain. Two more mutants, Em5 and Em6, were subsequently made. Em5, which contains mutation of N15 to E, was constructed based on the molecular simulation studies showing that this residue may be essential for oligomerization of the protein (, four mutants, Em1, Em2, Em3, and Em4, were initially made by mutation of 3–7 leucine/valine residues to charged amino acid residues in the transmembrane domain. Two more mutants, Em5 and Em6, were subsequently made. Em5, which contains mutation of N15 to E, was constructed based on the molecular simulation studies showing that this residue may be essential for oligomerization of the protein (<xref rid="gr2" ref-type="fig">Fig. 2</xref>) () (Torres et al., 2005). Em6 was made by combination of the Em4 and C40/43/44-A (<xref rid="gr2" ref-type="fig">Fig. 2</xref>). Expression of these mutants showed the detection of polypeptides with apparent molecular masses ranging from 10 to 18 kDa (). Expression of these mutants showed the detection of polypeptides with apparent molecular masses ranging from 10 to 18 kDa (<xref rid="gr4" ref-type="fig">Fig. 4</xref>a). Interestingly, mutations introduced into Em2, Em3, EM4, and Em6 significantly change the migration rate of the corresponding mutant E protein on SDS-PAGE. The apparent molecular mass of these mutants is approximately 10 kDa, which is consistent with the predicted molecular weight for the Flag-tagged E protein (a). Interestingly, mutations introduced into Em2, Em3, EM4, and Em6 significantly change the migration rate of the corresponding mutant E protein on SDS-PAGE. The apparent molecular mass of these mutants is approximately 10 kDa, which is consistent with the predicted molecular weight for the Flag-tagged E protein (<xref rid="gr4" ref-type="fig">Fig. 4</xref>a). The fact that substitutions of the hydrophobic amino acid residues in the transmembrane domain of the E protein with charged amino acids significantly alter the migrating properties of the E protein in SDS-PAGE may reflect the changes in overall conformation and membrane association of these mutants compared to wild type E protein.a). The fact that substitutions of the hydrophobic amino acid residues in the transmembrane domain of the E protein with charged amino acids significantly alter the migrating properties of the E protein in SDS-PAGE may reflect the changes in overall conformation and membrane association of these mutants compared to wild type E protein.Fig. 4Mutational analysis of the transmembrane domain of SARS-CoV E protein. (a) HeLa cells were transfected with the Flag-tagged wild type and six mutant constructs containing mutations in the transmembrane domain of the E protein. Cell lysates were prepared 24 h posttransfection, polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. Numbers on the left indicate molecular masses in kilodaltons. (b) Entry of hygromycin B into HeLa cells expressing wild type and mutant E proteins. HeLa cells expressing the Flag-tagged wild type E (lanes 1, 2, and 3) and six mutant E constructs (lanes 4–21), respectively, were treated with 0, 0.5, and 1 mM of hygromycin B for 30 min at 12 h posttransfection, and radiolabeled with [35S] methionine–cysteine for 3 h. Cell lysates were prepared and the expression of E protein was detected by immunoprecipitation with anti-Flag antibody under mild washing conditions. SARS-CoV N protein was coexpressed with wild type and mutant E protein, and the expression of N protein was detected by immunoprecipitation with polyclonal anti-N antibodies. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. The percentages of E and N proteins detected in the presence of hygromycin B were determined by densitometry and indicated at the bottom. Numbers on the left indicate molecular masses in kilodaltons.'], 'gr3': ['In the membrane permeability assay shown in <xref rid="gr3" ref-type="fig">Fig. 3</xref>b, 0.5 and 1 mM of hygromycin B were used. The use of lower concentrations of hygromycin B is to ensure the detection of subtle changes on membrane permeability induced by the mutant constructs. Meanwhile, SARS-CoV N protein was cotransfected into HeLa cell together with wild type and mutant E proteins to aid assessment of the inhibitory effect of protein synthesis by hygromycin B. Expression of wild type and mutant E protein showed that similar levels of inhibition of protein synthesis by hygromycin B were obtained (b, 0.5 and 1 mM of hygromycin B were used. The use of lower concentrations of hygromycin B is to ensure the detection of subtle changes on membrane permeability induced by the mutant constructs. Meanwhile, SARS-CoV N protein was cotransfected into HeLa cell together with wild type and mutant E proteins to aid assessment of the inhibitory effect of protein synthesis by hygromycin B. Expression of wild type and mutant E protein showed that similar levels of inhibition of protein synthesis by hygromycin B were obtained (<xref rid="gr3" ref-type="fig">Fig. 3</xref>b). When 0.5 and 1 mM of hygromycin B were added to the culture medium, wild type and mutant E constructs render similar levels of inhibition to the expression of both N and E proteins (b). When 0.5 and 1 mM of hygromycin B were added to the culture medium, wild type and mutant E constructs render similar levels of inhibition to the expression of both N and E proteins (<xref rid="gr3" ref-type="fig">Fig. 3</xref>b). These results suggest that, contrary to the previous results observed in bacterial cells, these cysteine residues do not render significant effects on the membrane permeabilizing activity of the E protein. The reason for this discrepancy is uncertain, but it may reflect differences in posttranslational modifications, membrane association, subcellular localization, and translocation of the E protein in prokaryotic and eukaryotic cells.b). These results suggest that, contrary to the previous results observed in bacterial cells, these cysteine residues do not render significant effects on the membrane permeabilizing activity of the E protein. The reason for this discrepancy is uncertain, but it may reflect differences in posttranslational modifications, membrane association, subcellular localization, and translocation of the E protein in prokaryotic and eukaryotic cells.'], 'gr4': ['In the hygromycin B permeability assays, cells transfected with Em1, Em2, and Em5 constructs showed a similar degree of inhibition on protein synthesis as in cells expressing wild type E protein (<xref rid="gr4" ref-type="fig">Fig 4</xref>b). In cells expressing Em3 and Em4, much less inhibition of protein synthesis by hygromycin B was observed compared to cells expressing wild type E protein (b). In cells expressing Em3 and Em4, much less inhibition of protein synthesis by hygromycin B was observed compared to cells expressing wild type E protein (<xref rid="gr4" ref-type="fig">Fig. 4</xref>b). No obvious inhibition of protein synthesis was observed in cells expressing Em6 and N protein (b). No obvious inhibition of protein synthesis was observed in cells expressing Em6 and N protein (<xref rid="gr4" ref-type="fig">Fig. 4</xref>b). These results confirm that the transmembrane domain is essential for the membrane permeabilizing activity of the protein, and further suggest that dramatic mutations of the transmembrane domain are required to disrupt this function. The combination of mutations in the transmembrane domain and the three cysteine residues abolishes the membrane permeabilizing activity of E protein, suggesting that these cysteine residues and may play certain roles in the membrane association and permeabilizing activity of the E protein.b). These results confirm that the transmembrane domain is essential for the membrane permeabilizing activity of the protein, and further suggest that dramatic mutations of the transmembrane domain are required to disrupt this function. The combination of mutations in the transmembrane domain and the three cysteine residues abolishes the membrane permeabilizing activity of E protein, suggesting that these cysteine residues and may play certain roles in the membrane association and permeabilizing activity of the E protein.', 'Mutations introduced into the transmembrane domain of SARS-CoV E protein in Em2 as well as in Em3 and Em4 drastically change the migration properties of the E protein in SDS-PAGE (<xref rid="gr4" ref-type="fig">Fig. 4</xref>a). It suggests that these mutations would have significantly altered the overall folding, hydrophobicity, and membrane association properties of the E protein. However, this mutant shows very similar properties in subcellular localization, membrane association, and membrane-permeabilizing activity as wild type E protein. The high tolerance of E protein to such dramatic mutations indicates that maintenance of these properties would be essential for the functionality of E protein in coronavirus life cycles. This possibility would warrant more systematic studies by using an infectious clone system.a). It suggests that these mutations would have significantly altered the overall folding, hydrophobicity, and membrane association properties of the E protein. However, this mutant shows very similar properties in subcellular localization, membrane association, and membrane-permeabilizing activity as wild type E protein. The high tolerance of E protein to such dramatic mutations indicates that maintenance of these properties would be essential for the functionality of E protein in coronavirus life cycles. This possibility would warrant more systematic studies by using an infectious clone system.'], 'gr5': ['To characterize the membrane association property of the SARS-CoV E protein, HeLa cells expressing the Flag-tagged E protein were fractionated into membrane and cytosol fractions, and the presence of the E protein in each fraction was analyzed by Western blot. As shown in <xref rid="gr5" ref-type="fig">Fig. 5</xref>\n, the protein was almost exclusively located in the membrane fraction. Western blot analysis of the same fractions with anti-GM130 antibody (Abcam) showed the detection of an unknown host protein of approximately 60 kDa that is exclusively located in the membrane fraction (\n, the protein was almost exclusively located in the membrane fraction. Western blot analysis of the same fractions with anti-GM130 antibody (Abcam) showed the detection of an unknown host protein of approximately 60 kDa that is exclusively located in the membrane fraction (<xref rid="gr5" ref-type="fig">Fig. 5</xref>). Similarly, fractionation of HeLa cells expressing the Flag-tagged IBV E protein also showed exclusive detection of the protein in the membrane fraction (). Similarly, fractionation of HeLa cells expressing the Flag-tagged IBV E protein also showed exclusive detection of the protein in the membrane fraction (<xref rid="gr5" ref-type="fig">Fig. 5</xref>).).Fig. 5Determination of SARS-CoV E protein as an integral membrane protein. HeLa cells expressing the Flag-tagged SARS-CoV and IBV E proteins, respectively, were harvested at 12 h posttransfection, broken by 20 stokes with a Dounce cell homogenizer, and fractionated into cytosol (C) and membrane (M) fractions after removal of cell debris and nuclei. The membrane fraction was treated with 1% Triton X-100, 100 mM Na2CO3 (pH 11), and 1 M KCl, respectively, and further fractionated into soluble (S) and pellet (P) fractions. Polypeptides were separated by SDS-PAGE and analyzed by Western blot using either anti-Flag antibody or anti-GM130 antibody (Abcam). Numbers on the left indicate molecular masses in kilodaltons.', 'The membrane fraction was then treated with either 1% Triton X-100, 100 mM Na2CO3 pH 11 (high pH), or 1 M KCl (high salt), and centrifuged to separate the soluble contents (S) from the pellets (P). Treatment of the membrane fraction with 1 M KCl showed that both SARS-CoV and IBV E proteins were solely detected in the pellets (<xref rid="gr5" ref-type="fig">Fig. 5</xref>). Treatment of the same membrane pellets with 1% Triton X-100 and high pH led to the detection of the E proteins in both the supernatants and the pellets (). Treatment of the same membrane pellets with 1% Triton X-100 and high pH led to the detection of the E proteins in both the supernatants and the pellets (<xref rid="gr5" ref-type="fig">Fig. 5</xref>). As an integral membrane protein control, anti-GM130 antibodies detected the protein exclusively in the supernatants after treatment of the membrane fraction with Triton X-100 (). As an integral membrane protein control, anti-GM130 antibodies detected the protein exclusively in the supernatants after treatment of the membrane fraction with Triton X-100 (<xref rid="gr5" ref-type="fig">Fig. 5</xref>). In samples treated with both high pH and high salt, the protein was detected in the pellets only (). In samples treated with both high pH and high salt, the protein was detected in the pellets only (<xref rid="gr5" ref-type="fig">Fig. 5</xref>), confirming that the procedures and conditions used to fractionate the cell lysates and to treat the membrane fractions are appropriate.), confirming that the procedures and conditions used to fractionate the cell lysates and to treat the membrane fractions are appropriate.'], 'gr6': ['Oligomerization of viroporin is thought to be critical for the formation and expansion of the hydrophilic pore in the lipid bilayers. To determine the oligomerization status of the SARS-CoV E protein, the E protein with a His-tag at the C-terminus was expressed in insect cells using a baculovirus expression system and purified by Ni-NTA purification system. The purified E protein was concentrated and subjected to cross-linking with three different concentrations of glutaraldehyde, a short self-polymerizing reagent that reacts with lysine, tyrosine, histidine, and tryptophan. Cross-linking with glutaraldehyde showed the detection of dimer, trimer, tetramer, pentamer, and other higher-order oligomers/aggregates of the E protein under either non-reducing (<xref rid="gr6" ref-type="fig">Fig. 6</xref>\n, lanes 1–3) or reducing (\n, lanes 1–3) or reducing (<xref rid="gr6" ref-type="fig">Fig. 6</xref>, lanes 4–6) conditions. It was noted that more higher-order oligomers/aggregates were detected under non-reducing conditions when higher concentrations of the cross-linking reagent were used (, lanes 4–6) conditions. It was noted that more higher-order oligomers/aggregates were detected under non-reducing conditions when higher concentrations of the cross-linking reagent were used (<xref rid="gr6" ref-type="fig">Fig. 6</xref>, lanes 1–3). These results indicate that both interchain disulfide bond formation and hydrophobic interaction are contributing to the oligomerization of the E protein. More detailed characterization of the oligomerization status of the SARS-CoV E protein was hampered by the low expression efficiency of the protein in the system., lanes 1–3). These results indicate that both interchain disulfide bond formation and hydrophobic interaction are contributing to the oligomerization of the E protein. More detailed characterization of the oligomerization status of the SARS-CoV E protein was hampered by the low expression efficiency of the protein in the system.Fig. 6Oligomerization of SARS-CoV E protein. The His-tagged E protein expressed in Sf9 insect cells was purified using Ni-NTA purification system (Qiagen), and incubated with three different concentrations of glutaraldehyde (0.1, 0.25, and 0.5 mM) for 1 h at room temperature. The reaction was quenched by adding 100 mM glycine. Polypeptides were separated on SDS-15% polyacrylamide gel in the presence or absence of 1% β-mercaptoethanol, and analyzed by Western blot with anti-His antibody. Different oligomers of the E protein are indicated on the right. Numbers on the left indicate molecular masses in kilodaltons.'], 'gr7': ['The E protein from coronavirus MHV and IBV was previously shown to undergo modification by palmitoylation (11, 43). To verify if SARS-CoV E protein is palmitoylated, two independent experiments were performed. First, treatment of the E protein with 1M hydroxylamine showed the reduced detection of the more slowly migrating isoforms (<xref rid="gr7" ref-type="fig">Fig. 7</xref>a, lanes 1 and 2). As a control, treatment of the IBV E protein with the same reagent abolished the detection of upper bands (a, lanes 1 and 2). As a control, treatment of the IBV E protein with the same reagent abolished the detection of upper bands (<xref rid="gr7" ref-type="fig">Fig. 7</xref>a, lanes 3 and 4). Second, the three cysteine residues in combinations of two or all three were mutated to alanine (a, lanes 3 and 4). Second, the three cysteine residues in combinations of two or all three were mutated to alanine (<xref rid="gr2" ref-type="fig">Fig. 2</xref>). Wild type and mutant E proteins were then expressed in HeLa cells and labeled with [). Wild type and mutant E proteins were then expressed in HeLa cells and labeled with [3H] palmitic acid or [35S] methionine–cysteine. As shown in <xref rid="gr7" ref-type="fig">Fig. 7</xref>b, wild type and all mutant E proteins were efficiently labeled with [b, wild type and all mutant E proteins were efficiently labeled with [35S] methionine–cysteine (<xref rid="gr7" ref-type="fig">Fig. 7</xref>b, upper panel). In cells labeled with [b, upper panel). In cells labeled with [3H] palmitate, wild type and the three mutants with mutations of different combinations of two cysteine residues (C40/44-A, C40/43-A and C43/44-A) were efficiently detected (<xref rid="gr7" ref-type="fig">Fig. 7</xref>b, lower panel, lanes 1–4). However, the construct with mutation of all three cysteine residues (C40/43/44-A) was not labeled (b, lower panel, lanes 1–4). However, the construct with mutation of all three cysteine residues (C40/43/44-A) was not labeled (<xref rid="gr7" ref-type="fig">Fig. 7</xref>b, lower panel, lane 5). As a positive control, the IBV E protein was also efficiently labeled by [b, lower panel, lane 5). As a positive control, the IBV E protein was also efficiently labeled by [3H] palmitate (<xref rid="gr7" ref-type="fig">Fig. 7</xref>b, lower panel, lane 6). These results confirm that SARS-CoV E protein is modified by palmitoylation at all three cysteine residues.b, lower panel, lane 6). These results confirm that SARS-CoV E protein is modified by palmitoylation at all three cysteine residues.Fig. 7Palmitoylation of SARS-CoV E protein. (a) Total cell lysates prepared from HeLa cells expressing the Flag-tagged SARS-CoV E protein (lanes 1 and 2) and IBV E protein (lanes 3 and 4) were treated either with 1 M Tris–HCl (lanes 1 and 3) or 1M hydroxylamine (lanes 2 and 4). Polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. Numbers on the left indicate molecular masses in kilodaltons. (b) HeLa cells expressing the Flag-tagged wild type SARS-CoV E protein (lane 1), mutant C40/44-A (lane 2), C40/44-A (lane 3), C43/44-A (lane4), C40/43/44-A (lane 5), and IBV E protein (lane 6) were radiolabeled with [35S] methionine–cysteine (upper panel) and [3H] palmitic acid (lower panel). Cell lysates were prepared and subjected to immunoprecipitation with anti-Flag antibody. Polypeptides were separated by SDS-PAGE and visualized by autoradiography. Numbers on the left indicate molecular masses in kilodaltons.'], 'gr8a': ['To further analyze the membrane association properties of the E protein, its subcellular localization was studied by indirect immunofluorescence. HeLa cells overexpressing the Flag-tagged E protein were fixed with 4% paraformaldehyde at 12 h postinfection and stained with anti-Flag monoclonal antibody (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>a). In cells permeabilized with 0.2% Triton X-100, the Flag-tagged E protein is mainly localized to the perinuclear regions of the cells (a). In cells permeabilized with 0.2% Triton X-100, the Flag-tagged E protein is mainly localized to the perinuclear regions of the cells (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>a, panel A). The staining patterns largely overlap with calnexin, an ER resident protein (panels B and C). It was also noted that some granules and punctated staining patterns are not well merged with the calnexin staining patterns. They may represent aggregates of the E protein.a, panel A). The staining patterns largely overlap with calnexin, an ER resident protein (panels B and C). It was also noted that some granules and punctated staining patterns are not well merged with the calnexin staining patterns. They may represent aggregates of the E protein.Fig. 8Subcellular localization and membrane association of wild type and mutant SARS-CoV E protein. (a) HeLa cells expressing the Flag-tagged E protein (A–C), and BHK cells expressing the Flag-tagged (D–F) and untagged (G–I) SARS-CoV E were stained with either anti-Flag (A–F) or anti-E (G–I) antibodies at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. The same HeLa cells were also stained with anti-calnexin antibody (B), and the same BHK cells were also stained with anti-p230 trans Golgi antibodies (panels E and H). Panels C, F, and I show the overlapping images. (b) BHK cells expressing the Flag-tagged wild type (E) and mutant E protein (Em1, Em2, Em3, Em4, Em5, and Em6) were stained with anti-Flag antibody at 12 h posttransfection after permeabilizing with 0.2% Triton X-100. (c) HeLa cells expressing the Flag-tagged wild type and mutant E protein were harvested at 12 h posttransfection, broken by 20 stokes with a Dounce cell homogenizer, and fractionated into cytosol (C) and membrane (M) fractions after removal of cell debris and nuclei. Polypeptides were separated by SDS-PAGE and analyzed by Western blot using the anti-Flag antibody. The percentages of E protein detected in the membrane fraction were determined by densitometry and indicated on the right. Numbers on the left indicate molecular masses in kilodaltons.', 'The exact subcellular localization of a coronavirus E protein is an issue of debate in the current literature (Corse and Machamer, 2003). Although clear ER localization of the coronavirus IBV E protein was observed at early time points in a time course experiment using an overexpression system (Lim and Liu, 2001), no such localization patterns were observed as reported by Corse and Machamer (2003). To clarify that the above observed ER localization pattern may be due to the high expression level of the protein in HeLa cells using the vaccinia/T7 system, the subcellular localization of the SARS-CoV E protein in another cell type with lower expression efficiency of the protein was carried out. As shown in <xref rid="gr8a" ref-type="fig">Fig. 8</xref>a, expression of the Flag-tagged SARS-CoV in BHK cells stably expressing the T7 RNA polymerase (a, expression of the Flag-tagged SARS-CoV in BHK cells stably expressing the T7 RNA polymerase (Buchholz et al., 1999) showed that the protein exhibits typical Golgi localization patterns (panels D–F). Expression of the untagged SARS-CoV E protein in the same cell type also shows very similar Golgi localization patterns as the Flag-tagged protein (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>a, panels G–I). These results suggest that the predominant ER localization patterns in HeLa cells observed above may be due to the cell type used and the very high expression levels of the protein in individual cells with the vaccinia/T7 expression system.a, panels G–I). These results suggest that the predominant ER localization patterns in HeLa cells observed above may be due to the cell type used and the very high expression levels of the protein in individual cells with the vaccinia/T7 expression system.', 'The subcellular localization of wild type and six mutants, Em1, Em2, Em3, Em4, Em5, and Em6, was then studied in BHK cells. The localization patterns of Em1, Em2, and Em5 were similar to wild type E protein, showing predominant Golgi localization patterns (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>b). In cells expressing Em3 and Em4, more diffuse staining patterns throughout the cytoplasm were observed (b). In cells expressing Em3 and Em4, more diffuse staining patterns throughout the cytoplasm were observed (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>b). It suggests that these mutations may change the membrane association properties of the protein, leading to the alteration of the subcellular localization of the protein. In cells expressing Em6, a diffuse localization pattern was observed (b). It suggests that these mutations may change the membrane association properties of the protein, leading to the alteration of the subcellular localization of the protein. In cells expressing Em6, a diffuse localization pattern was observed (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>b).b).', 'The membrane association properties of wild type and mutant E proteins were further confirmed by fractionation of HeLa cells expressing E protein into membrane and cytosol fractions, and the presence of E protein in each fraction was analyzed by Western blot. As shown in <xref rid="gr8a" ref-type="fig">Fig. 8</xref>c, 95.28% of wild type E protein was detected in the membrane fraction. The percentages of the mutant E protein detected in the similarly prepared membrane fraction were 93.67% for Em1, 89.85% for Em2, 62.60% for Em3, 58.58%for Em4, 93.64% for Em5, and 55.46% for Em6 (c, 95.28% of wild type E protein was detected in the membrane fraction. The percentages of the mutant E protein detected in the similarly prepared membrane fraction were 93.67% for Em1, 89.85% for Em2, 62.60% for Em3, 58.58%for Em4, 93.64% for Em5, and 55.46% for Em6 (<xref rid="gr8a" ref-type="fig">Fig. 8</xref>c).c).'], 'gr9': ['To test if the E protein may be associated with lipid rafts, the low-density, detergent-insoluble membrane fraction was isolated from HeLa cells overexpressing the SARS-CoV E protein. As shown in <xref rid="gr9" ref-type="fig">Fig. 9</xref>\n, the majority of the E protein was detected at the bottom fractions (lanes 9–11). However, a certain proportion of E protein associated with lipid rafts was detected in fractions 3, 4, and 5 (\n, the majority of the E protein was detected at the bottom fractions (lanes 9–11). However, a certain proportion of E protein associated with lipid rafts was detected in fractions 3, 4, and 5 (<xref rid="gr9" ref-type="fig">Fig. 9</xref>, lanes 3–5). As a marker for lipid rafts, the GM1 was detected in fractions 4 and 5 (, lanes 3–5). As a marker for lipid rafts, the GM1 was detected in fractions 4 and 5 (<xref rid="gr9" ref-type="fig">Fig. 9</xref>, lanes 3 and 4)., lanes 3 and 4).Fig. 9Association of SARS-CoV E protein with lipid rafts. HeLa cells expressing the Flag-tagged SARS E protein were lysed with 1% Triton, and centrifuged to remove insoluble material and nuclei. The supernatants were fractionated by ultracentrifugation with a sucrose gradient, and 11 fractions were collected. The presence of the SARS-CoV E protein in each fraction was analyzed by Western blot using anti-Flag antibody, and the presence of GM1 was determined by dot blot. Numbers on the left indicate molecular masses in kilodaltons.']}
|
Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein
|
[
"SARS-CoV",
"E protein",
"Membrane association",
"Permeabilizing activity",
"Palmitoylation"
] |
Virology
|
1149490800
|
A diverse group of cytolytic animal viruses encodes small, hydrophobic proteins to modify host cell membrane permeability to ions and small molecules during their infection cycles. In this study, we show that expression of the SARS-CoV E protein in mammalian cells alters the membrane permeability of these cells. Immunofluorescent staining and cell fractionation studies demonstrate that this protein is an integral membrane protein. It is mainly localized to the ER and the Golgi apparatus. The protein can be translocated to the cell surface and is partially associated with lipid rafts. Further biochemical characterization of the protein reveals that it is posttranslationally modified by palmitoylation on all three cysteine residues. Systematic mutagenesis studies confirm that the membrane permeabilizing activity of the SARS-CoV E protein is associated with its transmembrane domain.
|
[
"Amino Acid Sequence",
"Amino Acid Substitution",
"Animals",
"Cell Line",
"Cell Membrane",
"Cell Membrane Permeability",
"Cricetinae",
"DNA Mutational Analysis",
"Endoplasmic Reticulum",
"Golgi Apparatus",
"HeLa Cells",
"Humans",
"Immunohistochemistry",
"Membrane Microdomains",
"Microscopy, Fluorescence",
"Molecular Sequence Data",
"Mutagenesis, Site-Directed",
"Mutation, Missense",
"Palmitoyl Coenzyme A",
"Protein Processing, Post-Translational",
"Protein Structure, Tertiary",
"Viral Envelope Proteins",
"Viroporin Proteins"
] |
other
|
PMC7111751
| null |
44
|
[
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] |
Virology. 2006 Jun 5; 349(2):264-275
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NO-CC CODE
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Subcellular localization of TGEV N protein in IEC. Cells were transfected with N-GFP expression vector or GFP vector.(A) Cells were stained by Hoechst33342 and ER-Tracker ™ Red. (B) Cells were stained with anti-N antibody, followed by goat anti-mouse antibody. Bar = 20 μm for all the figures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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gr2_lrg
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2
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fae21facd70d324ce7d9dcb2d983062313d24e134caf9de39e82389e107a13fa
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gr2_lrg.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
689,
958
] |
[{'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC7111826/gr4_lrg.jpg', 'caption': 'The effect of TGEV N protein in IEC on NF-κB activity and the expression of GRP78, IL-8, Bcl-2, IL-8.Cells expressing GFP or N-GFP for 48\u202fh. (A) The level of GRP78 expression was determined by western blot. (B) Real-time PCR analysis of GRP78 mRNA levels. (C) The level of NF-κB expression was determined by western blot. (D) NF-κB p65 activation was determined using the ELISA assay. (E) The expression of IL-8 in N-GFP expressing IEC or untransfected cells (treated or untreated with MG132) culture supernatants were measured by ELISA. (F) IL-8 mRNA levels were analysed by Real-time PCR assay. (G) The level of Bcl-2 expression was determined by western blot. (H) Real-time PCR analysis of Bcl-2 mRNA levels. β-actin was used as an internal loading control. The results are mean\u202f±\u202fSD and representative of three independent experiments.', 'hash': 'eae96fa601ec879ac90be41a2bcce361e19fa17ecb17b01f511fdd7f0f6733e9'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC7111826/gr1_lrg.jpg', 'caption': 'The expression products and protein degradation characteristics of TGEV N protein in IEC.Cells were transfected with N-GFP expression vector or GFP vector and treated with MG132 for 24\u202fh. (A) The cells were subjected to Western blot analysis using anti-GFP antibodies. (B) The cells were subjected to Western blot analysis using TGEV N protein antibodies. (C) Protein degradation characteristics were observed by fluorescence microscopy. All the data shown are representative of three independent experiments.', 'hash': 'a84ee163d685aa2d3855b95e44a2721a9cf590e87004422f04253e1cae9699f6'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC7111826/gr3_lrg.jpg', 'caption': 'Cell cycle arrest and the expression of cyclin A induced by TGEV N protein. Cells were transfected with N-GFP expression vector or GFP ector for 48\u202fh.(A) Flow cytometry analysis of cells by propidium iodide staining. (B) The percentage of cells in each phase of the cell cycle from flow cytometry data. (C) The level of cyclin A expression was determined by western blot. β-actin was used as an internal loading control. (D) Real-time PCR analysis of cyclin A mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The results are mean\u202f±\u202fSD from three independent experiments. * p\u202f<\u202f0.05 versus the control groupversus the control group (the cells expressing GFP and untransfected IEC cells).', 'hash': '9a1fb8f114a28e8e4b48fb50dc248564fa8ece22df0613059ad2c3ef4a65bffa'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC7111826/gr2_lrg.jpg', 'caption': 'Subcellular localization of TGEV N protein in IEC. Cells were transfected with N-GFP expression vector or GFP vector.(A) Cells were stained by Hoechst33342 and ER-Tracker ™ Red. (B) Cells were stained with anti-N antibody, followed by goat anti-mouse antibody. Bar\u202f=\u202f20\u202fμm for all the figures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)', 'hash': 'fae21facd70d324ce7d9dcb2d983062313d24e134caf9de39e82389e107a13fa'}]
|
{'gr1_lrg': ['The Western blot analysis demonstrated that the protein molecular mass produced by the cells transfected with GFP-N plasmid is approximately 70\u202fkDa, as was detected with anti-GFP monoclonal antibodies (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>A) and anti-TGEV N protein antibodies (A) and anti-TGEV N protein antibodies (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B). The molecular mass of GFP is approximately 27\u202fkDa, indicating that N protein, whose molecular mass is approximately 43\u202fkDa, was successfully expressed. Meantime, no signal was detected from untransfected cells.B). The molecular mass of GFP is approximately 27\u202fkDa, indicating that N protein, whose molecular mass is approximately 43\u202fkDa, was successfully expressed. Meantime, no signal was detected from untransfected cells.Fig. 1The expression products and protein degradation characteristics of TGEV N protein in IEC.Cells were transfected with N-GFP expression vector or GFP vector and treated with MG132 for 24\u202fh. (A) The cells were subjected to Western blot analysis using anti-GFP antibodies. (B) The cells were subjected to Western blot analysis using TGEV N protein antibodies. (C) Protein degradation characteristics were observed by fluorescence microscopy. All the data shown are representative of three independent experiments.Fig. 1', 'Fluorescence microscopyhe were used to observe N proteins\' degradation characteristics (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>C) and its degradation level was detected by Western blot assays (C) and its degradation level was detected by Western blot assays (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>A, B). The results show that GFP-N protein level in the cells treated with MG132 was higher than that in untreated cells, while there were no GFP protein expression differences between MG132 treated and untreated ones.A, B). The results show that GFP-N protein level in the cells treated with MG132 was higher than that in untreated cells, while there were no GFP protein expression differences between MG132 treated and untreated ones.'], 'gr2_lrg': ['Confocal fluorescence microscopy were used to investigate the subcellular localization of N protein. And results show that GFP-N proteins distribute predominantly in the cytoplasm, while the GFP protein was localised in the whole cell (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>A, B).A, B).Fig. 2Subcellular localization of TGEV N protein in IEC. Cells were transfected with N-GFP expression vector or GFP vector.(A) Cells were stained by Hoechst33342 and ER-Tracker ™ Red. (B) Cells were stained with anti-N antibody, followed by goat anti-mouse antibody. Bar\u202f=\u202f20\u202fμm for all the figures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Fig. 2'], 'gr3_lrg': ['To investigate in which particular phase TGEV N-effected cell cycle arrest occurred, cell cycle profiles were analysed by flow cytometry (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>A) and cyclin protein were examined by RT-PCR and Western-blot assay (A) and cyclin protein were examined by RT-PCR and Western-blot assay (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C, D). Besides, in order to confirm the percentage of cells in each phases of the G0/G1, S, and G2/M(C, D). Besides, in order to confirm the percentage of cells in each phases of the G0/G1, S, and G2/M(<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>B), histograms quantitative analysis was employed. These data suggest that the DNA content of S-phase increased, while the G0/G1 and G2/M phases showed slight changes and that TGEV N protein prolonged the S-phase cell cycle and prevented GFP-N expressing cells from entering the G2/M phase. The results show that TGEV N protein could cause S-phase prolongation. As shown in B), histograms quantitative analysis was employed. These data suggest that the DNA content of S-phase increased, while the G0/G1 and G2/M phases showed slight changes and that TGEV N protein prolonged the S-phase cell cycle and prevented GFP-N expressing cells from entering the G2/M phase. The results show that TGEV N protein could cause S-phase prolongation. As shown in <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C, comparing with control cells, the Cyclin A protein level was significantly decreased in N protein expressing cells. To further support these findings, real-time quantitative PCR assay were uesd to determine the cyclin A mRNA level. The results show that, in the GFP-N expressing cells, cyclin A mRNA levels are significantly lower than that in control cells (C, comparing with control cells, the Cyclin A protein level was significantly decreased in N protein expressing cells. To further support these findings, real-time quantitative PCR assay were uesd to determine the cyclin A mRNA level. The results show that, in the GFP-N expressing cells, cyclin A mRNA levels are significantly lower than that in control cells (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>D), suggesting that S-phase prolongation induced by TGEV N protein is closely related to cyclin A protein degradation and can down-regulate cyclin A transcription.D), suggesting that S-phase prolongation induced by TGEV N protein is closely related to cyclin A protein degradation and can down-regulate cyclin A transcription.Fig. 3Cell cycle arrest and the expression of cyclin A induced by TGEV N protein. Cells were transfected with N-GFP expression vector or GFP ector for 48\u202fh.(A) Flow cytometry analysis of cells by propidium iodide staining. (B) The percentage of cells in each phase of the cell cycle from flow cytometry data. (C) The level of cyclin A expression was determined by western blot. β-actin was used as an internal loading control. (D) Real-time PCR analysis of cyclin A mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The results are mean\u202f±\u202fSD from three independent experiments. * p\u202f<\u202f0.05 versus the control groupversus the control group (the cells expressing GFP and untransfected IEC cells).Fig. 3'], 'gr4_lrg': ['GRP78 is widely used as a key regulator for ER stress. The levels of GRP78 expression in transfected and untransfected cells were detected by Western blot assays (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>A) and real-time PCR assay (A) and real-time PCR assay (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>B). The results show that GRP78 and expression level in transfected cells expressing N protein are both significantly higher than that in untransfected cells. Western Blot assay (B). The results show that GRP78 and expression level in transfected cells expressing N protein are both significantly higher than that in untransfected cells. Western Blot assay (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>C) and ELISA assay (C) and ELISA assay (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>D) results show that the NF-κB expression level presents the same trends as GRP78.D) results show that the NF-κB expression level presents the same trends as GRP78.Fig. 4The effect of TGEV N protein in IEC on NF-κB activity and the expression of GRP78, IL-8, Bcl-2, IL-8.Cells expressing GFP or N-GFP for 48\u202fh. (A) The level of GRP78 expression was determined by western blot. (B) Real-time PCR analysis of GRP78 mRNA levels. (C) The level of NF-κB expression was determined by western blot. (D) NF-κB p65 activation was determined using the ELISA assay. (E) The expression of IL-8 in N-GFP expressing IEC or untransfected cells (treated or untreated with MG132) culture supernatants were measured by ELISA. (F) IL-8 mRNA levels were analysed by Real-time PCR assay. (G) The level of Bcl-2 expression was determined by western blot. (H) Real-time PCR analysis of Bcl-2 mRNA levels. β-actin was used as an internal loading control. The results are mean\u202f±\u202fSD and representative of three independent experiments.Fig. 4', 'In this research, ELISA assay (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>E) and real-time PCR assay (E) and real-time PCR assay (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>F) results showed that TGEV N up-regulates IL-8 expression in IEC, suggesting that TGEV N expression result in ER stress, and NF-κB activation, are responsible for the up-regulation of IL-8.F) results showed that TGEV N up-regulates IL-8 expression in IEC, suggesting that TGEV N expression result in ER stress, and NF-κB activation, are responsible for the up-regulation of IL-8.', 'In this research, Western blot(<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>G) and quantitative real-time PCR (G) and quantitative real-time PCR (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>H) were used, showing that the expression of Bcl-2 in N-GFP protein expressing cells is higher than that in untransfected cells. The results suggest that TGEV N protein is able to up-regulate Bcl-2 expression both at the gene, and protein, levels which might be induced by ER stress response through the NF-κB signalling pathway.H) were used, showing that the expression of Bcl-2 in N-GFP protein expressing cells is higher than that in untransfected cells. The results suggest that TGEV N protein is able to up-regulate Bcl-2 expression both at the gene, and protein, levels which might be induced by ER stress response through the NF-κB signalling pathway.']}
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Transmissible gastroenteritis virus N protein causes endoplasmic reticulum stress, up-regulates interleukin-8 expression and its subcellular localization in the porcine intestinal epithelial cell
| null |
Res Vet Sci
|
1534057200
|
This essay focuses on transmissible gastroenteritis virus (TGEV), which is an enteropathogenic virus related to contagious and acute diseases in suckling piglets. Previous literature suggests that the TGEV nucleocapsid protein (N) plays a significant role in viral transcriptional process, however, there is a need to examine other functions of TGEV N protein in the porcine intestinal epithelial cell (IEC) which is the target cell of TGEV. In the present study, we investigated the degradation, subcellular localisation, and function of TGEV N protein by examining its effects on cycle progression, endoplasmic reticulum (ER) stress, interleukin-8 (IL-8) expression, and cell survival. The results showed that TGEV N protein localised in the cytoplasm, inhibited IEC growth, prolonged the S-phase cell cycle by down-regulating cell cycle protein cyclin A, and was mainly degraded through the proteasome pathway. Moreover, TGEV N protein induced ER stress and activated NF-κB, which was responsible for the up-regulation of IL-8 and Bcl-2 expression. This report mainly considers the functions of TGEV N protein in IEC. To be specific, in IEC, TGEV N protein induces cell cycle prolongation at the S-phase, ER stress and up-regulates IL-8 expression. These results provide a better understanding of the functions and structural mechanisms of TGEV N protein.
|
[
"Animals",
"Endoplasmic Reticulum Stress",
"Epithelial Cells",
"Gastroenteritis, Transmissible, of Swine",
"Interleukin-8",
"Intestines",
"Swine",
"Transmissible gastroenteritis virus"
] |
other
|
PMC7111826
| null |
27
|
[
"{'Citation': 'Baker R.G., Hayden M.S., Ghosh S. NF-kB, inflammation, and metabolic disease. Cell Metab. 2011;13:11–22.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3040418'}, {'@IdType': 'pubmed', '#text': '21195345'}]}}",
"{'Citation': 'Calvo E. Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells. J. Gen. Virol. 2005;86:2255–2267.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16033973'}}}",
"{'Citation': 'Chang R., Zhang Q., He Y., Tong D., Xu X. Expression of protein N of porcine transmissible gastroenteritis in swine intestinal epithelial cells and effect and influence on cell cycle. Prog. Vet. Med. 2015;36(8):7–10.'}",
"{'Citation': 'de Groot R.J., Baker S.G., Baric R.S., Enjuanes L., Gorbalenya A.E. Coronaviridae. In: King A.M.Q., Adams M.J., Carstens E.B., Lefkowitz E.J., editors. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press; San Diego: 2011. pp. 774–796.'}",
"{'Citation': 'Diemer C., Schneider M., Seebach J., Quaas J., Frösner G., Schätz H.M. Cell type-specific cleavage of nucleocapsid protein by effector caspases during SARS coronavirus infection. J. Mol. Biol. 2008;376:23–34.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7094231'}, {'@IdType': 'pubmed', '#text': '18155731'}]}}",
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"{'Citation': 'Ding L., Huang Y., Du Qian, Dong F., Zhao X.M., Zhang W.L., Xu X.G., Tong D.W. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling. Biochem. Biophys. Res. Co. 2014;445:497–503.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7092941'}, {'@IdType': 'pubmed', '#text': '24548406'}]}}",
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] |
Res Vet Sci. 2018 Aug 12; 119:109-115
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NO-CC CODE
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Experiment 3 results. (A) Classification of the stimulus category, ignoring the position (error bars, 95% c.i.). (B) A flat map of face-sensitive cortex from a representative subject showing the distribution of the categories to which each voxel maximally contributed. (C) Distribution of the proportion of voxels that maximally contributed to each of the three categories in the OFA and FFA (error bars, 95% c.i.). Asterisks indicate the level of significance for independent t-tests in (A) and paired t-tests in (C) (*p < 0.05, **p < 0.01, ***p < 0.001).
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fpsyg-01-00028-g010
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2
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a94f2dad12a0f1456c4121d9b7344bc94e541970852a8019658931491cad3b5f
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fpsyg-01-00028-g010.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
441,
871
] |
[{'image_id': 'fpsyg-01-00028-g001', 'image_file_name': 'fpsyg-01-00028-g001.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g001.jpg', 'caption': 'Stimuli from all three experiments. (A) In Experiment 1, synthetic face stimuli (Wilson et al., 2002) contained either the head shape (Outline condition), internal facial features (Features condition), or both the features and outline (Whole Face condition). (B) In Experiment 2 the stimuli were created by independently scrambling the internal features and head outlines within squares of 16 and 24 pixels, respectively, which were constrained to lie within the locations occupied by the unscrambled contours. Stimuli for all categories had identical Fourier power spectra and contrast energy. (C) The stimuli in Experiment 3 were constructed in an identical fashion as Experiment 1, but the images were reduced in size and assigned to one of four quadrants in the visual display.', 'hash': '45383d5be590555592d95e05cee8dcc974b75a53855f289348ef16035cc0d31a'}, {'image_id': 'fpsyg-01-00028-g006', 'image_file_name': 'fpsyg-01-00028-g006.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g006.jpg', 'caption': 'Experiment 1 results. (A) The multi-class SVM analysis within specific ROIs indicates that classification of each stimulus category was significantly above chance in both hemispheres of the OFA and FFA (error bars, 95% c.i.). (B) The color-coded distributions of voxels according to the stimulus category to which they maximally contributed are shown here on the flat map of face-sensitive cortex from a representative subject. Anatomical labels are as follows: superior temporal sulcus (STS), inferotemporal sulcus (ITS), inferior-occipital gyrus (IOG), collateral sulcus (CoS), fusiform gyrus (FG). (C) The relative proportion of voxels that maximally contributed to the three stimulus categories within the OFA and the FFA (error bars, 95% c.i.). Asterisks indicate the level of significance for independent t-tests in (A) and paired t-tests in (C) (*p\u2009<\u20090.05, **p\u2009<\u20090.01, ***p\u2009<\u20090.001).', 'hash': 'f29ad9376f4aff381d3378796c5d9d79abf1132003eafeb5454f563ff699036a'}, {'image_id': 'fpsyg-01-00028-g008', 'image_file_name': 'fpsyg-01-00028-g008.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g008.jpg', 'caption': 'Mean classification of stimulus category in Experiment 1 based on different numbers of voxels in early visual cortex (V1–V3). The rightmost square symbols indicate where the number of V1–V3 voxels matched the number of voxels in the FFA (Table 1). The rightmost circle and diamond symbols indicate the classification performance within the OFA and FFA. (error bars, 95% c.i.).', 'hash': '6bd17867a0965acb257f1fca4efcab514f6752726ebe1006cf3bfe0ba5c2e763'}, {'image_id': 'fpsyg-01-00028-g010', 'image_file_name': 'fpsyg-01-00028-g010.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g010.jpg', 'caption': 'Experiment 3 results. (A) Classification of the stimulus category, ignoring the position (error bars, 95% c.i.). (B) A flat map of face-sensitive cortex from a representative subject showing the distribution of the categories to which each voxel maximally contributed. (C) Distribution of the proportion of voxels that maximally contributed to each of the three categories in the OFA and FFA (error bars, 95% c.i.). Asterisks indicate the level of significance for independent t-tests in (A) and paired t-tests in (C) (*p\u2009<\u20090.05, **p\u2009<\u20090.01, ***p\u2009<\u20090.001).', 'hash': 'a94f2dad12a0f1456c4121d9b7344bc94e541970852a8019658931491cad3b5f'}, {'image_id': 'fpsyg-01-00028-g009', 'image_file_name': 'fpsyg-01-00028-g009.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g009.jpg', 'caption': 'Experiment 2 results. (A) The mean response level to the different scrambled stimulus categories (21 samples each; error bars, 1 s.e.). (B) Results of the multi-class SVM analysis within early visual areas and face-selective cortex for Experiment 1 (◊) and Experiment 2 (□) (error bars, 95% c.i.). (C) Categorization accuracy of classifiers trained to discriminate fMRI responses to scrambled stimuli that were then tested on independent fMRI responses to unscrambled stimuli (error bars, 95% c.i.). (D) The relative proportion of voxels that maximally contributed to the three stimulus categories within the OFA and the FFA (error bars, 95% c.i.). Asterisks indicate the level of significance for paired t-tests in (B–D) and independent t-tests relative to chance in (C) (*p\u2009<\u20090.05, **p\u2009<\u20090.01, ***p\u2009<\u20090.001).', 'hash': '4c4f2375d09214975a87c3eea72e14b84e7c5637163d22d5559862eaafd39620'}, {'image_id': 'fpsyg-01-00028-g007', 'image_file_name': 'fpsyg-01-00028-g007.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g007.jpg', 'caption': 'Exploration of interactions between the ROI and the average response level to the three stimulus categories. (A) Mean activation in all ROIs (error bars, 1 s.e.). (B) Classification of the stimulus category when either the normalized ROI response in each ROI was used as input to a pattern classifier, or the entire normalized voxel-wise pattern of activation across all ROIs was used as input to a pattern classifier (error bars, 95% c.i.). Asterisks indicate the level of significance for both independent t-tests and paired t-tests (*p\u2009<\u20090.05, **p\u2009<\u20090.01, ***p\u2009<\u20090.001).', 'hash': 'd5a6e5840d0cb0ba03784fb2bcda404e4d4387e4f0c35403615b9b33987eccb1'}, {'image_id': 'fpsyg-01-00028-g003', 'image_file_name': 'fpsyg-01-00028-g003.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g003.jpg', 'caption': 'Demonstration of the effects of alternative normalization procedures on actual and simulated data. (A) Actual data from an individual subject is shown before normalization and following each of two different normalization procedures. Classification performance (PC\u2009=\u2009percent correct) remained high for both types of normalization. (B) The simulated data incorporated additive and multiplicative scaling of a single Gaussian spatial pattern of activation. Classification performance was preserved when normalization was conducted on each individual sample. Classification fell to chance levels when normalization was conducted on the mean response pattern of each category.', 'hash': 'f9815ac9d9efe8133bc03ae6e2e3771b0da57aab6989ba7c7929e61f362151de'}, {'image_id': 'fpsyg-01-00028-g004', 'image_file_name': 'fpsyg-01-00028-g004.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g004.jpg', 'caption': 'Steps in building the multi-class classifier for stimulus category and assessing classification accuracy. (A) During the training stage, samples from all but one scan of the data set were used (1) Pair-wise classifiers were built by inputting all training samples of a particular pair of categories into a SVM. The SVM returned a set of weights (one for each voxel) and a bias, which together determined a decision boundary for the two stimulus categories. (2) A single classifier was established for each stimulus category by summing together the weights and biases of the relevant pair-wise classifiers, with proper inversion of the sign of the weights. The three category classifiers together comprised the multi-class classifier. (B) During the testing stage, the samples from the left-out scan were independently classified through a matrix multiplication of the trained classifier weights and the sample response vector. The resulting product was adjusted by the biases and the maximum response was taken as the guess for the category to which a particular sample belonged. The entire process (A, B) was then repeated, leaving out a different scan each time, until all samples were part of a test set exactly once.', 'hash': '0dc6c13d7ea3db6f22e15466e6e4517d7a9741288d07133cade8c6adff89c58c'}, {'image_id': 'fpsyg-01-00028-g005', 'image_file_name': 'fpsyg-01-00028-g005.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g005.jpg', 'caption': 'Stages in determining the category to which a particular voxel contributed most positively. For clarity, a small set of 25 example voxels is shown here. (A) The fMRI responses (percent signal change from baseline) for the 25 voxels were averaged across all training samples and categories. (B) The sign of the mean fMRI activation was recorded for each voxel. (C) The multi-class classifiers from all training sets (see Figure 4) were averaged to determine the mean weights for each category, indicating the relative contribution of each voxel to the discriminatory information for a particular category classifier. (D) The mean classifier weights were multiplied by the sign of the normalized fMRI data (B) on a voxel-by-voxel basis, so that positive weighted responses represented evidence for a particular category, and negative weighted responses represented evidence against a particular category. For the set of 25 example voxels shown here, the sign of the weights for voxels 13–25 are therefore flipped. (E) Each voxel was then assigned to the category to which it contributed the most positively. (F) The proportion of voxels in each category was tallied.', 'hash': '69b1460880c6732957a21d641be2ae1e8351a636dd21d7d2f1d676231fc1d2e3'}, {'image_id': 'fpsyg-01-00028-g002', 'image_file_name': 'fpsyg-01-00028-g002.jpg', 'image_path': '../data/media_files/PMC3153747/fpsyg-01-00028-g002.jpg', 'caption': 'Experiment 1 average BOLD activation pre- and post-normalization in the combined OFA and FFA region of interest. (A) The activation within each voxel to the three categories, rank ordered based on the percent signal change after averaging across all categories on a voxel-by-voxel basis. Note that the response to the head outlines (red curve) is consistently lower than the response to the other two categories. (B) The activation within each voxel to the three categories, after normalization. Note that there are no longer differences in the mean level of activation across the three stimulus categories. However, differences in the pattern of activation across categories may still remain, which could provide the information necessary for reliable pattern classification.', 'hash': 'fc9ad85f2390f7c008f428dadc1dd34915f8ec64b0352fca4ecd8f32f4e17e12'}]
|
{'fpsyg-01-00028-g001': ['Synthetic face stimuli were constructed from a database of 80 grayscale photographs (Wilson et al., 2002). In Experiment 1, we analyzed data from eight participants that were previously acquired in the context of a single-trial event-related fMRI adaptation study (Betts and Wilson, 2009). Whole faces, features, or outlines (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>A) were centrally presented for 5\u2009s at the beginning of each trial. The average whole face was 10° tall and 7.5° wide. Observers performed a two-interval, forced choice task in response to an 8% change in stimulus size that occurred after 2.5\u2009s. The activation within each voxel for these event-related trials was defined as the percent signal change from baseline averaged over 3.75\u2009s, beginning 3.75\u2009s after stimulus onset to compensate for the delay of the hemodynamic response function. Baseline was defined as the average activation of the 3.75\u2009s prior to stimulus onset. Each stimulus type was randomly presented four times per scan in each of nine scans, for a total of 36 repetitions per condition. In Experiment 2 we presented blocks of synthetic face stimuli in which specific regions of the stimuli were scrambled in a grid-like fashion (Figure A) were centrally presented for 5\u2009s at the beginning of each trial. The average whole face was 10° tall and 7.5° wide. Observers performed a two-interval, forced choice task in response to an 8% change in stimulus size that occurred after 2.5\u2009s. The activation within each voxel for these event-related trials was defined as the percent signal change from baseline averaged over 3.75\u2009s, beginning 3.75\u2009s after stimulus onset to compensate for the delay of the hemodynamic response function. Baseline was defined as the average activation of the 3.75\u2009s prior to stimulus onset. Each stimulus type was randomly presented four times per scan in each of nine scans, for a total of 36 repetitions per condition. In Experiment 2 we presented blocks of synthetic face stimuli in which specific regions of the stimuli were scrambled in a grid-like fashion (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>B). Stimuli were created by independently scrambling the internal features and head outlines within squares of 16 and 24 pixels, respectively, which were constrained to lie within the locations occupied by the unscrambled contours. Stimuli for all categories had identical Fourier power spectra and contrast energy. Participants completed a 1-back face identity matching task. Stimulus blocks were 15\u2009s in length and alternated with 15\u2009s of a full field of mean luminance that contained a white fixation cross. The activation within each voxel for these blocks was defined as the percent signal change from baseline averaged over 11.25\u2009s, beginning 6.25\u2009s after stimulus onset. Baseline was defined as the average activation of the 3.75\u2009s prior to stimulus onset. Subjects viewed three repetitions of four stimulus conditions in each of seven scans, for a total of 21 repetitions per condition. Experiment 3 used the same stimulus set as Experiment 1, but the faces were reduced to approximately 5° tall and were presented 3.75° in the periphery in one of four stimulus locations (Figure B). Stimuli were created by independently scrambling the internal features and head outlines within squares of 16 and 24 pixels, respectively, which were constrained to lie within the locations occupied by the unscrambled contours. Stimuli for all categories had identical Fourier power spectra and contrast energy. Participants completed a 1-back face identity matching task. Stimulus blocks were 15\u2009s in length and alternated with 15\u2009s of a full field of mean luminance that contained a white fixation cross. The activation within each voxel for these blocks was defined as the percent signal change from baseline averaged over 11.25\u2009s, beginning 6.25\u2009s after stimulus onset. Baseline was defined as the average activation of the 3.75\u2009s prior to stimulus onset. Subjects viewed three repetitions of four stimulus conditions in each of seven scans, for a total of 21 repetitions per condition. Experiment 3 used the same stimulus set as Experiment 1, but the faces were reduced to approximately 5° tall and were presented 3.75° in the periphery in one of four stimulus locations (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>C). The stimuli were blocked according to stimulus category (Whole Faces, Features, and Outlines) and position (Upper Left, Upper Right, Lower Left, and Lower Right quadrant). To maintain central fixation, subjects performed a 1-of-3 color discrimination task at the fovea during both stimulus and fixation blocks (15\u2009s per block). Activation for each voxel was defined as the percent signal change from baseline averaged over 11.25\u2009s, beginning 6.25\u2009s after stimulus onset. Baseline activation was defined in the same manner as Experiments 1 and 2. The participants viewed one repetition of each stimulus/category combination per scan (12 stimulus blocks per scan) for five scans, which provided a total of 20 category and 15 position repetitions.C). The stimuli were blocked according to stimulus category (Whole Faces, Features, and Outlines) and position (Upper Left, Upper Right, Lower Left, and Lower Right quadrant). To maintain central fixation, subjects performed a 1-of-3 color discrimination task at the fovea during both stimulus and fixation blocks (15\u2009s per block). Activation for each voxel was defined as the percent signal change from baseline averaged over 11.25\u2009s, beginning 6.25\u2009s after stimulus onset. Baseline activation was defined in the same manner as Experiments 1 and 2. The participants viewed one repetition of each stimulus/category combination per scan (12 stimulus blocks per scan) for five scans, which provided a total of 20 category and 15 position repetitions.', 'To investigate the representation of faces and face parts in the human visual cortex, we used fMRI to measure brain activation within the FFA and OFA regions of interest to synthetic face stimuli comprised of whole faces, the internal facial features, and the head outlines (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>A) (Wilson et al., A) (Wilson et al., 2002; Betts and Wilson, 2009). Normalization of the response across voxels ensured that classification was based on differences in the spatial pattern of the response across voxels rather than differences in mean overall response to the three stimulus categories (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g002">2</xref>). A linear, multi-class SVM classifier was created for each region of interest using the leave-one-scan-out procedure (Kamitani and Tong, ). A linear, multi-class SVM classifier was created for each region of interest using the leave-one-scan-out procedure (Kamitani and Tong, 2005; Figure <xref ref-type="fig" rid="fpsyg-01-00028-g004">4</xref>). The classification accuracy for the spatial patterns within these areas was assessed separately for each participant, and statistics were performed on the average proportion correct across participants (Figure ). The classification accuracy for the spatial patterns within these areas was assessed separately for each participant, and statistics were performed on the average proportion correct across participants (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>A). Classification for all stimulus categories was significantly above chance (33% correct) in the OFA and FFA regions in both hemispheres. For example, in the left hemisphere OFA, outlines were distinguished from features and whole faces on 65% of trials (A). Classification for all stimulus categories was significantly above chance (33% correct) in the OFA and FFA regions in both hemispheres. For example, in the left hemisphere OFA, outlines were distinguished from features and whole faces on 65% of trials (t(7)\u2009=\u20095.24, p\u2009<\u20090.01), features were distinguished from whole faces and outlines on 55% of trials (t(7)\u2009=\u20093.64, p\u2009<\u20090.01), and whole faces were distinguished from features and outlines on 61% of trials (t(7)\u2009=\u20094.54, p\u2009<\u20090.01). Classification performance, averaged across category and hemisphere, did not significantly differ between the OFA (64%) and the FFA (61%) regions (t(7)\u2009=\u20090.59, p\u2009>\u20090.55), indicating that similar levels of discriminatory information for the facial categories were present in both regions.', 'We first scrambled the stimulus regions corresponding to the feature and outlines to create the following three stimulus categories: (1) scrambled features, intact outline (SF/IO); (2) Intact Features, Scrambled Outline (IF/SO); and (3) Intact Features, Intact Outline (IF/IO) (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>B). The average Fourier amplitude spectra of the randomly selected set of eight faces used within a scan was then applied to each of the stimuli to ensure that all stimuli had the same spatial frequency and contrast energy. Additionally, a white rectangular grid was superimposed on the stimuli to minimize the effects of new local edges created during the scrambling procedure.B). The average Fourier amplitude spectra of the randomly selected set of eight faces used within a scan was then applied to each of the stimuli to ensure that all stimuli had the same spatial frequency and contrast energy. Additionally, a white rectangular grid was superimposed on the stimuli to minimize the effects of new local edges created during the scrambling procedure.', 'Just how similar are the patterns of activation in face-sensitive cortex to the scrambled and unscrambled versions of the stimuli? This is akin to asking to what extent does face-irrelevant stimulus information affect processing of the individual face components. To address this question, we trained SVM multi-class classifiers on the OFA and FFA activation patterns elicited by the scrambled stimuli (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>B) in six subjects, and then tested these classifiers on fMRI responses to unscrambled versions of the stimuli (Figure B) in six subjects, and then tested these classifiers on fMRI responses to unscrambled versions of the stimuli (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>A) obtained during an independent set of scans. It is worth noting that while all of the experimental scans were run on the same day for three of the subjects, scrambled and unscrambled runs were taken 3 months apart for two subjects, and 20 months apart for one subject, with the generalization performance of similar magnitudes regardless of the temporal proximity of the scans. As shown in Figure A) obtained during an independent set of scans. It is worth noting that while all of the experimental scans were run on the same day for three of the subjects, scrambled and unscrambled runs were taken 3 months apart for two subjects, and 20 months apart for one subject, with the generalization performance of similar magnitudes regardless of the temporal proximity of the scans. As shown in Figure <xref ref-type="fig" rid="fpsyg-01-00028-g009">9</xref>C, significant generalization was possible in the OFA (percent correct\u2009=\u200950%, C, significant generalization was possible in the OFA (percent correct\u2009=\u200950%, t(5)\u2009=\u20095.65, p\u2009<\u20090.01), but not in the FFA (percent correct\u2009=\u200938%, t(4)\u2009=\u2009\u20092.12, p\u2009>\u20090.10). This indicates that the pattern of activation was more affected in the FFA than the OFA when face-irrelevant stimulus information was added, even though similar levels of classification were found in both areas for scrambled and unscrambled versions of the stimuli separately. Examining the relative distribution of voxels contributing maximally to the different category classifiers (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g009">9</xref>D), there was again a greater proportion of voxels contributing to the Head Outline classifier within the OFA than within the FFA (D), there was again a greater proportion of voxels contributing to the Head Outline classifier within the OFA than within the FFA (t(7)\u2009=\u20092.67, p\u2009<\u20090.05), but the relative proportion of voxels contributing to the Whole Face classifier was not significantly different between the OFA and the FFA (t(7)\u2009=\u20090.01). Based on all of the results of Experiment 2 taken together, it appears that the discriminatory patterns of activation had a greater effect on the representation of the whole face than on the face components when scrambled stimuli were used, consistent with the representation of whole faces being more selective to stimulus conditions than the representation of the head outlines or internal features.', 'We conducted a final experiment to rule out the possibility that the observed interaction between ROI and the distribution of voxels contributing to the Head Outline and Whole Face classifiers simply reflects a differential foveal versus peripheral processing bias in OFA and FFA. We scanned an additional eight subjects (five of whom were naïve; three F) who viewed unscrambled stimuli, as in Experiment 1, though reduced in size and presented in the periphery (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g001">1</xref>C), while performing a moderately demanding fixation task. Presentation of the stimuli was in one of the four quadrants of the visual field on any given block, but position varied across blocks. One block of each category at each position was presented per scan, for five scans, resulting in a total of 20 samples per category and 15 samples per position for the SVM analysis. Given the known position sensitivity in the OFA and FFA (Schwarzlose et al., C), while performing a moderately demanding fixation task. Presentation of the stimuli was in one of the four quadrants of the visual field on any given block, but position varied across blocks. One block of each category at each position was presented per scan, for five scans, resulting in a total of 20 samples per category and 15 samples per position for the SVM analysis. Given the known position sensitivity in the OFA and FFA (Schwarzlose et al., 2008), the peripheral stimulus presentation, and the focus of attention on an unrelated central fixation task, it is not surprising that classification of the stimulus category was substantially reduced compared to Experiments 1 and 2. However, the category classification performance was still above chance levels (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g010">10</xref>A; OFA percent correct\u2009=\u200939%, A; OFA percent correct\u2009=\u200939%, p\u2009<\u20090.01; FFA percent correct\u2009=\u200943%, p\u2009<\u20090.05; combined OFA and FFA percent correct\u2009=\u200945%, p\u2009<\u20090.01). More importantly, even with the reduced bias for features being presented centrally and outlines peripherally through this stimulus manipulation, a similar pattern of voxel distributions to Experiment 1 was found (Figures <xref ref-type="fig" rid="fpsyg-01-00028-g010">10</xref>B,C; ROI\u2009× Category interaction of the voxel distribution: B,C; ROI\u2009× Category interaction of the voxel distribution: F(2,14)\u2009=\u20097.47, p\u2009<\u20090.01), with proportionally more voxels contributing to the Head Outline classifier in OFA compared to FFA (t(7)\u2009=\u20093.79, p\u2009<\u20090.01), and proportionally more voxels contributing to the Whole Face classifier in FFA compared to OFA (t(7)\u2009=\u20093.30, p\u2009<\u20090.05). These results again indicate that the OFA is more involved in processing the building blocks of faces, and particularly information regarding head shape, whereas the FFA is more involved in processing the final construction of a face. The finding that the distribution of voxels in the OFA and FFA was similarly biased with very different stimulus manipulations, different experimental methods, and across a total of 16 participants, indicates that the tendency towards representing outlines in the OFA and whole faces in the FFA is a robust phenomenon.'], 'fpsyg-01-00028-g002': ['The data were normalized prior to the pattern classification analysis as follows. The signal for every sample, which was calculated from either a single event-related trial in Experiment 1 or a complete block of trials in Experiments 2 and 3, was converted to the percent signal change from baseline on a voxel-wise basis. The resulting values were then scaled such that the mean activation across the voxels for each sample was set to 0 and the variance was set to 1. In this way, any information due to mean activation differences between the different categories was removed, and only differences in the spatial pattern of activation could be used to classify the particular pattern of brain activation resulting from viewing a particular category. The effect of normalization is illustrated in Figure <xref ref-type="fig" rid="fpsyg-01-00028-g002">2</xref>. Figure . Figure <xref ref-type="fig" rid="fpsyg-01-00028-g002">2</xref>A shows the activation within each voxel to the three categories, rank ordered by the mean percent signal change after averaging across all categories. Note that the response to the Outline condition (red line) was consistently lower than to Whole Face or Features prior to normalization, which produced lower mean responses to the Outline stimuli than the other two conditions. Normalization eliminated the mean differences across conditions and centered the mean activation to zero for all categories (Figure A shows the activation within each voxel to the three categories, rank ordered by the mean percent signal change after averaging across all categories. Note that the response to the Outline condition (red line) was consistently lower than to Whole Face or Features prior to normalization, which produced lower mean responses to the Outline stimuli than the other two conditions. Normalization eliminated the mean differences across conditions and centered the mean activation to zero for all categories (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g002">2</xref>B).B).'], 'fpsyg-01-00028-g003': ['Normalization of each sample prior to pattern classification analysis was done primarily to remove differences in mean activation levels across categories, in order to explicitly test whether the different categories of face-relevant stimuli resulted in distinct spatial patterns of activation. It should be noted that differences in mean activation levels that could arise from attentional factors were therefore also controlled by the normalization procedure (Wojciulik et al., 1998). However, as a technical aside, non-uniform differences in mean activation levels across voxels (such as with a family of Gaussian distributions, where some voxels have consistently higher activation than others; Figure <xref ref-type="fig" rid="fpsyg-01-00028-g003">3</xref>B) could cause discriminable patterns of activation following normalization. Despite identical underlying patterns of activation, responses to the three stimulus categories could be discriminated solely on the basis of additive and/or multiplicative scaling factors. Therefore, an alternative, albeit more complicated, method of normalization was also used to verify the results. The alternative method entailed subtracting the mean and dividing through by the root mean square of the response across voxels, either on an individual sample basis or on the mean pattern of activation of each category (Figure B) could cause discriminable patterns of activation following normalization. Despite identical underlying patterns of activation, responses to the three stimulus categories could be discriminated solely on the basis of additive and/or multiplicative scaling factors. Therefore, an alternative, albeit more complicated, method of normalization was also used to verify the results. The alternative method entailed subtracting the mean and dividing through by the root mean square of the response across voxels, either on an individual sample basis or on the mean pattern of activation of each category (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g003">3</xref>). For the actual data (Figure ). For the actual data (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g003">3</xref>A) classification of the category was above chance before normalization, and stayed above chance after both types of normalization (average percent correct, PC, is reported averaged across categories and subjects). Simulated data (Figure A) classification of the category was above chance before normalization, and stayed above chance after both types of normalization (average percent correct, PC, is reported averaged across categories and subjects). Simulated data (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g003">3</xref>B) included additive and multiplicative scaling of a single Gaussian spatial pattern of activation. Following independent normalization of each individual sample, classification was possible because of residual differences in the variance across samples. However, when normalization was done on the mean response pattern of each category, averaged across samples, with the same scaling then applied to each sample of a particular category, classification of the category was no longer possible (chance\u2009=\u200933%). This indicates that the additional normalization procedure successfully removed differences in patterns of activation that were due simply to additive and multiplicative scaling. Therefore, the successful classification of the category in the real data, which was found for both types of normalization, can be taken as true differences in the patterns of activation, irrespective of differences in mean response amplitude. Comparable levels of classification for the experimental data were obtained using the initial and alternative normalization procedures. The description of the procedures and the results are therefore based on the simpler, more straight forward initial normalization procedure described in Figure B) included additive and multiplicative scaling of a single Gaussian spatial pattern of activation. Following independent normalization of each individual sample, classification was possible because of residual differences in the variance across samples. However, when normalization was done on the mean response pattern of each category, averaged across samples, with the same scaling then applied to each sample of a particular category, classification of the category was no longer possible (chance\u2009=\u200933%). This indicates that the additional normalization procedure successfully removed differences in patterns of activation that were due simply to additive and multiplicative scaling. Therefore, the successful classification of the category in the real data, which was found for both types of normalization, can be taken as true differences in the patterns of activation, irrespective of differences in mean response amplitude. Comparable levels of classification for the experimental data were obtained using the initial and alternative normalization procedures. The description of the procedures and the results are therefore based on the simpler, more straight forward initial normalization procedure described in Figure <xref ref-type="fig" rid="fpsyg-01-00028-g002">2</xref>..'], 'fpsyg-01-00028-g004': ['A linear, multi-class SVM classifier was established using the leave-one-scan-out procedure detailed in Kamitani and Tong (2005) using custom-built Matlab code combined with freely distributed support vector machine (SVM) functions from Canu et al. (2005). The specific procedure will be detailed utilizing an example from Experiment 1, though it was the same for all experiments. First, the samples from one entire scan (i.e., 12 samples consisting of four repetitions of each of the three conditions) were removed from the data set. The remaining samples were designated as training samples. Second, pair-wise classifiers were built by establishing the discriminatory information between two specified categories (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g004">4</xref>A1), using all training samples of a particular pair of categories as input to the SVM. The SVM returned a set of weights, one for each voxel, and a bias, which together determined a decision boundary for the two stimulus categories. Positive outputs represented one stimulus category and negative outputs represented the other. The weights for each pair were then normalized to have a length of one (μ\u2009=\u20090, σ\u2009=\u20091). The divisive scaling of the normalization weights was also applied to the bias. Third, a multi-class linear classifier was established by summing together the weights and biases of the relevant pair-wise classifiers, with proper inversion of the sign of the weights based on whether the category was represented by a positive or negative response in the output of the pair-wise classifier (Figure A1), using all training samples of a particular pair of categories as input to the SVM. The SVM returned a set of weights, one for each voxel, and a bias, which together determined a decision boundary for the two stimulus categories. Positive outputs represented one stimulus category and negative outputs represented the other. The weights for each pair were then normalized to have a length of one (μ\u2009=\u20090, σ\u2009=\u20091). The divisive scaling of the normalization weights was also applied to the bias. Third, a multi-class linear classifier was established by summing together the weights and biases of the relevant pair-wise classifiers, with proper inversion of the sign of the weights based on whether the category was represented by a positive or negative response in the output of the pair-wise classifier (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g004">4</xref>A2). Thus, each category had a single set of weights and a bias which together represented the multi-class linear classifier for each category for that particular set of training samples.A2). Thus, each category had a single set of weights and a bias which together represented the multi-class linear classifier for each category for that particular set of training samples.', 'The performance of the multi-class linear classifiers was tested using the samples that were originally removed from the data set (the 12 test trials from one entire scan) (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g004">4</xref>B). Each sample was classified through a matrix multiplication of the trained multi-class classifier weights and the vector of voxel responses. The resulting product for each sample was adjusted by the bias. The maximum positive response was taken as the guess for the category to which that particular sample belonged. The accuracy of the guess was recorded for each sample. The entire process was then repeated, i.e., a different scan was left out, a new multi-class linear classifier was created on the remaining data, and the accuracy of the classifier responses to the test trials were recorded, until all samples were part of a test set exactly once. The proportion of correctly classified samples was determined for each individual subject, and the average proportion correct across subjects was used to establish statistical significance in relation to chance.B). Each sample was classified through a matrix multiplication of the trained multi-class classifier weights and the vector of voxel responses. The resulting product for each sample was adjusted by the bias. The maximum positive response was taken as the guess for the category to which that particular sample belonged. The accuracy of the guess was recorded for each sample. The entire process was then repeated, i.e., a different scan was left out, a new multi-class linear classifier was created on the remaining data, and the accuracy of the classifier responses to the test trials were recorded, until all samples were part of a test set exactly once. The proportion of correctly classified samples was determined for each individual subject, and the average proportion correct across subjects was used to establish statistical significance in relation to chance.', 'Stages in determining the category to which a particular voxel contributed most positively. For clarity, a small set of 25 example voxels is shown here. (A) The fMRI responses (percent signal change from baseline) for the 25 voxels were averaged across all training samples and categories. (B) The sign of the mean fMRI activation was recorded for each voxel. (C) The multi-class classifiers from all training sets (see Figure <xref ref-type="fig" rid="fpsyg-01-00028-g004">4</xref>) were averaged to determine the mean weights for each category, indicating the relative contribution of each voxel to the discriminatory information for a particular category classifier. ) were averaged to determine the mean weights for each category, indicating the relative contribution of each voxel to the discriminatory information for a particular category classifier. (D) The mean classifier weights were multiplied by the sign of the normalized fMRI data (B) on a voxel-by-voxel basis, so that positive weighted responses represented evidence for a particular category, and negative weighted responses represented evidence against a particular category. For the set of 25 example voxels shown here, the sign of the weights for voxels 13–25 are therefore flipped. (E) Each voxel was then assigned to the category to which it contributed the most positively. (F) The proportion of voxels in each category was tallied.'], 'fpsyg-01-00028-g005': ['Multiple steps were necessary to assess the distribution of classifier weights within a region of interest, though each was simple and straightforward (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>). First, the normalized fMRI data were averaged across all samples and categories on a voxel-wise basis (Figure ). First, the normalized fMRI data were averaged across all samples and categories on a voxel-wise basis (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>A), and the resulting signs of the voxels recorded (Figure A), and the resulting signs of the voxels recorded (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>B). The sign of a particular voxel therefore indicates whether the fMRI signal was greater (i.e., positive) or less (i.e., negative) than the mean response of all voxels. Next, the multi-class linear classifiers for each training set were averaged to determine the mean weights for each category (Figure B). The sign of a particular voxel therefore indicates whether the fMRI signal was greater (i.e., positive) or less (i.e., negative) than the mean response of all voxels. Next, the multi-class linear classifiers for each training set were averaged to determine the mean weights for each category (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>C). This was done because the trained weights varied slightly between training sets, though they were highly similar due to the large number of shared samples between sets. Furthermore, the trained weights were separately normalized for each category to remove differences in the variance, to ensure that only the relative magnitude of the weight for a particular voxel in contributing to the overall category classifier was compared across categories. The mean classifier weights were then multiplied by the signs of the normalized fMRI data so that positive weighted responses represented evidence for a particular category, and negative weighted responses represented evidence against a particular category (Figure C). This was done because the trained weights varied slightly between training sets, though they were highly similar due to the large number of shared samples between sets. Furthermore, the trained weights were separately normalized for each category to remove differences in the variance, to ensure that only the relative magnitude of the weight for a particular voxel in contributing to the overall category classifier was compared across categories. The mean classifier weights were then multiplied by the signs of the normalized fMRI data so that positive weighted responses represented evidence for a particular category, and negative weighted responses represented evidence against a particular category (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>D). Lastly, each voxel was assigned to the category to which it contributed the most positively (Figure D). Lastly, each voxel was assigned to the category to which it contributed the most positively (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>E), and the distribution of categories across the voxels was determined for each individual subject (Figure E), and the distribution of categories across the voxels was determined for each individual subject (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>F).F).', 'Do the classified voxels truly represent three separate categories of voxels, or are the features and outline voxels simply a subset of a broad category of voxels that responds to whole faces? The former would be consistent with a heterogeneous population of three distinct types of neurons, as schematized in Figure 5 of Betts and Wilson (2009). The latter would be consistent with a homogenous population of neurons within the OFA and FFA that respond best to whole faces, but may partially respond to the other two categories of stimuli. The nature of the analysis used here, which forces a winner-take-all categorization on each voxel regarding the category classifier it contributes to the most (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>E), cannot distinguish between these possibilities. We therefore examined similarities in the relative responses to the different categories across voxels. The relative response was determined for each subject by subtracting out the mean response level on a voxel-by-voxel basis to all categories of stimuli. Similarities in the relative response to the different categories were then determined by correlating the relative response for all pairs of categories. If all voxels responded strongest to whole faces and less strongly to head outlines and internal features, one would expect positive correlations between whole faces and the other two stimulus types, and a negative correlation between head outlines and internal features. On average across subjects, we found no correlation (E), cannot distinguish between these possibilities. We therefore examined similarities in the relative responses to the different categories across voxels. The relative response was determined for each subject by subtracting out the mean response level on a voxel-by-voxel basis to all categories of stimuli. Similarities in the relative response to the different categories were then determined by correlating the relative response for all pairs of categories. If all voxels responded strongest to whole faces and less strongly to head outlines and internal features, one would expect positive correlations between whole faces and the other two stimulus types, and a negative correlation between head outlines and internal features. On average across subjects, we found no correlation (r\u2009=\u2009−0.17, t(7)\u2009=\u20090.70, n.s.) between the relative response of whole faces and internal features, but both the whole faces and internal features were negatively correlated with the relative response of head outlines (r\u2009=\u2009−0.68, t(7)\u2009=\u20095.04, p\u2009<\u20090.01, and r\u2009=\u2009−0.72, t(7)\u2009=\u20095.56, p\u2009<\u20090.001, respectively). These correlations are inconsistent with a single homogenous population of neurons/voxels. Rather, the results support three distinct types of voxels that are populated to different degrees by three proposed types of neurons, i.e., neurons selectively preferring head outlines, internal features, and whole faces.'], 'fpsyg-01-00028-g006': ['The significant classification shown in Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>A demonstrates that the differential patterns of activation were reliable, even after normalization removed the differences in the mean amplitude response. These results support the conclusions of previous fMRI adaptation studies that suggest whole faces and face parts are represented by different populations of neurons within face-selective visual cortex (Harris and Aguirre, A demonstrates that the differential patterns of activation were reliable, even after normalization removed the differences in the mean amplitude response. These results support the conclusions of previous fMRI adaptation studies that suggest whole faces and face parts are represented by different populations of neurons within face-selective visual cortex (Harris and Aguirre, 2008; Betts and Wilson, 2009), but demonstrate it in a much more direct manner.', 'Significant classification indicates spatially distributed neural populations responsive to whole faces, internal facial features, and head outlines in both the OFA and FFA. We also asked whether the output of the linear pattern classifiers could reveal any differences in the way the OFA and FFA process the three categories of facial stimuli. First, a single SVM classifier for each of the three categories was constructed after combining all of the voxels in the left and right hemisphere OFA and FFA regions. Classification for all stimulus categories was marginally improved, with 73%, 60%, and 69% accuracy for outlines, features, and whole faces (t(7)\u2009>\u20096.0, p\u2009<\u20090.001 in all categories; Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>A, rightmost bars). Each voxel was then labeled according to the category to which it contributed the strongest supportive weighting (Kamitani and Tong, A, rightmost bars). Each voxel was then labeled according to the category to which it contributed the strongest supportive weighting (Kamitani and Tong, 2005) (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>B; see also Figure B; see also Figure <xref ref-type="fig" rid="fpsyg-01-00028-g005">5</xref>). Next, the relative proportion of voxels contributing to each of the three categories was determined separately for the OFA and FFA regions by regrouping the voxels in the single SVM classifier into their respective ROIs for individual subjects. Finally, the representative proportion of voxels maximally contributing to each category was determined by averaging across subjects (Figure ). Next, the relative proportion of voxels contributing to each of the three categories was determined separately for the OFA and FFA regions by regrouping the voxels in the single SVM classifier into their respective ROIs for individual subjects. Finally, the representative proportion of voxels maximally contributing to each category was determined by averaging across subjects (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>C).C).', 'Inspection of the cortical maps of one subject (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>B) revealed a distinct difference in the distribution of red (Outline) and blue (Whole Face) voxels in the OFA and FFA. This pattern of results, with more red voxels in the OFA and more blue voxels in the FFA, was consistent across subjects. A repeated measures ANOVA, with ROI and stimulus category as factors, confirmed a significant interaction between ROI and the proportion of voxels contributing to each classifier (B) revealed a distinct difference in the distribution of red (Outline) and blue (Whole Face) voxels in the OFA and FFA. This pattern of results, with more red voxels in the OFA and more blue voxels in the FFA, was consistent across subjects. A repeated measures ANOVA, with ROI and stimulus category as factors, confirmed a significant interaction between ROI and the proportion of voxels contributing to each classifier (F(2,28)\u2009=\u20097.27, p\u2009<\u20090.001), without main effects for either ROI or stimulus category (F\u2009<\u20091). The interaction is explained by the fact that a greater proportion of voxels contributed to the Head Outline classifier in the OFA than the FFA (t(7)\u2009=\u20094.72, p\u2009<\u20090.01) and a greater proportion of voxels contributed to the Whole Face classifier in the FFA than the OFA (t(7)\u2009=\u20094.91, p\u2009<\u20090.01). The results also show that within the OFA, a greater proportion of voxels contributed to the Head Outline classifier than the Whole Face classifier (t(7)\u2009=\u20093.86, p\u2009<\u20090.01), and within the FFA, the opposite was true, as the greater proportion of voxels contributed to the Whole Face classifier (t(7)\u2009=\u20095.04, p\u2009<\u20090.01). The proportion of voxels assigned to the Internal Features classifier was not significantly different between areas (t(7)\u2009=\u20090.41, p\u2009>\u20090.65).'], 'fpsyg-01-00028-g007': ['Can the ability to classify the stimulus categories in the combined FFA and OFA ROI be explained simply by an interaction between the individual ROIs and the mean activation to the different stimulus categories? This would be implied if, for instance, the OFA responded the most to outlines while the FFA responded the most to whole faces. Inspection of the mean activation response, however, reveals that all ROIs yielded consistent relative responses to the different stimulus categories (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g007">7</xref>A). Although outlines consistently elicited the A). Although outlines consistently elicited the lowest response (main effect of category, F(2,14)\u2009=\u20098.74, p\u2009<\u20090.01), there was no significant interaction between ROI and percent signal change to the different stimulus categories (F(6,42)\u2009=\u20091.60, p\u2009>\u20090.50). Overall response levels, therefore, could not explain why the OFA contributes disproportionately to the Outline category compared to the FFA.', 'To test for reliable differences in the relative responses to the different stimulus categories across ROIs on an individual subject basis, a new analysis was run with the average activation for voxels within a particular ROI to each stimulus category as the input to the SVM. This was done after normalization of the response across all voxels on a trial-by-trial basis to ensure that there were no differences in mean response to the different categories. Although the category could be classified at 46% accuracy, this was significantly lower than the classification performance of 70% correct when the entire pattern of activation across all voxels was maintained (t(7)\u2009=\u20096.58, p\u2009<\u20090.001, Figure <xref ref-type="fig" rid="fpsyg-01-00028-g007">7</xref>B). As classification performance was significantly lower following the averaging of the response across voxels, we concluded that differences in the spatial distribution of activation within the ROIs, rather than the mean amplitude itself, drove the differential contributions to the category classifiers across the OFA and FFA described in Figure B). As classification performance was significantly lower following the averaging of the response across voxels, we concluded that differences in the spatial distribution of activation within the ROIs, rather than the mean amplitude itself, drove the differential contributions to the category classifiers across the OFA and FFA described in Figure <xref ref-type="fig" rid="fpsyg-01-00028-g006">6</xref>C.C.'], 'fpsyg-01-00028-g008': ['A basic property of the stimuli in Experiment 1, and of face organization in general, is that when a face is fixated, the facial features are central and the outline is peripheral. Thus, visual areas that are organized retinotopically would be expected to distinguish between these categories based solely on the spatial configuration of the facial elements. We tested this hypothesis by running the classification procedure on the activation in V1, V2, and V3. These early visual areas contain a much greater number of voxels compared to the FFA and OFA (Table 1). To control for the influence of ROI size, we repeated the classification process using several different sized subsets of randomly selected voxels, as well as a subset that matched the number of voxels in the OFA and FFA (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g008">8</xref>). A single random subset of voxels was selected for each subject, though results did not vary significantly when multiple subsets were averaged for each subject prior to group analyses. The results demonstrate (1) classification of stimulus category was possible in early visual areas, indicating clear differences in low-level stimulus features, (2) classification was possible using only a small subset of voxels (above chance for all retinotopic cortex ROIs using 10 or more voxels), (3) classification performance asymptotes by around 50 voxels. Classification performance was better in early retinotopic areas compared to face-sensitive areas even after matching the number of voxels for each subject to the number of voxels in FFA.). A single random subset of voxels was selected for each subject, though results did not vary significantly when multiple subsets were averaged for each subject prior to group analyses. The results demonstrate (1) classification of stimulus category was possible in early visual areas, indicating clear differences in low-level stimulus features, (2) classification was possible using only a small subset of voxels (above chance for all retinotopic cortex ROIs using 10 or more voxels), (3) classification performance asymptotes by around 50 voxels. Classification performance was better in early retinotopic areas compared to face-sensitive areas even after matching the number of voxels for each subject to the number of voxels in FFA.'], 'fpsyg-01-00028-g009': ['Both the OFA and FFA responded strongly to the IF/IO condition (which is similar to the Whole Face condition used in the above experiment, except with band-pass filtered noise and a grid added), but robust activation was also elicited by the IF/SO and SF/IO conditions (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g009">9</xref>A). In fact, no response differences were observed between the stimulus categories (A). In fact, no response differences were observed between the stimulus categories (F(2,12)\u2009=\u20090.91, p\u2009>\u20090.40). The fMRI responses to a fourth condition, in which both the features and outlines were scrambled, were significantly lower than any of the other three conditions, indicating that the scrambling procedure successfully interfered with face processing in the OFA and FFA. Furthermore, the response amplitudes were all in the same range in early visual areas (V1–V3), as would be expected from the controlled low-level stimulus information. As predicted, category classification was heavily disrupted by the scrambling procedure in early visual cortex, as variations in the spatial position of local stimulus contrast no longer served as a strong cue to stimulus category (Figure <xref ref-type="fig" rid="fpsyg-01-00028-g009">9</xref>B). However, classification performance in face-selective visual areas remained similar to the levels observed in Experiment 1, indicating that the perception of the intact face components could still sufficiently activate the neural populations responding to outlines, features, and whole faces. This clearly demonstrates that the classification performance observed in Experiment 1 was not simply due to a residual retinotopic bias in higher visual areas.B). However, classification performance in face-selective visual areas remained similar to the levels observed in Experiment 1, indicating that the perception of the intact face components could still sufficiently activate the neural populations responding to outlines, features, and whole faces. This clearly demonstrates that the classification performance observed in Experiment 1 was not simply due to a residual retinotopic bias in higher visual areas.']}
|
Decoding of Faces and Face Components in Face-Sensitive Human Visual Cortex
|
[
"vision",
"face perception",
"functional magnetic resonance imaging",
"fusiform face area",
"occipital face area",
"multi-voxel pattern classification"
] |
Front Psychol
|
1278572400
|
Science begins with the question, what do I want to know? Science becomes science, however, only when this question is justified and the appropriate methodology is chosen for answering the research question. Research question should precede the other questions; methods should be chosen according to the research question and not vice versa. Modern quantitative psychology has accepted method as primary; research questions are adjusted to the methods. For understanding thinking in modern quantitative psychology, two epistemologies should be distinguished: structural-systemic that is based on Aristotelian thinking, and associative-quantitative that is based on Cartesian-Humean thinking. The first aims at understanding the structure that underlies the studied processes; the second looks for identification of cause-effect relationships between the events with no possible access to the understanding of the structures that underlie the processes. Quantitative methodology in particular as well as mathematical psychology in general, is useless for answering questions about structures and processes that underlie observed behaviors. Nevertheless, quantitative science is almost inevitable in a situation where the systemic-structural basis of behavior is not well understood; all sorts of applied decisions can be made on the basis of quantitative studies. In order to proceed, psychology should study structures; methodologically, constructive experiments should be added to observations and analytic experiments.
|
[] |
other
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PMC3153747
| null |
65
|
[
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] |
Front Psychol. 2010 Jul 8; 1:28
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NO-CC CODE
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Induction of apoptosis by over-expression of M protein in HEK293T cells. (A–L) M protein expression induced nuclear condensation in HEK293T cells. Expression of M protein was only detected in transfected cells (G, in green), but not in untransfected (A and J) and empty vector (D) controls. Hoechst 33342 was used to label cell nuclei (B, E, H and K, in blue). Cells expressed with M protein showed nuclear condensation (I, arrowheads) at 48 h post-transfection. Untransfected cells treated with 1 μM staurosporine for 8 h also displayed nuclear condensation (K, arrowheads), while untreated (B) and empty vector (E) controls showed normal nuclear morphology. (M) The percentage of cells showed nuclear condensation was quantified. Results were plotted as percentage of cells showed nuclear condensation and expressed as means + SEM of three independent experiments. At least 100 cells were counted in each experiment. Scale bars represent 16 μm. ∗p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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gr2
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2
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ed785f942c970a5885ff583a310788848cf9ca33c2ee14ec87dd9cdb0310b5fd
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gr2.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
591,
920
] |
[{'image_id': 'gr7', 'image_file_name': 'gr7.jpg', 'image_path': '../data/media_files/PMC7094499/gr7.jpg', 'caption': 'Down-regulation of Akt protein phosphorylation in transgenic Drosophila over-expressing M protein. (A) Western blot analysis showed that M expression reduced Akt protein phosphorylation without altering total Akt protein level. β-tubulin was used as loading control. Phospho- (B) and total-Akt protein (C) levels were quantified and normalized against the β-tubulin loading control. Results were plotted as the percentage change of band intensity and expressed as means\xa0+\xa0SEM of five independent experiments. ∗p\xa0<\xa00.05.', 'hash': 'b5d7fe1efcc8126b2f47eff362f338ae8d7f4e5072ef994c2b60e2d6def82c0f'}, {'image_id': 'gr1', 'image_file_name': 'gr1.jpg', 'image_path': '../data/media_files/PMC7094499/gr1.jpg', 'caption': 'Expression and subcellular localization of SARS-CoV Membrane protein in HEK293T cells. (A) mRNA expression level of the SARS-CoV M gene was examined by RT-PCR. Cells transfected with pcDNA3.1(+)-Membrane plasmid showed M mRNA expression at 48\xa0h post-transfection, while no expression was detected in the untransfected and empty vector controls. GAPDH was used as loading control. RT(+) and RT(−) represent the presence and absence of reverse transcriptase enzyme in the RT reaction, respectively. (B–G) Subcellular localization of M protein was determined by immunofluorescence staining. The M protein displayed a punctate cytoplasmic staining pattern (E, in green) and partially localized (G) with the Golgi body (F, in red). Untransfected cells showed no expression of Membrane protein (B). Scale bars represent 16\xa0μm.', 'hash': '3aac3a28e0e08ebd6b1e4d8975df611e840922d9faca753fe3575810331e72b4'}, {'image_id': 'gr6', 'image_file_name': 'gr6.jpg', 'image_path': '../data/media_files/PMC7094499/gr6.jpg', 'caption': 'Suppression of M-induced apoptosis by phosphoinositide-dependent kinase-1 over-expression in Drosophila. (A–C) PDK-1 over-expression suppressed M-induced rough-eye phenotype. gmr-GAL4 alone control showed no dominant eye phenotype (A). The M-induced rough-eye phenotype (B) was suppressed by over-expression of PDK-1 (C). In the presence of GAL4, PDK-1EP837 causes over-expression of endogenous PDK-1[35]. (D–F) PDK-1 over-expression suppressed M-induced apoptosis as shown by acridine orange (AO) staining. The elevated number of AO-positive cells caused by M protein over-expression in imaginal eye discs (E) was reduced upon the over-expression of PDK-1 (F). Arrows indicate morphogenetic furrows. (G) The number of AO-positive cells in each genotype was quantified. Results were plotted as the number of AO-positive cells and expressed as means\xa0+\xa0SEM of three-independent experiments. At least nine imaginal eye discs were analyzed in each genotype. ∗p\xa0<\xa00.05 versus gmr-GAL4; #p\xa0<\xa00.05 versus gmr-GAL4 UAS-Membrane.', 'hash': '5d5cc4689c30af79729b3fdba9f5abfa6c53c8babc491db95052a1e27e97a09d'}, {'image_id': 'gr5', 'image_file_name': 'gr5.jpg', 'image_path': '../data/media_files/PMC7094499/gr5.jpg', 'caption': 'Induction of apoptosis by over-expression of M protein in Drosophila. (A–E) gmr-GAL4 alone control showed no dominant eye phenotype (A). A rough-eye phenotype was observed upon M protein over-expression (B), and was suppressed by co-expression with anti-apoptotic genes DIAP1 (C), P35 (D), and apo-cytochrome cdc3EP2305 (E). (F–J) M over-expression induced apoptosis in third instar imaginal eye discs as indicated by the increase in the number of acridine orange (AO)-stained cells (G). Induction of apoptosis by M protein was suppressed by the co-expression of anti-apoptotic genes DIAP1 (H), P35 (I), and apo-cytochrome cdc3EP2305 (J). Arrows indicate morphogenetic furrows. (K) The number of AO-positive cells in each genotype was quantified. Results were plotted as the number of AO-positive cells and expressed as means\xa0+\xa0SEM of three-independent experiments. At least nine imaginal eye discs were analyzed in each genotype. ∗p\xa0<\xa00.05 versus gmr-GAL4; #p\xa0<\xa00.05 versus gmr-GAL4 UAS-Membrane.', 'hash': 'ad8705d5cfcd9791d609493f65c6568e9dc97f5b7a0a3267e105bedcdf8c9869'}, {'image_id': 'gr2', 'image_file_name': 'gr2.jpg', 'image_path': '../data/media_files/PMC7094499/gr2.jpg', 'caption': 'Induction of apoptosis by over-expression of M protein in HEK293T cells. (A–L) M protein expression induced nuclear condensation in HEK293T cells. Expression of M protein was only detected in transfected cells (G, in green), but not in untransfected (A and J) and empty vector (D) controls. Hoechst 33342 was used to label cell nuclei (B, E, H and K, in blue). Cells expressed with M protein showed nuclear condensation (I, arrowheads) at 48\xa0h post-transfection. Untransfected cells treated with 1\xa0μM staurosporine for 8\xa0h also displayed nuclear condensation (K, arrowheads), while untreated (B) and empty vector (E) controls showed normal nuclear morphology. (M) The percentage of cells showed nuclear condensation was quantified. Results were plotted as percentage of cells showed nuclear condensation and expressed as means\xa0+\xa0SEM of three independent experiments. At least 100 cells were counted in each experiment. Scale bars represent 16\xa0μm. ∗p\xa0<\xa00.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)', 'hash': 'ed785f942c970a5885ff583a310788848cf9ca33c2ee14ec87dd9cdb0310b5fd'}, {'image_id': 'gr3', 'image_file_name': 'gr3.jpg', 'image_path': '../data/media_files/PMC7094499/gr3.jpg', 'caption': 'Cytochrome c and caspases are involved in M-induced apoptosis. (A–F) M protein expression induced mis-localization of cytochrome c protein in HEK293T cells. When cells were either transfected with M (C) for 48\xa0h or treated with 1\xa0μM staurosporine for 8\xa0h (E), mis-localization of cytochrome c protein was observed. Untransfected cells were used as control (A). Respective phase-contrast images are shown in B, D and F. Scale bars represent 16\xa0μm. (G) M-induced nuclear condensation was inhibited by caspase inhibitors (at 50\xa0μM) z-DQMD-fmk (caspase-3 inhibitor V), z-IETD-fmk (caspase-8 inhibitor II) and z-LEHD-fmk (caspase-9 inhibitor I). Results were plotted as percentage of cells showed nuclear condensation and expressed as means\xa0+\xa0SEM of three independent experiments. At least 100 cells were counted in each experiment. ∗p\xa0<\xa00.05.', 'hash': '6be03715a21045d09e08f3761e0e04c270bbdcf3140c271eee2672ec5a355571'}, {'image_id': 'gr4', 'image_file_name': 'gr4.jpg', 'image_path': '../data/media_files/PMC7094499/gr4.jpg', 'caption': 'Induction of rough-eye phenotype by over-expression of M protein in Drosophila. (A–C) Over-expression of the M protein in eye tissues resulted in rough-eye phenotype as characterized by loss of regularity of the adult external eye structure (C), whereas the gmr-GAL4 driver alone control (A) and over-expression of the EGFP protein (B) showed no dominant external eye phenotype. (D–F) Subcellular localization of M protein in the Drosophila third instar imaginal eye disc tissues. The M protein showed a distinct punctate cytoplasmic expression pattern (F, in green), whereas the expression EGFP control protein showed homogeneous intracellular staining (E, in green). Cell nuclei were stained by propidium iodide (in red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)', 'hash': '0ae05b32a297f4c92e9ec06ff5c2408a8193e649670fec93f483d3238e6bda06'}]
|
{'gr1': ['To study the function of the SARS-CoV Membrane protein, the M ORF of the CUHK-Su10 SARS-CoV isolate was subcloned into a mammalian expression vector pcDNA3.1(+). By RT-PCR, mRNA expression of the M gene was detected 48\xa0h post-transfection (<xref rid="gr1" ref-type="fig">Fig. 1</xref>\nA). Immunofluorescence was performed to determine the subcellular localization of the M protein. We observed that M protein displayed a punctate localization in the cytoplasm (\nA). Immunofluorescence was performed to determine the subcellular localization of the M protein. We observed that M protein displayed a punctate localization in the cytoplasm (<xref rid="gr1" ref-type="fig">Fig. 1</xref>E) and was partially co-localized with the Golgi body (E) and was partially co-localized with the Golgi body (<xref rid="gr1" ref-type="fig">Fig. 1</xref>G, inset).G, inset).Fig. 1Expression and subcellular localization of SARS-CoV Membrane protein in HEK293T cells. (A) mRNA expression level of the SARS-CoV M gene was examined by RT-PCR. Cells transfected with pcDNA3.1(+)-Membrane plasmid showed M mRNA expression at 48\xa0h post-transfection, while no expression was detected in the untransfected and empty vector controls. GAPDH was used as loading control. RT(+) and RT(−) represent the presence and absence of reverse transcriptase enzyme in the RT reaction, respectively. (B–G) Subcellular localization of M protein was determined by immunofluorescence staining. The M protein displayed a punctate cytoplasmic staining pattern (E, in green) and partially localized (G) with the Golgi body (F, in red). Untransfected cells showed no expression of Membrane protein (B). Scale bars represent 16\xa0μm.', 'The SARS-CoV Membrane protein is the most abundant protein embedded in the viral envelope [25]. The M protein promotes membrane fusion, regulates viral replication, and is also responsible for the packing of viral RNA into matured virions [26], [33]. It has been shown that M is concentrated in the Golgi body of viral-infected cells [45]. Time-lapse microscopy further shows that the M protein is transported in and out of the Golgi body via trafficking vesicles in mammalian cells [46], which further demonstrates its dynamic trafficking properties. In the present study, we showed that M protein partially localized with the Golgi body (<xref rid="gr1" ref-type="fig">Fig. 1</xref>G) which is consistent with previous findings G) which is consistent with previous findings [46], [47]. The incomplete co-localization of M with the Golgi body may be explained by the dynamic shuttling properties of the M protein as previously observed [46].'], 'gr2': ['When the cell nuclei of M-transfected HEK293 cells were stained with Hoechst dye, we found that ∼56% of the cell nuclei were condensed as indicated by size reduction (<xref rid="gr2" ref-type="fig">Fig. 2</xref>\nI and M) whereas only around 10% of the control cells had condensed nuclei (\nI and M) whereas only around 10% of the control cells had condensed nuclei (<xref rid="gr2" ref-type="fig">Fig. 2</xref>B, E and M). Since nuclear condensation is a hallmark feature of apoptosis, we reasoned that M over-expression induced apoptosis in HEK293T cells. To confirm this, we treated untransfected HEK293T cells with staurosporine, a commonly used apoptosis inducer, and nuclear condensation was also observed (B, E and M). Since nuclear condensation is a hallmark feature of apoptosis, we reasoned that M over-expression induced apoptosis in HEK293T cells. To confirm this, we treated untransfected HEK293T cells with staurosporine, a commonly used apoptosis inducer, and nuclear condensation was also observed (<xref rid="gr2" ref-type="fig">Fig. 2</xref>K and M).K and M).Fig. 2Induction of apoptosis by over-expression of M protein in HEK293T cells. (A–L) M protein expression induced nuclear condensation in HEK293T cells. Expression of M protein was only detected in transfected cells (G, in green), but not in untransfected (A and J) and empty vector (D) controls. Hoechst 33342 was used to label cell nuclei (B, E, H and K, in blue). Cells expressed with M protein showed nuclear condensation (I, arrowheads) at 48\xa0h post-transfection. Untransfected cells treated with 1\xa0μM staurosporine for 8\xa0h also displayed nuclear condensation (K, arrowheads), while untreated (B) and empty vector (E) controls showed normal nuclear morphology. (M) The percentage of cells showed nuclear condensation was quantified. Results were plotted as percentage of cells showed nuclear condensation and expressed as means\xa0+\xa0SEM of three independent experiments. At least 100 cells were counted in each experiment. Scale bars represent 16\xa0μm. ∗p\xa0<\xa00.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)', 'It is well known that SARS-CoV infection induces apoptosis in viral-infected cells [4], [11], [13], [14], [15] and the pro-apoptotic properties of a number of individual viral proteins have recently been revealed [12], [16], [17], [18], [19], [20], [21], [22]. Common morphological apoptotic features including cell shrinkage, condensed chromatin and apoptotic body formation are all observed in SARS-CoV viral-infected cells [13]. Swelling of mitochondria is also found in viral-infected cells [13] which indicates a mitochondrial involvement in SARS-CoV-induced apoptosis. At the mechanistic level, SARS-CoV infection has been shown to interfere with the Bcl-2 anti-apoptotic pathway [15]. In this study, we observed that both the in vitro (<xref rid="gr2" ref-type="fig">Fig. 2</xref>) and ) and in vivo (<xref rid="gr5" ref-type="fig">Fig. 5</xref>) over-expression of M protein induced apoptotic cell death. We also observed nuclear condensation () over-expression of M protein induced apoptotic cell death. We also observed nuclear condensation (<xref rid="gr2" ref-type="fig">Fig. 2</xref>H) and mis-localization of mitochondrial cytochrome H) and mis-localization of mitochondrial cytochrome c protein in M-expressing cells (<xref rid="gr3" ref-type="fig">Fig. 3</xref>C). Our findings are therefore well in line with the previously described pro-apoptotic properties of SARS-CoV.C). Our findings are therefore well in line with the previously described pro-apoptotic properties of SARS-CoV.'], 'gr3': ['Since cytochrome c release from the mitochondria represents one form of apoptotic cell death, we performed cytochrome c immunofluorescence staining to examine whether mitochondrial release of cytochrome c was detected in M-transfected cells. Unlike the untransfected control (<xref rid="gr3" ref-type="fig">Fig. 3</xref>\nA), we found that cells either transfected with \nA), we found that cells either transfected with M (<xref rid="gr3" ref-type="fig">Fig. 3</xref>C) or treated with staurosporine (C) or treated with staurosporine (<xref rid="gr3" ref-type="fig">Fig. 3</xref>E) showed extensive mis-localization of cytochrome E) showed extensive mis-localization of cytochrome c protein which is indicative of mitochondrial release of cytochrome c protein [39]. The appearance of M-transfected (<xref rid="gr3" ref-type="fig">Fig. 3</xref>D) and staurosporine-treated cells (D) and staurosporine-treated cells (<xref rid="gr3" ref-type="fig">Fig. 3</xref>F) were also morphologically distinct from the untransfected control (F) were also morphologically distinct from the untransfected control (<xref rid="gr3" ref-type="fig">Fig. 3</xref>B). We further showed that B). We further showed that M-induced nuclear condensation in HEK293T cells could be suppressed by caspase-3, -8 and -9 inhibitors (<xref rid="gr3" ref-type="fig">Fig. 3</xref>G).G).Fig. 3Cytochrome c and caspases are involved in M-induced apoptosis. (A–F) M protein expression induced mis-localization of cytochrome c protein in HEK293T cells. When cells were either transfected with M (C) for 48\xa0h or treated with 1\xa0μM staurosporine for 8\xa0h (E), mis-localization of cytochrome c protein was observed. Untransfected cells were used as control (A). Respective phase-contrast images are shown in B, D and F. Scale bars represent 16\xa0μm. (G) M-induced nuclear condensation was inhibited by caspase inhibitors (at 50\xa0μM) z-DQMD-fmk (caspase-3 inhibitor V), z-IETD-fmk (caspase-8 inhibitor II) and z-LEHD-fmk (caspase-9 inhibitor I). Results were plotted as percentage of cells showed nuclear condensation and expressed as means\xa0+\xa0SEM of three independent experiments. At least 100 cells were counted in each experiment. ∗p\xa0<\xa00.05.', 'We next questioned whether M-induced apoptosis would act through the route of cytochrome c in flies as in HEK293T cells (<xref rid="gr3" ref-type="fig">Fig. 3</xref>C). It has been reported that over-expression of the apo-form of cytochrome C). It has been reported that over-expression of the apo-form of cytochrome c (without heme group) can block apoptosis [40]. We made use of an EP insert line dc3\nEP2305 to over-express the endogenous apo-cytochrome c gene dc3\n[16] and asked if dc3 over-expression could suppress M-induced apoptosis in Drosophila. Both the rough-eye phenotype (<xref rid="gr5" ref-type="fig">Fig. 5</xref>B) and elevated number of AO-positive apoptotic cells in eye discs (B) and elevated number of AO-positive apoptotic cells in eye discs (<xref rid="gr5" ref-type="fig">Fig. 5</xref>G and K) were largely suppressed upon co-expression of M with apo-cytochrome G and K) were largely suppressed upon co-expression of M with apo-cytochrome c (<xref rid="gr5" ref-type="fig">Fig. 5</xref> E, J and K). E, J and K).'], 'gr4': ['We previously used a transgenic Drosophila model [16] to demonstrate the pro-apoptotic role of the SARS-CoV 3a protein [17]. In this study, we established UAS-M transgenic fly lines, and over-expressed the SARS-CoV M protein in the Drosophila eye using GAL4/UAS transgene expression system [36]. Over-expression of M caused a rough-eye phenotype in adult flies, as shown by the loss of regularity of external eye morphology (<xref rid="gr4" ref-type="fig">Fig. 4</xref>\nC), while the over-expression of the EGFP control protein (\nC), while the over-expression of the EGFP control protein (<xref rid="gr4" ref-type="fig">Fig. 4</xref>B) did not show any obvious dominant eye malformation phenotype. To determine the subcellular localization of M in B) did not show any obvious dominant eye malformation phenotype. To determine the subcellular localization of M in Drosophila, we performed immunofluorescence staining in third instar larval eye discs (<xref rid="gr4" ref-type="fig">Fig. 4</xref>D and F). Similar to mammalian cells (D and F). Similar to mammalian cells (<xref rid="gr1" ref-type="fig">Fig. 1</xref>E), M displayed a punctate cytoplasmic localization in fly cells (E), M displayed a punctate cytoplasmic localization in fly cells (<xref rid="gr4" ref-type="fig">Fig. 4</xref>F), whereas the EGFP control protein showed homogeneous intracellular localization (F), whereas the EGFP control protein showed homogeneous intracellular localization (<xref rid="gr4" ref-type="fig">Fig. 4</xref>E).E).Fig. 4Induction of rough-eye phenotype by over-expression of M protein in Drosophila. (A–C) Over-expression of the M protein in eye tissues resulted in rough-eye phenotype as characterized by loss of regularity of the adult external eye structure (C), whereas the gmr-GAL4 driver alone control (A) and over-expression of the EGFP protein (B) showed no dominant external eye phenotype. (D–F) Subcellular localization of M protein in the Drosophila third instar imaginal eye disc tissues. The M protein showed a distinct punctate cytoplasmic expression pattern (F, in green), whereas the expression EGFP control protein showed homogeneous intracellular staining (E, in green). Cell nuclei were stained by propidium iodide (in red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)', 'We performed a forward genetic modifier screen in Drosophila in order to gain further insights into the mechanistic details of M action in vivo. By analyzing the modifying effects of overlapping genomic deletion lines, from both the Exelixis [41] and DrosDel [42] collections, on the M-induced rough-eye phenotype (<xref rid="gr4" ref-type="fig">Fig. 4</xref>C); 30 modifying genomic regions were identified (C.M. Chan, W.M. Chan, C.S. Chan, C.W. Ma, K.W. Ching, L.L. Ho, K.M. Lau, H.Y. Chan, unpublished observations). Detailed characterization of one of these modifying regions (61B1-C1) is described here. The 61B1-C1 genomic region is uncovered by a deletion line C); 30 modifying genomic regions were identified (C.M. Chan, W.M. Chan, C.S. Chan, C.W. Ma, K.W. Ching, L.L. Ho, K.M. Lau, H.Y. Chan, unpublished observations). Detailed characterization of one of these modifying regions (61B1-C1) is described here. The 61B1-C1 genomic region is uncovered by a deletion line Df(3L)ED201\n[42]. We further found that one of the EP insert lines in the 61B1-C1 region, PDK-1\nEP837, dominantly suppressed the M-induced rough-eye phenotype (<xref rid="gr6" ref-type="fig">Fig. 6</xref>\nB and C). The \nB and C). The PDK-1\nEP837 insert is located at the 5′ upstream region of the phosphoinositide-dependent kinase-1 (PDK-1) gene transcription unit, and had previously been shown to cause over-expression of endogenous PDK-1\n[35].Fig. 6Suppression of M-induced apoptosis by phosphoinositide-dependent kinase-1 over-expression in Drosophila. (A–C) PDK-1 over-expression suppressed M-induced rough-eye phenotype. gmr-GAL4 alone control showed no dominant eye phenotype (A). The M-induced rough-eye phenotype (B) was suppressed by over-expression of PDK-1 (C). In the presence of GAL4, PDK-1EP837 causes over-expression of endogenous PDK-1[35]. (D–F) PDK-1 over-expression suppressed M-induced apoptosis as shown by acridine orange (AO) staining. The elevated number of AO-positive cells caused by M protein over-expression in imaginal eye discs (E) was reduced upon the over-expression of PDK-1 (F). Arrows indicate morphogenetic furrows. (G) The number of AO-positive cells in each genotype was quantified. Results were plotted as the number of AO-positive cells and expressed as means\xa0+\xa0SEM of three-independent experiments. At least nine imaginal eye discs were analyzed in each genotype. ∗p\xa0<\xa00.05 versus gmr-GAL4; #p\xa0<\xa00.05 versus gmr-GAL4 UAS-Membrane.'], 'gr5': ['Since over-expression of many genes would result in rough-eye phenotype in Drosophila, we therefore performed standard acridine orange (AO) staining [38] in third instar larval imaginal eye discs to examine the pro-apoptotic roles of M in vivo. Acridine orange is a dye that specifically stains apoptotic cells, and we found that the number of AO-positive cells in M-over-expressing eye discs (<xref rid="gr5" ref-type="fig">Fig. 5</xref>\nG and K) was significantly higher than that of the control (\nG and K) was significantly higher than that of the control (<xref rid="gr5" ref-type="fig">Fig. 5</xref>F and K). To further confirm the pro-apoptotic role of M, we co-expressed two anti-apoptotic factors, P35 (a viral caspase inhibitor) and the F and K). To further confirm the pro-apoptotic role of M, we co-expressed two anti-apoptotic factors, P35 (a viral caspase inhibitor) and the Drosophila inhibitor of apoptosis 1 (DIAP1) protein, independently with M. As expected, both the rough-eye phenotype (<xref rid="gr5" ref-type="fig">Fig. 5</xref>B–D) and elevated number of AO-positive cells (B–D) and elevated number of AO-positive cells (<xref rid="gr5" ref-type="fig">Fig. 5</xref>G–I and K) were mostly suppressed back to the control level, respectively.G–I and K) were mostly suppressed back to the control level, respectively.Fig. 5Induction of apoptosis by over-expression of M protein in Drosophila. (A–E) gmr-GAL4 alone control showed no dominant eye phenotype (A). A rough-eye phenotype was observed upon M protein over-expression (B), and was suppressed by co-expression with anti-apoptotic genes DIAP1 (C), P35 (D), and apo-cytochrome cdc3EP2305 (E). (F–J) M over-expression induced apoptosis in third instar imaginal eye discs as indicated by the increase in the number of acridine orange (AO)-stained cells (G). Induction of apoptosis by M protein was suppressed by the co-expression of anti-apoptotic genes DIAP1 (H), P35 (I), and apo-cytochrome cdc3EP2305 (J). Arrows indicate morphogenetic furrows. (K) The number of AO-positive cells in each genotype was quantified. Results were plotted as the number of AO-positive cells and expressed as means\xa0+\xa0SEM of three-independent experiments. At least nine imaginal eye discs were analyzed in each genotype. ∗p\xa0<\xa00.05 versus gmr-GAL4; #p\xa0<\xa00.05 versus gmr-GAL4 UAS-Membrane.', 'Since PDK-1 over-expression was able to rescue M-induced rough-eye phenotype, we investigated if PDK-1 suppressed the phenotype through inhibiting apoptosis. We showed that over-expression of SARS-CoV M induced apoptosis in vivo as indicated by the increased number of AO-positive cells (<xref rid="gr5" ref-type="fig">Figs. 5</xref>G and G and <xref rid="gr6" ref-type="fig">6</xref>E). When M was co-expressed with PDK-1, the number of AO-positive cells was largely reduced (E). When M was co-expressed with PDK-1, the number of AO-positive cells was largely reduced (<xref rid="gr6" ref-type="fig">Fig. 6</xref>F and G).F and G).', 'In apoptotic cells, holo-cytochrome c protein released from mitochondria triggers the formation of apoptosome and activates the caspase cascades to promote apoptosis [58]. It was reported that the expression of apo-cytochrome c protein (without heme group) is able to compete with holo-cytochrome c (with heme group) for Apaf1, which in turn blocks caspase activation and inhibits apoptosis [40]. We showed that over-expression of apo-cytochrome c blocked M-induced apoptosis (<xref rid="gr5" ref-type="fig">Fig. 5</xref>E, J and K, E, J and K, [16]). Caspase activation has been shown to be involved in SARS-CoV-induced apoptosis [15]. Previous studies also revealed that SARS-CoV viral proteins are able to activate individual caspases. For example, N activates caspase-3 and -7 [12], 3a activates caspase-8 [17], and 7a activates caspase-3 [18]. To test the involvement of caspases in M-induced apoptosis, we treated M-expressing cells with caspase inhibitors and found that M-induced nuclear condensation could be prevented by inhibitors specific for caspase-3, -8 and -9 (<xref rid="gr3" ref-type="fig">Fig. 3</xref>G). G). Drosophila inhibitor of apoptosis 1 (DIAP1) and the baculoviral caspase inhibitor protein P35 are effective broad-range inhibitors of caspase activation in Drosophila\n[59], [60]. When we co-expressed M with either DIAP1 (<xref rid="gr5" ref-type="fig">Fig. 5</xref>H and K) or P35 (H and K) or P35 (<xref rid="gr5" ref-type="fig">Fig. 5</xref>I and K) I and K) in vivo, we found that M-induced apoptosis was suppressed. The incomplete inhibition of apoptosis by specific caspases-3, -8 and -9 inhibitors (<xref rid="gr3" ref-type="fig">Fig. 3</xref>G) underscores the involvement of other caspases in M-induced apoptosis.G) underscores the involvement of other caspases in M-induced apoptosis.'], 'gr7': ['Both Akt and PDK-1 kinases play a central role in the cell survival pathway [43]. Since the Akt pathway can modulate apoptosis [44], we therefore investigated whether over-expression of Drosophila Akt1 could suppress M-induced apoptosis. Unlike PDK-1, the M-induced rough-eye phenotype was not suppressed by Akt1 over-expression (data not shown). Since the phosphorylation state of Akt is an essential cell survival indicator, we then argued if M-over-expression would affect Akt1 phosphorylation. Using phospho-specific Akt1 (Ser505) antibodies, we found that over-expression of M down-regulated the phosphorylation states of Akt1 (<xref rid="gr7" ref-type="fig">Fig. 7</xref>\nA and B) without affecting total Akt1 protein levels (\nA and B) without affecting total Akt1 protein levels (<xref rid="gr7" ref-type="fig">Fig. 7</xref>A and C).A and C).Fig. 7Down-regulation of Akt protein phosphorylation in transgenic Drosophila over-expressing M protein. (A) Western blot analysis showed that M expression reduced Akt protein phosphorylation without altering total Akt protein level. β-tubulin was used as loading control. Phospho- (B) and total-Akt protein (C) levels were quantified and normalized against the β-tubulin loading control. Results were plotted as the percentage change of band intensity and expressed as means\xa0+\xa0SEM of five independent experiments. ∗p\xa0<\xa00.05.', 'The Akt pathway is a cellular pro-survival signaling cascade [61], and is known to regulate mitochondria-mediated apoptosis [62], [63], [64], [65]. Caspase-9 is one of the phosphorylation targets of Akt, and the protease activity of caspase-9 is reduced upon Akt phosphorylation [66]. In addition, it is known that caspase-8 activity is also regulated indirectly through Akt [67]. The Akt pathway has been implicated in viral infections [24] including SARS-CoV [5], [9], [11]. A weak activation of the Akt cellular pro-survival pathway has been observed in viral-infected cells as evidenced by transient phosphorylation of Akt, GSK-3β and PKCζ kinases [5], [9], [11]. Such transient activation would reflect the inability of viral-infected cells to continuously combat with viral infection. In this report, we showed that over-expression of the M protein caused down-regulation of Akt phosphorylation without affecting total Akt protein level (<xref rid="gr7" ref-type="fig">Fig. 7</xref>). Such action would therefore contribute to the reduction of the cell survival signal, which in turn lead to the apoptotic induction.). Such action would therefore contribute to the reduction of the cell survival signal, which in turn lead to the apoptotic induction.'], 'gr6': ['From our forward genetic modifier screen, we identified PDK-1 kinase, an upstream effector in the Akt signaling pathway, as a dominant suppressor of M-induced rough-eye phenotype (<xref rid="gr6" ref-type="fig">Fig. 6</xref>C) and apoptosis (C) and apoptosis (<xref rid="gr6" ref-type="fig">Fig. 6</xref>F). In addition, we found that heterozygous loss-of-function mutation of F). In addition, we found that heterozygous loss-of-function mutation of PDK-1 (PDK-1\n1, PDK-1\n2\n[35]) showed no modification on the M-induced rough-eye phenotype (data not shown). This indicates that PDK-1 showed no haplo-insufficient effect on M function. The human homolog of PDK-1 gene is expressed in the lung which further implicates its role in SARS-CoV infection (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.459691) [68].']}
|
The SARS-Coronavirus Membrane protein induces apoptosis through modulating the Akt survival pathway
|
[
"{'italic': 'c', '#text': 'Cytochrome'}",
"{'italic': 'Drosophila'}",
"HEK293T",
"PDK-1",
"Severe Acute Respiratory Syndrome"
] |
Arch Biochem Biophys
|
1173942000
|
A number of viral gene products are capable of triggering apoptotic cell death through interfering with cellular signaling cascades, including the Akt kinase pathway. In this study, the pro-apoptotic role of the SARS-CoV Membrane (M) structural protein is described. We found that the SARS-CoV M protein induced apoptosis in both HEK293T cells and transgenic Drosophila. We further showed that M protein-induced apoptosis involved mitochondrial release of cytochrome c protein, and could be suppressed by caspase inhibitors. Over-expression of M caused a dominant rough-eye phenotype in adult Drosophila. By performing a forward genetic modifier screen, we identified phosphoinositide-dependent kinase-1 (PDK-1) as a dominant suppressor of M-induced apoptotic cell death. Both PDK-1 and Akt kinases play essential roles in the cell survival signaling pathway. Altogether, our data show that SARS-CoV M protein induces apoptosis through the modulation of the cellular Akt pro-survival pathway and mitochondrial cytochrome c release.
|
[
"Animals",
"Animals, Genetically Modified",
"Apoptosis",
"Cell Line",
"Cell Survival",
"Coronavirus M Proteins",
"Drosophila",
"Humans",
"Kidney",
"Oncogene Protein v-akt",
"Phosphorylation",
"Severe acute respiratory syndrome-related coronavirus",
"Signal Transduction",
"Viral Matrix Proteins"
] |
other
|
PMC7094499
| null |
69
|
[
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] |
Arch Biochem Biophys. 2007 Mar 15; 459(2):197-207
|
NO-CC CODE
|
|
Mutant PFN1 produces ubiquitinated insoluble aggregatesa, Western blot analysis of transfected N2A cells subject to NP-40-soluble (S) and insoluble (I) fractionation. b, Transfected cells were treated with MG132 and processed as in (a). Hash marks indicate 25 kDa and 37 kDa markers. Transfected N2A cells (c) and PMNs (d) were stained with V5, HA (PMNs) and ubiquitin (N2A) antibodies. Example aggregates are enlarged in the inset in (d). e, Transfected N2A cells displaying insoluble aggregates were counted and analyzed using one-way ANOVA testing with Dunnett’s multiple test comparison (n=127-135 transfected cells from 3 independent experiments). *P<0.05, ***P<0.001, n.s. P>0.05. Error bars indicate SEM. f, Transfected PMNs stained with V5 and TDP-43 antibodies. Scale bars: 5 μm (c), 10 μm (d, f)
|
nihms-382266-f0002
|
2
|
cfd6a722659be21fc9fecf31a473ee7e88537435ca4c69c286284356c2b5827d
|
nihms-382266-f0002.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
415,
599
] |
[{'image_id': 'nihms-382266-f0001', 'image_file_name': 'nihms-382266-f0001.jpg', 'image_path': '../data/media_files/PMC3575525/nihms-382266-f0001.jpg', 'caption': 'Exome sequencing identifies PFN1 gene mutations in familial ALSa-c, Familial ALS pedigrees harboring PFN1 mutations are shown. Asterisks indicate samples subjected to exome sequencing. To prevent identification of individual family members, the gender of each subject and information on the lower generation are withheld. Genotypes of available DNA samples for the indicated PFN1 mutation are shown (w=wild-type, m=mutant). The genotype of sample III:2 in Family #2 (+) was inferred from the genotypes of spouse and progeny (not shown). d, The evolutionary conservation of PFN1 mutations is shown. For each, the mutated amino acid is colored in red.', 'hash': 'af5c834755306175817589b50f6738bdcb86a6c3ba22c7866691c91816c16089'}, {'image_id': 'nihms-382266-f0002', 'image_file_name': 'nihms-382266-f0002.jpg', 'image_path': '../data/media_files/PMC3575525/nihms-382266-f0002.jpg', 'caption': 'Mutant PFN1 produces ubiquitinated insoluble aggregatesa, Western blot analysis of transfected N2A cells subject to NP-40-soluble (S) and insoluble (I) fractionation. b, Transfected cells were treated with MG132 and processed as in (a). Hash marks indicate 25 kDa and 37 kDa markers. Transfected N2A cells (c) and PMNs (d) were stained with V5, HA (PMNs) and ubiquitin (N2A) antibodies. Example aggregates are enlarged in the inset in (d). e, Transfected N2A cells displaying insoluble aggregates were counted and analyzed using one-way ANOVA testing with Dunnett’s multiple test comparison (n=127-135 transfected cells from 3 independent experiments). *P<0.05, ***P<0.001, n.s. P>0.05. Error bars indicate SEM. f, Transfected PMNs stained with V5 and TDP-43 antibodies. Scale bars: 5 μm (c), 10 μm (d, f)', 'hash': 'cfd6a722659be21fc9fecf31a473ee7e88537435ca4c69c286284356c2b5827d'}, {'image_id': 'nihms-382266-f0003', 'image_file_name': 'nihms-382266-f0003.jpg', 'image_path': '../data/media_files/PMC3575525/nihms-382266-f0003.jpg', 'caption': 'Mutant PFN1 inhibits axon outgrowtha, PFN1-actin interaction region (PDB accession: 2BTF) using the PyMOL Molecular Graphics System (v. 1.4). Magenta: actin; Yellow: PFN1; Green: actin-binding PFN1 residues; Red: ALS-linked mutated PFN1 residues. b, Transfected HEK293 cells were immunoprecipitated with a V5 antibody and then immunoblotted with antibodies for either V5 or actin. c, PMNs transfected with wild-type or mutant V5-PFN1 and a GFP expressing construct were stained to detect V5-PFN1. d, Cumulative distribution of axon lengths relative to the mean of wild-type PFN1 transfected cells was plotted. The axon tip, indicated by arrows, is enlarged in the inset in (d), right panel. P values are given in the legend (n=104-161 cells from 4 independent experiments). Scale bar: 100 μm.', 'hash': 'c1b16bb919e3c4ce36fd02d79d28a8147630f9872c4875dd00a06c87d9c1a70b'}, {'image_id': 'nihms-382266-f0004', 'image_file_name': 'nihms-382266-f0004.jpg', 'image_path': '../data/media_files/PMC3575525/nihms-382266-f0004.jpg', 'caption': 'Mutant PFN1 reduces growth cone size and F-/G-actin expressiona, PMNs were transfected with either wild-type or mutant V5-PFN1. At 3 days post-transfection, cells were fixed and stained to detect V5-PFN1, F-actin (Phalloidin, red) and G-actin (DNase I, green). The growth cone region of representative cells is shown. Scale bar: 10 μm. The growth cone area (b) and F-/G-actin expression (c) of transfected cells was determined and plotted. Comparisons to the wild-type V5-PFN1 transfected cells were made using one-way ANOVA testing. *P<0.05, **P<0.01, ***P<0.001 (n=27-35 cells from 3 independent experiments). Error bars indicate SEM.', 'hash': '77b1fb8245926b35fd8fe63876e01442e3ac83ca7259569bbc5884f34a6a5eea'}]
|
{'nihms-382266-f0001': ['To identify causative genes for familial ALS, we performed exome capture followed by deep sequencing on two large ALS families (<xref ref-type="fig" rid="nihms-382266-f0001">Fig. 1a-b</xref>) of Caucasian (Family #1) and Sephardic Jewish (Family #2) origin. Both display a dominant inheritance mode and are negative for known ALS-causing mutations, including the newly identified hexanucleotide repeat expansion in ) of Caucasian (Family #1) and Sephardic Jewish (Family #2) origin. Both display a dominant inheritance mode and are negative for known ALS-causing mutations, including the newly identified hexanucleotide repeat expansion in c9orf726,8,9 (Supplementary Fig.1). For each family, two affected members with maximum genetic distance were selected for exome sequencing. A high level of coverage (>150X) was achieved with an average of 1.1 × 1010 and 2.3 × 1010 base pairs sequenced for members of Families #1 and #2, respectively (Supplementary Table 1 and 2). To identify candidate causative mutations, variants were identified and filtered, as in previous exome sequencing reports5, using several criteria: the variant is observed in both family members, alters amino acid sequence, is not excluded by linkage analysis (Supplementary Fig. 2), and is absent from dbSNP132, the 1000 Genomes Project (May 2011 release), or the NHLBI ESP Exome Variant Server (5,379 sequenced exomes). Remaining variants were confirmed by Sanger sequencing and tested for Mendelian segregation in all affected family members. The resulting number of candidate causative mutations identified was two within Family #1 and three within Family #2 (Supplementary Table 3 and 4). Interestingly, the two families harbor different mutations (C71G and M114T) within a single common gene: profilin 1 (PFN1), located on chromosome 17p13.2. PFN1 is a 140 amino acid protein and major growth regulator of filamentous (F)-actin through its binding of monomeric (G)-actin10. Based on these data, we tested the hypothesis that PFN1 gene mutations cause familial ALS.', '<xref ref-type="fig" rid="nihms-382266-f0001">Figure 1</xref> shows sequence analysis of all available members of Family #1 and #2. All four affected members of Family #1 for which DNA was available possess the PFN1 C71G variant. A single obligate carrier of the C71G variant (III:13) did not develop disease; however, death occurred before this family’s average age of onset ( shows sequence analysis of all available members of Family #1 and #2. All four affected members of Family #1 for which DNA was available possess the PFN1 C71G variant. A single obligate carrier of the C71G variant (III:13) did not develop disease; however, death occurred before this family’s average age of onset (Supplementary Table 5). All unaffected Family #1 members displayed the wild-type genotype. Within Family #2, all eight affected members for which DNA was available harbored the M114T variant. Based on genotypes of spouse and progeny (not shown), we confirmed that a ninth affected family member (III:2) also carries the mutation. Of 7 unaffected members, 5 do not harbor the M114T variant. One unaffected mutation carrier is currently in their mid-40s (III:15) and a second obligate carrier (II:4) was asymptomatic into their 70s. Our results suggest that these mutations have a high degree of penetrance. Affected-only linkage analysis of the PFN1 variants in Family #1 and #2 yielded LOD scores of 1.80 and 2.71, respectively.', 'To determine whether PFN1 mutations cause familial ALS, the entire 3 exon coding region was sequenced in a panel of 272 additional FALS cases prescreened for common causative mutations. Five additional familial ALS cases harboring alterations in the PFN1 gene (Supplementary Fig. 3-4) were identified. Interestingly, the C71G alteration originally identified in Family #1 was discovered in two additional families. For one of these families (Family #3), DNA was available for three additional affected family members. Sequencing of these samples revealed that the mutation co-segregates with ALS (<xref ref-type="fig" rid="nihms-382266-f0001">Fig. 1c</xref>). A single unaffected member of the family (IV:2) also harbors the mutation; the current age of this family member is mid 40s. Affected-only linkage analysis of Family #3 yielded a LOD score of 1.50 and a combined LOD score of 6.01 for Families #1 - #3. A second M114T mutation was identified in an ALS family of Italian origin (Family #4, ). A single unaffected member of the family (IV:2) also harbors the mutation; the current age of this family member is mid 40s. Affected-only linkage analysis of Family #3 yielded a LOD score of 1.50 and a combined LOD score of 6.01 for Families #1 - #3. A second M114T mutation was identified in an ALS family of Italian origin (Family #4, Supplementary Fig. 3). DNA available for one sibling was shown by sequencing to harbor this mutation. PFN1 variants were observed in two additional FALS cases: a consecutive base pair change (AA to GT) resulting in an E117G mutation, and a G to T transversion resulting in a G118V mutation (Supplementary Fig. 3 and 4). DNA was not available from additional family members for these cases. Sequencing of the PFN1 coding region in 816 sporadic ALS (SALS) samples identified two samples harboring the E117G mutation. No additional non-synonymous changes were identified in FALS and SALS samples (Supplementary Table 6). Haplotype analysis using surrounding SNPs suggests the C71G mutation derives from a single ancestral mutation (Supplementary Table 7).', 'In total, we identified 4 mutations in 7/274 FALS cases. In each case, the altered amino acid was evolutionarily conserved down to the level of zebrafish (<xref ref-type="fig" rid="nihms-382266-f0001">Fig. 1d</xref>), supporting the possibility that these mutations are pathogenic. The age of onset for FALS cases with ), supporting the possibility that these mutations are pathogenic. The age of onset for FALS cases with PFN1 mutation is 44.8 ± 7.4 (Supplementary Table 5). All PFN1 mutant cases displayed limb onset; no bulbar onset was observed (n=22, Supplementary Table 5). Given that bulbar onset represents ~25% of ALS cases11, this result suggests a common clinical phenotype among patients with PFN1 mutations.'], 'nihms-382266-f0002': ['Ubiquitinated, insoluble aggregates are pathological hallmarks of several neurodegenerative diseases including ALS, Parkinson’s disease, and Alzheimer’s disease. To investigate whether the observed PFN1 mutants form insoluble aggregates, western blot analysis of NP-40-soluble and insoluble fractions was performed on Neuro2A (N2A) cells transfected with wild-type or one of the 4 PFN1 mutants. PFN1 protein was present predominantly in the soluble fraction of cells transfected with the wild-type construct as compared to the insoluble fraction (<xref ref-type="fig" rid="nihms-382266-f0002">Fig. 2a</xref>). Conversely, a considerable proportion of the C71G, M114T and G118V mutant proteins were detected in the insoluble fraction. Furthermore, several higher molecular weight species were observed, indicative of SDS-resistant PFN1 oligomers. However, the E117G mutant displayed a pattern more similar to wild-type PFN1 with most of the expressed protein in the soluble fraction. Differential expression of ). Conversely, a considerable proportion of the C71G, M114T and G118V mutant proteins were detected in the insoluble fraction. Furthermore, several higher molecular weight species were observed, indicative of SDS-resistant PFN1 oligomers. However, the E117G mutant displayed a pattern more similar to wild-type PFN1 with most of the expressed protein in the soluble fraction. Differential expression of PFN1 constructs was ruled out by western blot analysis of whole cell lysates (Supplementary Figure 5). Analysis of lymphoblast cell lines derived from affected and unaffected members of Family #1 did not display any differences in PFN1 protein solubility (Supplementary Figure 6). Autopsy material was not available for any affected individual.', 'We extended these observations by staining the PFN1 protein in transfected cells. Wild-type PFN1 exhibited a diffuse cytoplasmic expression pattern in transfected N2A cells (<xref ref-type="fig" rid="nihms-382266-f0002">Fig. 2c</xref>), as previously reported), as previously reported12. In contrast, ALS-linked PFN1 mutants often assembled into cytoplasmic aggregates. Image analysis determined that 15-61% of mutant expressing cells contain cytoplasmic aggregates, including the E117G mutant, which showed minimal insoluble PFN1 protein by western blot analysis. No aggregates were observed for cells expressing wild-type PFN1 (<xref ref-type="fig" rid="nihms-382266-f0002">Fig. 2e</xref>). Co-staining revealed that these aggregates were also ubiquitinated. Primary motor neurons (PMNs) expressing the C71G, M114T, and G118V PFN1 mutants similarly demonstrated ubiquitinated aggregates, albeit at a lower percentage (). Co-staining revealed that these aggregates were also ubiquitinated. Primary motor neurons (PMNs) expressing the C71G, M114T, and G118V PFN1 mutants similarly demonstrated ubiquitinated aggregates, albeit at a lower percentage (<xref ref-type="fig" rid="nihms-382266-f0002">Fig. 2d</xref>); aggregates were not observed in cells expressing wild-type and E117G PFN1. Immunoprecipitation of the PFN1 protein followed by western blot analysis confirmed that the insoluble mutant PFN1 protein is polyubiquitinated (); aggregates were not observed in cells expressing wild-type and E117G PFN1. Immunoprecipitation of the PFN1 protein followed by western blot analysis confirmed that the insoluble mutant PFN1 protein is polyubiquitinated (Supplementary Figure 7).', 'To determine if ubiquitin-proteosome system impairment causes accumulation of mutant PFN1 aggregates, transfected N2A cells were exposed to the proteasome inhibitor MG132. ALS-linked PFN1 mutants, including E117G, displayed increased insoluble protein levels and increased levels of higher molecular weight species by western blot analysis. Minimal insoluble protein was observed for the wild-type PFN1 protein (<xref ref-type="fig" rid="nihms-382266-f0002">Fig. 2b</xref>). PFN1 staining in N2A cells and PMNs confirmed these results. Cells expressing C71G, M114T and G118V mutant PFN1 displayed numerous, large aggregates following MG132 treatment. E117G mutants displayed a moderate aggregate level, and the wild-type protein displayed minimal levels (). PFN1 staining in N2A cells and PMNs confirmed these results. Cells expressing C71G, M114T and G118V mutant PFN1 displayed numerous, large aggregates following MG132 treatment. E117G mutants displayed a moderate aggregate level, and the wild-type protein displayed minimal levels (Supplementary Fig. 8-9).', 'Given mutant PFN1’s propensity to form aggregates, we investigated whether other ALS-related proteins may be present within these aggregates. Thus, we transfected cells with mutant PFN1 and tested for alterations in the cellular localization of the ALS-related proteins FUS and TDP-43. Additionally, we also tested for alterations in the spinal muscular atrophy related protein, SMN,13 due to its ability to bind PFN1. No co-aggregation of either FUS or SMN with mutant PFN1 was observed (Supplementary Fig. 10-11). However, ~30-40% of cells contained cytoplasmic PFN1 aggregates co-stained with TDP-43 (<xref ref-type="fig" rid="nihms-382266-f0002">Fig. 2f</xref>). These results suggest that mutant PFN1 may contribute to ALS pathogenesis by inducing aggregation of TDP-43. Based on these observations, we investigated whether aggregates of TDP-43 contain PFN1 by staining spinal cord tissues from 18 SALS cases displaying TDP-43 pathology and 6 non-ALS controls without TDP-43 pathology (). These results suggest that mutant PFN1 may contribute to ALS pathogenesis by inducing aggregation of TDP-43. Based on these observations, we investigated whether aggregates of TDP-43 contain PFN1 by staining spinal cord tissues from 18 SALS cases displaying TDP-43 pathology and 6 non-ALS controls without TDP-43 pathology (Supplementary Fig. 12). Abnormal PFN1 pathology was not discovered in SALS cases, suggesting that TDP-43 aggregation does not induce PFN1 aggregation. Expression of the C-terminal fragment of TDP-43, which produces insoluble aggregates, in primary motor neurons also failed to co-aggregate wild-type PFN1 supporting this observation (Supplementary Fig. 13).'], 'nihms-382266-f0003': ['Evaluation of the PFN1-actin complex crystal structure revealed that all ALS-linked mutations lie in close proximity to actin binding residues of PFN1 (<xref ref-type="fig" rid="nihms-382266-f0003">Fig. 3a</xref>))14. Therefore, we investigated whether the ALS-linked mutations display a decreased level of bound actin. Towards this end, we performed IP/western blot analysis of cells transfected with wild-type and mutant PFN1. As a control, we also transfected cells with a construct expressing a synthetic H120E PFN1 mutant protein. This alteration is located at a critical residue previously shown to abolish PFN1 binding to actin12. We observed that C71G, M114T, G118V and H120E mutants displayed reduced levels of bound actin relative to wild-type PFN1 (<xref ref-type="fig" rid="nihms-382266-f0003">Fig. 3b</xref>). The E117G mutant did not display a reduction of bound actin relative to wild-type PFN1.). The E117G mutant did not display a reduction of bound actin relative to wild-type PFN1.', 'Previous reports have shown that PFN1 protein alterations inhibit neurite outgrowth12,15. We investigated whether ALS-linked PFN1 mutants inhibit neurite outgrowth by measuring axonal length in PMNs transfected with wild-type or mutant PFN1. As a positive control, we also transfected PMNs with the H120E expressing construct. In addition to lacking actin binding ability, the H120E protein inhibits neurite outgrowth12. As expected, the H120E expressing cells displayed a pronounced decrease in axon length relative to the wild-type construct (<xref ref-type="fig" rid="nihms-382266-f0003">Fig. 3c</xref>). Three ALS-linked PFN1 mutations (C71G, M114T, and G118V) also displayed a significant decrease in axon outgrowth (). Three ALS-linked PFN1 mutations (C71G, M114T, and G118V) also displayed a significant decrease in axon outgrowth (<xref ref-type="fig" rid="nihms-382266-f0003">Fig. 3c,d</xref>). In particular, the G118V-associated reduction is similar to that observed with the H120E construct. Axon outgrowth inhibition was observed with the E117G mutant but did not reach statistical significance. These results suggest that mutations in PFN1 may contribute to ALS pathogenicity in part by inhibiting axon dynamics.). In particular, the G118V-associated reduction is similar to that observed with the H120E construct. Axon outgrowth inhibition was observed with the E117G mutant but did not reach statistical significance. These results suggest that mutations in PFN1 may contribute to ALS pathogenicity in part by inhibiting axon dynamics.'], 'nihms-382266-f0004': ['The regulation of actin dynamics in the growth cone is necessary for axon outgrowth. Defects in PFN1 are associated with growth cone arrest and reduced axon outgrowth in embryonic motor neurons of Drosophila15. To determine whether ALS-linked PFN1 mutants have a similar phenotype, PMNs transfected with wild-type and two mutant PFN1 constructs (C71G and G118V) were stained to detect F- and G-actin localization patterns in the highly dynamic and actin-rich growth cone. These mutants were selected due to their greater influence on axon outgrowth. PFN1 mutant expression in PMNs led to a significantly reduced growth cone size (~43-52%) relative to wild-type PFN1 (<xref ref-type="fig" rid="nihms-382266-f0004">Fig. 4b</xref>). Also, mutant PFN1 expression significantly altered growth cone morphology. In wild-type PFN1 expressing cells, growth cones had elaborate structures with several F-actin rich filopodia (). Also, mutant PFN1 expression significantly altered growth cone morphology. In wild-type PFN1 expressing cells, growth cones had elaborate structures with several F-actin rich filopodia (<xref ref-type="fig" rid="nihms-382266-f0004">Fig. 4a</xref>), while virtually no filopodia were visible in the mutant PFN1 growth cones. Similar results were observed for the synthetic H120E mutant defective in actin binding. The growth cones in mutant expressing PMNs also displayed a lower ratio of F-/G-actin relative to wild-type expressing PMNs (), while virtually no filopodia were visible in the mutant PFN1 growth cones. Similar results were observed for the synthetic H120E mutant defective in actin binding. The growth cones in mutant expressing PMNs also displayed a lower ratio of F-/G-actin relative to wild-type expressing PMNs (<xref ref-type="fig" rid="nihms-382266-f0004">Fig. 4c</xref>). In particular, the C71G mutant expressing PMNs displayed an F-/G-actin ratio of 24.4% relative to wild-type expressing PMNs. These results suggest that mutant PFN1 can inhibit the conversion of G-actin to F-actin within the growth cone region, thus affecting its morphology.). In particular, the C71G mutant expressing PMNs displayed an F-/G-actin ratio of 24.4% relative to wild-type expressing PMNs. These results suggest that mutant PFN1 can inhibit the conversion of G-actin to F-actin within the growth cone region, thus affecting its morphology.']}
|
Mutations in the Profilin 1 Gene Cause Familial Amyotrophic Lateral Sclerosis
| null |
Nature
|
1345705200
|
Evidence suggests that probiotic bacteria modulate both innate and adaptive immunity in the host, and in some situations can result in reduced severity of common illnesses, such as acute rotavirus infection and respiratory infections. Responses to vaccination are increasingly being used to provide high quality information on the immunomodulatory effects of dietary components in humans. The present review focuses on the effect of probiotic administration upon vaccination response. The majority of studies investigating the impact of probiotics on responses to vaccination have been conducted in healthy adults, and at best they show modest effects of probiotics on serum or salivary IgA titres. Studies in infants and in elderly subjects are very limited, and it is too early to draw any firm conclusions regarding the potential for probiotics to act as adjuvants in vaccination. Although some studies are comparable in terms of duration of the intervention, age and characteristics of the subjects, most differ in terms of the probiotic selected. Further well designed, randomized, placebo-controlled studies are needed to understand fully the immunomodulatory properties of probiotics, whether the effects exerted are strain-dependent and age-dependent and their clinical relevance in enhancing immune protection following vaccination.
|
[
"Adjuvants, Immunologic",
"Adult",
"Age Factors",
"Aged",
"Animals",
"Humans",
"Infant",
"Probiotics",
"Vaccination",
"Vaccines"
] |
other
|
PMC3575525
| null |
37
|
[
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"{'Citation': 'Bunout D, Barrera G, Hirsch S, Gattas V, de la Maza MP, Haschke F, Steenhout P, Klassen P, Hager C, Avendaño M, Petermann M, Muñoz C. Effects of a nutritional supplement on the immune response and cytokine production in free‐living Chilean elderly. JPEN J Parenter Enteral Nutr. 2004;28:348–354.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15449576'}}}",
"{'Citation': 'Boge T, Rémigy M, Vaudaine S, Tanguy J, van der Bourdet‐Sicard R, Werf S. A probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials. Vaccine. 2009;27:5677–5684.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19615959'}}}",
"{'Citation': 'Namba K, Hatano M, Yaeshima T, Takase M, Kunihiko S. Effects of Bifidobacterium longum BB536 administration on influenza infection, influenza vaccine antibody titre, and cell‐mediated immunity in the elderly. Biosci Biotechnol Biochem. 2010;74:939–945.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '20460726'}}}"
] |
Nature. 2012 Aug 23; 488(7412):499-503
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NO-CC CODE
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CD68 immunoreactivity of the cerebellar microglia in PLD4-deficient mice. (A–F) Double-labeling of cells by anti-Iba1 (red) and anti-CD68 (green) antibodies in the deep white matter (A–C) and folial white matter (D–F) of P5 cerebellum obtained from wild type (A, D) or PLD4-deficient (B, E) mice. (A'–E', A''–E'') Higher magnification images of representative Iba1+CD68+ cells from the white squares in A–E. Scale bars in B, E and B'', E'' are 100 µm and 10 µm, respectively. All Iba1-positive cells were also positive for CD68, however, the staining intensity of CD68 (see Materials and methods) in PLD4-deficient cells was significantly lower than in wild type cells (C, F). Thus, the phenotype of activated microglia in PLD4-deficient mice is different from that of wild type. Data were obtained from: 78 WT cells and 65 Ho cells in the deep white matter (C), and 25 WT cells and 32 Ho cells in the folia (F) (n = number of mice examined). Graphs indicate mean ± SEM. Statistical analysis was performed using a student t test. **P < 0.01.
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pjab-92-237-g003
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2
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622419638f6309497a59f9ea793a1107e71d8fac7db45947af6fc6e1c4f5faf7
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pjab-92-237-g003.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
767,
920
] |
[{'image_id': 'pjab-92-237-g005', 'image_file_name': 'pjab-92-237-g005.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g005.jpg', 'caption': "Relationship between myelination and microglial activation during mouse cerebellar development. (A–E) Progress of myelination in normal C57BL mouse cerebellum. Frozen sections obtained from P0 (A), P3 (B), P5 (C), P7 (D) and P10 (E) were immunostained with an anti-MBP antibody. Scale bar in E represents 200 µm. (F, F', F'') Double immunostaining of the white matter in cerebellar folia at P7 with anti-Iba1 (green) and anti-MBP (red) antibodies. Images are also shown with DIC overlays (F and F'). F' and F'' (single Iba1 image) indicate higher magnification of the white dotted squares in F. Note that myelination in the folial white matter is in progress and clusters of rounded activated microglia are mainly found at the myelination fronts (F') but not in already myelinated area (F''). Scale bars indicate 50 µm (F) and 20 µm (F', F'').", 'hash': 'c69f203575f1e86f0eb156224f35d250e5c88f9c23a99de729c0cad3ab7c2fdc'}, {'image_id': 'pjab-92-237-g002', 'image_file_name': 'pjab-92-237-g002.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g002.jpg', 'caption': 'Microglial activation in the cerebella of PLD4-deficient mice. (A, B) Comparison of gross brain structures in wild type (WT; A) and PLD4-deficient mice (Ho; B) at P7. Paraffin sections were stained with hematoxylin and eosin. High magnification images of cerebellar cortex (black squares) are shown in the right panels. No significant differences in gross brain anatomy were observed. Scale bars in B and in the higher magnification images at right represent 500 µm and 100 µm, respectively. (C–J) Iba1-positive microglia in P5 (C–F) and P7 (G–J) cerebella. Frozen sections of deep white matter (C, D, G, H) or folial white matter (E, F, I, J) of the cerebella obtained from wild type (C, E, G, I) and PLD4-deficient (D, F, H, J) mice were immunostained using an anti-Iba1 antibody. A representative Iba1-positive cell indicated by the white square in each panel is shown at higher magnification in the right lower corners. Note that microglia at P5 and P7 are the rounded activated type. Scale bars represent 100 µm (F, J) or 10 µm (in the white squares of F, J). (K, L) Intensity of Iba1 immunoreactivity in individual cells of the deep cerebellar white matter at P5 (K) and P7 (L). Graphs indicate the mean ± SEM. Data were obtained from: 92 WT cells and 98 Ho cells at P5, and 125 WT cells and 130 Ho cells in L (n = indicates number of mice examined). Statistical analysis was performed using a student t test. **P < 0.01.', 'hash': '964be7070fe9a477d330f3f682820b3ad652805b27bf0a8841bc4969ef9dfe6e'}, {'image_id': 'pjab-92-237-g003', 'image_file_name': 'pjab-92-237-g003.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g003.jpg', 'caption': "CD68 immunoreactivity of the cerebellar microglia in PLD4-deficient mice. (A–F) Double-labeling of cells by anti-Iba1 (red) and anti-CD68 (green) antibodies in the deep white matter (A–C) and folial white matter (D–F) of P5 cerebellum obtained from wild type (A, D) or PLD4-deficient (B, E) mice. (A'–E', A''–E'') Higher magnification images of representative Iba1+CD68+ cells from the white squares in A–E. Scale bars in B, E and B'', E'' are 100 µm and 10 µm, respectively. All Iba1-positive cells were also positive for CD68, however, the staining intensity of CD68 (see Materials and methods) in PLD4-deficient cells was significantly lower than in wild type cells (C, F). Thus, the phenotype of activated microglia in PLD4-deficient mice is different from that of wild type. Data were obtained from: 78 WT cells and 65 Ho cells in the deep white matter (C), and 25 WT cells and 32 Ho cells in the folia (F) (n = number of mice examined). Graphs indicate mean ± SEM. Statistical analysis was performed using a student t test. **P < 0.01.", 'hash': '622419638f6309497a59f9ea793a1107e71d8fac7db45947af6fc6e1c4f5faf7'}, {'image_id': 'pjab-92-237-g004', 'image_file_name': 'pjab-92-237-g004.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g004.jpg', 'caption': "Purkinje cells and astrocytes in the cerebella of PLD4-deficient mice. (A–D) Representative calbindin-positive Purkinje cells in wild type (WT; A, C) and PLD4-deficient (Ho; B, D) cerebella at P5 (A, B) and P7 (C, D). A' to D' are higher magnification images of the white squares in A to D. No apparent differences were observed between wild type and PLD4-deficient mice at either age. Scale bars indicate 100 µm (B, D) or 20 µm (B', D'). (E–J) GFAP immunoreactivity in astrocytes from the deep white matter of wild type (E, H) and PLD4-deficient (F, I) cerebella at P5 (E, F, G) and P7 (H, I, J). Higher magnification images of the white squares are shown in the lower right corners. Intensity of GFAP immunoreactivity in the deep white matter was quantified and is represented as intensity per unit area (G, J). Graphs in G and J indicate the mean ± SEM. Statistical analyses with student t tests showed no significant difference between the two genotypes at either age.", 'hash': '80833d424048d256c2763f0726bf7b8348f6beff0881869d2b9038c29c51d94c'}, {'image_id': 'pjab-92-237-g009', 'image_file_name': 'pjab-92-237-g009.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g009.jpg', 'caption': "Characteristics of microglia, Purkinje cells, and astrocytes in the cerebella of PLD4-deficient mice at P10. Representative images of Iba1-positive microglia in wild type (WT; A, B) and PLD4-deficient (Ho; C, D) cerebella at P10 were exhibited. Higher magnification of a representative Iba1-positive microglia is shown in the lower right corner. At P10 process-bearing ramified microglia were found in both types of mice. Purkinje cells in WT (E) and Ho (F) cerebella at P10 were immunostained by anti-calbindin antibody (E, F). E' and F' are higher magnification images of the white squares in E and F. GFAP immunoreactive astrocytes in the deep white matter (G, I) and folial white matter (H, J) of WT and Ho mice were immunostained by anti-GFAP antibody. Higher-magnification images of white squares (G, H, I, J) were indicated in the lower right corners. No apparent differences of calbindin and GFAP positive staining were observed between two genotypes. Scale bars indicate 100 µm (D, F, J) or 10 µm (in the white squares of D) or 20 µm (F', in the white squares of J).", 'hash': '80f2c482cd7787e53d8b7bfe03d886b222f33d700031bfde35a0d0edbdbf67e8'}, {'image_id': 'pjab-92-237-g007', 'image_file_name': 'pjab-92-237-g007.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g007.jpg', 'caption': 'Delayed myelination in the corpus callosum of P7 PLD4-deificent mice. (A, B) Sagittal sections of paraffin-embedded brains obtained from wild type (WT; A) and PLD4-deficient (Ho; B) mice were immunostained with an anti-MBP antibody. Brain sections from 4 mice of each type were examined, and representative images of the MBP-stained corpus callosum (forefront at right side) are shown. (C) Sagittal sections from three different regions (1 to 3) of each sample were identified based on the histological shape of the cerebellum. Sections from position No. 1 were characterized by having a cerebellum with 5 folia, position No. 2 had 6 folia, and position No. 3 had 7 folia. (D) MBP signals were restricted to the cell bodies of premyelinating oligodendrocytes, and these signals were diminished in myelinating oligodendrocytes. The numbers of premyelinating oligodendrocytes with MBP-positive cell bodies were counted in the corpus callosum at the three positions. Graphs indicate mean ± SEM. Two-way ANOVA with Bonferroni multiple comparison tests were used for statistical analyses. *P < 0.05, **P < 0.01.', 'hash': '677150e4074a8cceb84e38cba6387adcde6e29eb1766ccd691210280ef647b9f'}, {'image_id': 'pjab-92-237-g001', 'image_file_name': 'pjab-92-237-g001.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g001.jpg', 'caption': 'Generation of PLD4-deficient mice. (A) Genome structure of the PLD4 gene generated by homologous recombination of the STOP-tetO cassette in PLD4 knock-in (PLD4-deficient) mice. (B) Expression of PLD4 mRNA by quantitative real time PCR (qRT-PCR) in brain, spleen, liver and thymus of adult wild type (WT) and PLD4-deficient (Ho) mice. Graphs show the relative ratio of the level of PLD4 and β-actin mRNA from samples run in triplicate from 3 independent experiments. In wild type, PLD4 mRNA was expressed in brain and various reticuloendothelial tissues, including the spleen, liver and thymus. In PLD4-deficient mice, PLD4 mRNA was completely eliminated in these tissues. (C) PLD4 protein levels in adult wild type (WT), homozygote (Ho), and heterozygote (He) mice. Spleen homogenates (10 µg) with or without deglycosylation were separated by 10.5% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis was performed using an anti-PLD4 antibody. Since PLD4 has multiple glycosylation sites, various PLD4-related bands (70–75 kDa; indicated by *) were shown in non-treated samples. The arrow indicates deglycosylated PLD4 (∼45 kDa) by PNGase F (peptide-N-glycosidase F) treatment. The arrowhead indicates an unrelated protein product detected by the anti-PLD4 antibody. Note that this protein was also detected in PLD4-deficient spleen samples while PLD4 bands with or without sugars were completely eliminated. (D–F) Expression patterns of PLD1 (D), PLD2 (E), and PLD3 (F) mRNA in adult mice were examined by qRT-PCR. The quantitative analyses shown in B to F were obtained from three independent experiments. All experiments were performed in triplicate. Graphs indicate the mean ± standard error of the mean (SEM). Two-way ANOVA with Bonferroni multiple comparison tests were used for statistical analyses. ****P < 0.0001, **P < 0.01.', 'hash': 'a0629f80df6cc88da890f5de04ac09a35eb61ab1f43187c4b7bfd22f6c507037'}, {'image_id': 'pjab-92-237-g006', 'image_file_name': 'pjab-92-237-g006.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g006.jpg', 'caption': "Influence of PLD4 deficiency on cerebellar myelination. (A, B) Western blot for MBP in the cerebellum of P7 wild type (WT) and PLD4-deficient (Ho) mice. Cerebellar homogenates (10 µg) from individual mice (three mice from each group) were separated by 12% SDS-PAGE and Western blot was performed using an anti-MBP antibody. After stripping off the antibody, the same blot was restained with an anti-β-actin antibody for quantification. B. The total intensity of all MBP bands (14, 17, 18.5, and 21 kDa) was quantified for each animal and the ratio of MBP to β-actin for each genotype was calculated. Graph indicates mean ± SEM. Statistical analysis with a student t test shows no significant difference between the two groups. (C–F) Comparison of MBP immunoreactivity in wild type (C, E) and PLD4-deficient (D, F) cerebella at P5 (C, D) and P7 (E, F). Cell nuclei are stained with DAPI (blue). Representative images of 5 mice from each genotype were selected. As shown in Fig. 5C and D, myelination in the deep white matter is in progress at P5 and progresses to the folial white matter by P7. Images in C, D or E, F show the deep white matter tracts, or folial white matter, respectively. C'–F' are higher magnification views of the white squares in C–F without DAPI. At P5, MBP-positive myelin membranes were found in the deep white matter in wild type mice, while premyelinating oligodendrocytes with MBP-positive cell bodies (white arrowheads) were present in PLD4-deficient mice. At P7, myelin membranes were present in wild type folia, whereas premyelinating oligodendrocytes (white arrowheads) were still found in PLD4-deficeint mice in the same region. * represents the border between the cerebellum (upper) and the earlier myelinating brainstem (lower). Scale bars indicate 100 µm (D, F) and 20 µm (D', F'). (G, H) Double immunostaining of P7 wild type (G) and PLD4-deficient (H) cerebellar deep white matter around the cerebellar nuclei using anti-MBP (green) and anti-Iba1 (red) antibodies. The DAPI staining pattern is overlaid to indicate the cerebellar region. G' and H' are the same images as in G and H without DAPI. In mice of both genotypes, clusters of rounded Iba1-positive activated microglia are present in the area where myelination is progressing. Scale bar in H' represents 100 µm.", 'hash': '9b47730b53e767cd32202294704c89010d907f0f2d6e6a00c0e0419f9d47dc6a'}, {'image_id': 'pjab-92-237-g008', 'image_file_name': 'pjab-92-237-g008.jpg', 'image_path': '../data/media_files/PMC5114292/pjab-92-237-g008.jpg', 'caption': "Generation of PLD4-targeted knock-in mouse. A. Schematic diagram of PLD4-targeting. PLD4-targeting vector (PLD4 TV) included diphtheria toxin A subunit (DTA) was produced using Flexible Accelerated STOP Tetracycline Operator (tetO)-knock in (FAST) system (see Materials and methods). After homologous recombination in ES cells, the STOP-tetO cassette (3.5 kbp) was inserted into just upstream of ATG in PLD4 locus by PLD4 TV. The restriction sites, the restriction products (two-headed arrows), and the positions of probes (boxes) for Southern blot analysis were indicated. V, EcoRV site; K, KpnI site. Each color corresponds to the color in B. B. Genotyping of obtained two PLD4 knock in mouse lines by Southern blot analysis. In first screening of targeted ES cells by PLD4 TV, eight positive clones were selected from G418-resistant 116 colonies. After second screening, two clones were selected to produce chimera mice. Genomic DNAs from obtained two knock-in mice (KI/+_F1a and KI/+_F1b) were analyzed by Southern blotting. The restriction fragments indicating insertion mutation [14 kbp (EcoRV–5'-probe, blue arrowhead) and 5.3 kbp (KpnI–3'-probe, red arrowhead) fragments] (see panel A) were detected in both lines. EcoRV-digested DNA fragments derived from two KI/+ ES cell colonies were used as control (a and b).", 'hash': '65123f97845be366c09bdc3f9708bf3d7cad4fc80cb0b76ffcba99a3bc831f8d'}]
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{'pjab-92-237-g001': ['For preparation of the PLD4-targeting vector, a bacterial artificial chromosome (BAC) vector containing the mouse PLD4 gene was purchased from the Children’s Hospital Oakland Research Institute (Oakland, CA). A PLD4 target vector including the diphtheria toxin A subunit (DTA) was constructed using the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system.20) Briefly, a loxP-FRT-Neo-STOP-FRT-tetO-loxP cassette (3.5 kb, Fig. <xref ref-type="fig" rid="pjab-92-237-g001">1</xref>A) was inserted immediately upstream of the translation initiation site of the A) was inserted immediately upstream of the translation initiation site of the PLD4 gene in a BAC vector by conventional homologous recombination using two plasmids containing STOP-tetO cassette (pNeoSTOPtetO) and pBADTc TypeG. The PLD4 gene including regions 6 kb upstream and 4 kb downstream of the translation initiation site was then extracted using a retrieve vector, pMCS-DTA plasmid and constructed PLD4-targeting vector.', 'The PLD4-targeting vector containing a STOP-tetO cassette (Fig. <xref ref-type="fig" rid="pjab-92-237-g001">1</xref>A) was used for generation of the PLD4 knock-in mice.A) was used for generation of the PLD4 knock-in mice.20) For homologous recombination, the PLD4-targeting vector included sequences 6 kb upstream and 4 kb downstream of the PLD4 gene from the start codon. In front of the start codon located in exon 1, the inserted STOP-tetO cassette contained the following sequences in order: loxP, FRT, a neomycin resistant gene, STOP, FRT, tetO, and loxP. This targeting vector was introduced via electroporation into the ES cell line derived from C57BL/6 (RENKA) and the transduced cells were selected by G418.21,22) PLD4-knock-in ES cells were identified by Southern blot analysis (Supplemental Fig. <xref ref-type="fig" rid="pjab-92-237-g008">1</xref>B). The selected ES cell line was injected into eight-cell stage embryos of ICR mouse strain, and chimeric mice of 100% ES cells were crossed with ICR mice to confirm germline transmission. Mice with germline transmission were backcrossed with C57BL/6N mice. Genotypes of the generated PLD4 knock-in homozygous (PLD4-deficient) mice were determined by Southern blot analysis. The PLD4-deficient mice were born in predicted Mendelian ratios. As shown in Fig. B). The selected ES cell line was injected into eight-cell stage embryos of ICR mouse strain, and chimeric mice of 100% ES cells were crossed with ICR mice to confirm germline transmission. Mice with germline transmission were backcrossed with C57BL/6N mice. Genotypes of the generated PLD4 knock-in homozygous (PLD4-deficient) mice were determined by Southern blot analysis. The PLD4-deficient mice were born in predicted Mendelian ratios. As shown in Fig. <xref ref-type="fig" rid="pjab-92-237-g001">1</xref>B and B and <xref ref-type="fig" rid="pjab-92-237-g001">1</xref>C, qRT-PCR using specific primer sets and Western blot analyses demonstrated that PLD4 expression was completely suppressed at the mRNA as well as protein level in the adult homozygous animals. Expression patterns of PLD1, PLD2, and PLD3 were examined by qRT-PCR (Fig. C, qRT-PCR using specific primer sets and Western blot analyses demonstrated that PLD4 expression was completely suppressed at the mRNA as well as protein level in the adult homozygous animals. Expression patterns of PLD1, PLD2, and PLD3 were examined by qRT-PCR (Fig. <xref ref-type="fig" rid="pjab-92-237-g001">1</xref>D–F). The mRNA expression level of these PLD members were unchanged in the adult brains of PLD4-deficient mice, although a significant increase in the liver and decrease in the thymus was observed for PLD1 and PLD3, respectively.D–F). The mRNA expression level of these PLD members were unchanged in the adult brains of PLD4-deficient mice, although a significant increase in the liver and decrease in the thymus was observed for PLD1 and PLD3, respectively.'], 'pjab-92-237-g008': ['PLD4 knock-in mice were generated by the method described previously.21,22) The PLD4-targeting vector (0.05 pmol) was transfected into an ES cell line (1 × 105, RENKA) derived from ES cells of C57BL/6N mice21) by electroporation (Bio-RAD, Hercules, CA) (Supplemental Fig. <xref ref-type="fig" rid="pjab-92-237-g008">1</xref>A). Homologous recombinant cells were selected by G418 (175 µg/ml; Sigma-Aldrich Japan, Tokyo, Japan). Ten selected ES cells were individually isolated from each well of a 24-well plate, and the cell lines were prepared. Cells were confluent after 18 days, at which point half of the cells in each well were frozen, and the remaining cells were incubated in lysis buffer [50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS)] containing proteinase K (150 µg/mL) at 50 ℃ overnight. RNase A (10 µg/well) was added to the lysed cells and genomic DNA was isolated from each cell line using the PI-1200 DNA isolator (Kurabo, Osaka, Japan) for Southern blot analysis. Recombinant ES cell clones were identified by Southern blot analysis using a 5′-probe on EcoRV-digested genomic DNA for the first screening, and a 3′-probe on KpnI, EcoRI, or SpeI-digested genomic DNA and a 5′-probe on BstEII-digested genomic DNA for the second screening (sequence of each probe is available in Supporting information). Chimeric mice were generated using methods described previously.A). Homologous recombinant cells were selected by G418 (175 µg/ml; Sigma-Aldrich Japan, Tokyo, Japan). Ten selected ES cells were individually isolated from each well of a 24-well plate, and the cell lines were prepared. Cells were confluent after 18 days, at which point half of the cells in each well were frozen, and the remaining cells were incubated in lysis buffer [50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS)] containing proteinase K (150 µg/mL) at 50 ℃ overnight. RNase A (10 µg/well) was added to the lysed cells and genomic DNA was isolated from each cell line using the PI-1200 DNA isolator (Kurabo, Osaka, Japan) for Southern blot analysis. Recombinant ES cell clones were identified by Southern blot analysis using a 5′-probe on EcoRV-digested genomic DNA for the first screening, and a 3′-probe on KpnI, EcoRI, or SpeI-digested genomic DNA and a 5′-probe on BstEII-digested genomic DNA for the second screening (sequence of each probe is available in Supporting information). Chimeric mice were generated using methods described previously.22) Genotypes were verified by Southern blotting using a 5′-probe on EcoRV-digested genomic DNA and a 3′-probe on KpnI-digested genomic DNA (Supplemental Fig. <xref ref-type="fig" rid="pjab-92-237-g008">1</xref>).).'], 'pjab-92-237-g002': ['At P7, when PLD4 mRNA is typically upregulated in the cerebellum,2,6) no significant differences in gross brain structure were observed in PLD4-deficient mice compared to wild type (Fig. <xref ref-type="fig" rid="pjab-92-237-g002">2</xref>A, B). In the cerebellum, normal formation of the cortical layers including the molecular, external and internal granule layers was observed in PLD4-deficient mice (Fig. A, B). In the cerebellum, normal formation of the cortical layers including the molecular, external and internal granule layers was observed in PLD4-deficient mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g002">2</xref>B, right).B, right).', 'Microglial activation in cerebellum was examined by immunostaining with an anti-Iba1 antibody at P5 and P7. At P5, activated rounded microglia were spread diffusely throughout the cerebellar white matter in both wild type and PLD4-deficient mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g002">2</xref>C–F, K). At P7, rounded microglia that were strongly Iba1-positive could still be observed in the white matter that surrounds the deep cerebellar nuclei (deep white matter) of PLD4-deficient mice, whereas Iba1 staining intensity was reduced in most of the wild type microglia in the same area (Fig. C–F, K). At P7, rounded microglia that were strongly Iba1-positive could still be observed in the white matter that surrounds the deep cerebellar nuclei (deep white matter) of PLD4-deficient mice, whereas Iba1 staining intensity was reduced in most of the wild type microglia in the same area (Fig. <xref ref-type="fig" rid="pjab-92-237-g002">2</xref>G–J, L). At P10, process-bearing ramified microglia were found in both types of mice (Supplemental Fig. G–J, L). At P10, process-bearing ramified microglia were found in both types of mice (Supplemental Fig. <xref ref-type="fig" rid="pjab-92-237-g009">2</xref>).).'], 'pjab-92-237-g003': ['Microglial activation induces two phenotypes: M1 microglia and M2 microglia.26–29) M1 microglia show an inflammatory phenotype and secrete proinflammatory cytokines, including iNOS, TNFα, IL-1β, IFNγ and surface markers CD16, CD32, CD68, and CD86. In contrast, M2 microglia have an anti-inflammatory phenotype, and express different molecules such as IL-4, arginase 1, Ym1, CD163, CD206, and IL10.27,30–32) In order to determine the type of activated microglia present in the PLD4-deficient or wild type mice, sections were double-immunostained with an anti-Iba1 antibody and the microglial marker antibodies anti-CD68 or anti-arginase 1. Staining intensity of each CD68-positive cell was measured as described in Materials and methods. All Iba1-positive cells (red) were also positive for CD68 (green) in the deep white matter (Fig. <xref ref-type="fig" rid="pjab-92-237-g003">3</xref>A, B) and cerebellar folia (Fig. A, B) and cerebellar folia (Fig. <xref ref-type="fig" rid="pjab-92-237-g003">3</xref>D, E) of both genotypes at P5. However, the signal intensity of CD68 was significantly lower in the Iba1-positive cells of PLD4-deficient mice than in wild types at P5 (Fig. D, E) of both genotypes at P5. However, the signal intensity of CD68 was significantly lower in the Iba1-positive cells of PLD4-deficient mice than in wild types at P5 (Fig. <xref ref-type="fig" rid="pjab-92-237-g003">3</xref>C, F). Thus, wild type microglia showed high expression of CD68 while most of the PLD4-deficient microglia contained only low levels of CD68. No Iba1-positive microglia showed arginase 1 immunoreactivity in either genotype at P5 (data not shown).C, F). Thus, wild type microglia showed high expression of CD68 while most of the PLD4-deficient microglia contained only low levels of CD68. No Iba1-positive microglia showed arginase 1 immunoreactivity in either genotype at P5 (data not shown).'], 'pjab-92-237-g004': ['During development, microglia are actively involved in apoptosis of excessive neurons and axonal pruning during the final stages of neuronal circuit formation.8) Since inhibition of PLD4 by siRNA treatment caused partial reduction of phagocytic activity in microglia in vitro,6) we examined the development of Purkinje cells in PLD4-deficient mice. At P5 and P7, Calbindin immunostaining showed formation of the Purkinje cell layer with extension of the dendrites and axons (Fig. <xref ref-type="fig" rid="pjab-92-237-g004">4</xref>A–D, see also A\'–D\' for higher magnification views of the Purkinje cell layer). No obvious changes were observed in the two groups. In addition, the thickness of both the external and internal granule cell layers, as visualized by DAPI staining, were also unchanged (see DAPI staining of P7 cerebella in Fig. A–D, see also A\'–D\' for higher magnification views of the Purkinje cell layer). No obvious changes were observed in the two groups. In addition, the thickness of both the external and internal granule cell layers, as visualized by DAPI staining, were also unchanged (see DAPI staining of P7 cerebella in Fig. <xref ref-type="fig" rid="pjab-92-237-g002">2</xref>G–J). Calbindin-positive dendrites and axons were developed similarly at P10 (Supplemental Fig. G–J). Calbindin-positive dendrites and axons were developed similarly at P10 (Supplemental Fig. <xref ref-type="fig" rid="pjab-92-237-g009">2</xref>E, F).E, F).', 'Astrocytes and Bergmann glia were examined by immunostaining with an anti-GFAP antibody. The GFAP staining pattern and intensity was similar between PLD4-deficient and wild type mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g004">4</xref>E–J, Supplemental Fig. E–J, Supplemental Fig. <xref ref-type="fig" rid="pjab-92-237-g009">2</xref>G–J).G–J).'], 'pjab-92-237-g005': ['It is well known that activated microglia transiently appear during cerebellar development, and in our previous study, we showed that PLD4 expression is upregulated in these cells. In that study, a small number of PLD4 mRNA-positive cells first appeared in the proximal region of the white matter at P3, and became widely distributed within the folia from the deep white matter to the distal white matter at P5 and P7. By P10, they had dispersed into the gray matter.6) Since cerebellar myelination also occurs during the early postnatal period, we examined the myelination process by immunostaining with an anti-MBP antibody in C57BL/6 mice at various ages from P1 to P10 (Fig. <xref ref-type="fig" rid="pjab-92-237-g005">5</xref>A–E). MBP-positive oligodendrocytes appeared at P3 from the boundary of the brainstem. At P5, myelination occurred in the deep white matter around the cerebellar nucleus, then progressed into the white matter of the folia and spread to the tips by P7. Myelination ultimately reached the axons perforating into the internal granule cell layers at P10. These temporal changes were consistent with the presence of rounded Iba1-positive or PLD4-positive activated microglia (Fig. A–E). MBP-positive oligodendrocytes appeared at P3 from the boundary of the brainstem. At P5, myelination occurred in the deep white matter around the cerebellar nucleus, then progressed into the white matter of the folia and spread to the tips by P7. Myelination ultimately reached the axons perforating into the internal granule cell layers at P10. These temporal changes were consistent with the presence of rounded Iba1-positive or PLD4-positive activated microglia (Fig. <xref ref-type="fig" rid="pjab-92-237-g002">2</xref>).).6) Interestingly, double immunostaining with anti-Iba1 and anti-MBP antibodies clearly showed that a mass of activated microglia was often present at the myelination front in folia at P7 (Fig. <xref ref-type="fig" rid="pjab-92-237-g005">5</xref>F, F\'), while slender-shaped microglia were present in the already myelinated areas (Fig. F, F\'), while slender-shaped microglia were present in the already myelinated areas (Fig. <xref ref-type="fig" rid="pjab-92-237-g005">5</xref>F\'\').F\'\').', 'Transient microglial activation is a well-known phenomenon in the developing brain.7–10,38) Many studies have been conducted to look at the role of these transiently activated microglia during development, but these studies mainly focused on their involvement in neuronal events, including neurogenesis, neuronal death, neurite growth, and synaptic or axonal pruning.8,9) Our present study shows that activated microglia in the P7 cerebellum are often found at the myelination front, and microglia are transformed into a slender morphology in regions where MBP-positive myelin sheaths are present (Fig. <xref ref-type="fig" rid="pjab-92-237-g005">5</xref>). At the myelination front, activated microglia and oligodendrocytes with MBP-positive cell bodies (premyelinating oligodendrocytes) are found in close proximity. Thus, during early stages in the postnatal cerebellum, activated microglia are mainly present in the white matter near axons that will soon be myelinated. Although further studies using various differentiation-specific oligodendrocyte markers are needed to show how and in which stage myelination is affected in these mice, the delayed myelination observed in PLD4-deficient mice indicates a correlation between activated microglia and onset of myelination. This change is significant, and found also in corpus callosum, but only persists for several days during early neonatal development in this mutant line. Myelination is strictly regulated by local cues from axons or other glial cells.). At the myelination front, activated microglia and oligodendrocytes with MBP-positive cell bodies (premyelinating oligodendrocytes) are found in close proximity. Thus, during early stages in the postnatal cerebellum, activated microglia are mainly present in the white matter near axons that will soon be myelinated. Although further studies using various differentiation-specific oligodendrocyte markers are needed to show how and in which stage myelination is affected in these mice, the delayed myelination observed in PLD4-deficient mice indicates a correlation between activated microglia and onset of myelination. This change is significant, and found also in corpus callosum, but only persists for several days during early neonatal development in this mutant line. Myelination is strictly regulated by local cues from axons or other glial cells.17–19) In pathological conditions, microglia/macrophages can also promote remyelination.28,29,39) Although it is not clear how PLD4-deficient microglia affect the onset of myelination, phagocytosis of microglia may be important since down-regulation of PLD4 expression by siRNA treatment partially inhibited phagocytosis in vitro.6) In addition to cleaning up the pruned axonal branches or apoptotic cells prior to myelination, it is possible that activated microglia may also provide localized cues for oligodendrocytes to begin myelin formation either by direct contact or by secretion of humoral factors. Our data suggest that PLD4 may be involved in this series of events. Once this critical step is completed, oligodendrocytes may continue to myelinate without disturbance and could eventually catch up around P10 in PLD4-deficient mice (data not shown). Moreover, once myelin is formed, microglia are no longer activated, and switch to a resting state.', 'Influence of PLD4 deficiency on cerebellar myelination. (A, B) Western blot for MBP in the cerebellum of P7 wild type (WT) and PLD4-deficient (Ho) mice. Cerebellar homogenates (10 µg) from individual mice (three mice from each group) were separated by 12% SDS-PAGE and Western blot was performed using an anti-MBP antibody. After stripping off the antibody, the same blot was restained with an anti-β-actin antibody for quantification. B. The total intensity of all MBP bands (14, 17, 18.5, and 21 kDa) was quantified for each animal and the ratio of MBP to β-actin for each genotype was calculated. Graph indicates mean ± SEM. Statistical analysis with a student t test shows no significant difference between the two groups. (C–F) Comparison of MBP immunoreactivity in wild type (C, E) and PLD4-deficient (D, F) cerebella at P5 (C, D) and P7 (E, F). Cell nuclei are stained with DAPI (blue). Representative images of 5 mice from each genotype were selected. As shown in Fig. <xref ref-type="fig" rid="pjab-92-237-g005">5</xref>C and D, myelination in the deep white matter is in progress at P5 and progresses to the folial white matter by P7. Images in C, D or E, F show the deep white matter tracts, or folial white matter, respectively. C\'–F\' are higher magnification views of the white squares in C–F without DAPI. At P5, MBP-positive myelin membranes were found in the deep white matter in wild type mice, while premyelinating oligodendrocytes with MBP-positive cell bodies (white arrowheads) were present in PLD4-deficient mice. At P7, myelin membranes were present in wild type folia, whereas premyelinating oligodendrocytes (white arrowheads) were still found in PLD4-deficeint mice in the same region. * represents the border between the cerebellum (upper) and the earlier myelinating brainstem (lower). Scale bars indicate 100 µm (D, F) and 20 µm (D\', F\'). C and D, myelination in the deep white matter is in progress at P5 and progresses to the folial white matter by P7. Images in C, D or E, F show the deep white matter tracts, or folial white matter, respectively. C\'–F\' are higher magnification views of the white squares in C–F without DAPI. At P5, MBP-positive myelin membranes were found in the deep white matter in wild type mice, while premyelinating oligodendrocytes with MBP-positive cell bodies (white arrowheads) were present in PLD4-deficient mice. At P7, myelin membranes were present in wild type folia, whereas premyelinating oligodendrocytes (white arrowheads) were still found in PLD4-deficeint mice in the same region. * represents the border between the cerebellum (upper) and the earlier myelinating brainstem (lower). Scale bars indicate 100 µm (D, F) and 20 µm (D\', F\'). (G, H) Double immunostaining of P7 wild type (G) and PLD4-deficient (H) cerebellar deep white matter around the cerebellar nuclei using anti-MBP (green) and anti-Iba1 (red) antibodies. The DAPI staining pattern is overlaid to indicate the cerebellar region. G\' and H\' are the same images as in G and H without DAPI. In mice of both genotypes, clusters of rounded Iba1-positive activated microglia are present in the area where myelination is progressing. Scale bar in H\' represents 100 µm.'], 'pjab-92-237-g006': ['Since the activation of microglia is well correlated to the timing of myelination, especially at the myelination front, we examined the influence of PLD4-deficient microglia on myelination. Western blot analysis using an anti-MBP antibody showed no significant difference in P7 cerebella of wild-type and PLD4-deficient mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>A, B). In contrast, immunostaining with an anti-MBP antibody did show a difference between wild type and PLD4-deficient cerebella during the myelination process. In wild types, MBP-positive myelin membranes were found in the deep white matter at P5 (Fig. A, B). In contrast, immunostaining with an anti-MBP antibody did show a difference between wild type and PLD4-deficient cerebella during the myelination process. In wild types, MBP-positive myelin membranes were found in the deep white matter at P5 (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>C, C\'), whereas in PLD4-deficient mice, MBP-positive cell bodies of premyelinating oligodendrocytes were common and less myelin membrane staining was observed (Fig. C, C\'), whereas in PLD4-deficient mice, MBP-positive cell bodies of premyelinating oligodendrocytes were common and less myelin membrane staining was observed (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>D, D\', white arrowheads). At this stage of development, individual difference was observed in mice of both genotypes, however, 4 out of 5 mice from each group showed the same result as depicted in the figure, suggesting that these differences were significant. At P7, myelin membranes were found in the deep white matter around the cerebellar nuclei of both genotypes (Fig. D, D\', white arrowheads). At this stage of development, individual difference was observed in mice of both genotypes, however, 4 out of 5 mice from each group showed the same result as depicted in the figure, suggesting that these differences were significant. At P7, myelin membranes were found in the deep white matter around the cerebellar nuclei of both genotypes (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>G, H, green) and in the white matter of the folia in wild types (Fig. G, H, green) and in the white matter of the folia in wild types (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>E, E\'), whereas MBP-positive premyelinating oligodendrocyte cell bodies were frequently found in the folia of PLD4-deficient mice (Fig. E, E\'), whereas MBP-positive premyelinating oligodendrocyte cell bodies were frequently found in the folia of PLD4-deficient mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>F, F\', white arrowheads). Double immunostaining of P7 cerebella using anti-MBP and anti-Iba1 antibodies showed that in both types of mice rounded activated microglia were found adjacent to the myelinated areas in the folia and near the internal granule layers where myelination would occur in the near future (Fig. F, F\', white arrowheads). Double immunostaining of P7 cerebella using anti-MBP and anti-Iba1 antibodies showed that in both types of mice rounded activated microglia were found adjacent to the myelinated areas in the folia and near the internal granule layers where myelination would occur in the near future (Fig. <xref ref-type="fig" rid="pjab-92-237-g006">6</xref>G, G\', H, H\'). These results suggest a delay in the onset of myelination in PLD4-deficient mice compared with the timing in wild type mice.G, G\', H, H\'). These results suggest a delay in the onset of myelination in PLD4-deficient mice compared with the timing in wild type mice.'], 'pjab-92-237-g007': ['Similar results were obtained in the PLD4-deficient corpus callosum at P7 (Fig. <xref ref-type="fig" rid="pjab-92-237-g007">7</xref>). MBP-positive myelin was found in wild type mice whereas MBP signals were still restricted in their cell bodies and less myelin was observed in the PLD4-deficient mice (Fig. ). MBP-positive myelin was found in wild type mice whereas MBP signals were still restricted in their cell bodies and less myelin was observed in the PLD4-deficient mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g007">7</xref>A, B). For quantitative analysis, numbers of premyelinating oligodendrocytes with MBP-positive cell bodies were counted in the corpus callosum at three different positions in each brain (Fig. A, B). For quantitative analysis, numbers of premyelinating oligodendrocytes with MBP-positive cell bodies were counted in the corpus callosum at three different positions in each brain (Fig. <xref ref-type="fig" rid="pjab-92-237-g007">7</xref>C). In all the positions, significant numbers of MBP-positive cells remain premyelinating oligodendrocytes in the PLD-deficient mice (Fig. C). In all the positions, significant numbers of MBP-positive cells remain premyelinating oligodendrocytes in the PLD-deficient mice (Fig. <xref ref-type="fig" rid="pjab-92-237-g007">7</xref>D). Thus, the delay of myelination in the corpus callosum was more prominent than in the cerebellum, likely attributed to the later onset of myelination in corpus callosum.D). Thus, the delay of myelination in the corpus callosum was more prominent than in the cerebellum, likely attributed to the later onset of myelination in corpus callosum.']}
|
Microglial phospholipase D4 deficiency influences myelination during brain development
|
[
"phospholipase D",
"microglia",
"myelin",
"brain",
"development"
] |
Proc Jpn Acad Ser B Phys Biol Sci
|
1469775600
|
A long-standing goal of psychopathology research is to develop objective markers of symptomatic states, yet progress has been far slower than expected. Although prior reviews have attributed this state of affairs to diagnostic heterogeneity, symptom comorbidity and phenotypic complexity, little attention has been paid to the implications of intra-individual symptom dynamics and inter-relatedness for biomarker study designs. In this critical review, we consider the impact of short-term symptom fluctuations on widely used study designs that regress the 'average level' of a given symptom against biological data collected at a single time point, and summarize findings from ambulatory assessment studies suggesting that such designs may be sub-optimal to detect symptom-substrate relationships. Although such designs have a crucial role in advancing our understanding of biological substrates related to more stable, longer-term changes (for example, gray matter thinning during a depressive episode), they may be less optimal for the detection of symptoms that exhibit high frequency fluctuations, are susceptible to common reporting biases, or may be heavily influenced by the presence of other symptoms. We propose that a greater emphasis on intra-individual symptom chronometry may be useful for identifying subgroups of patients with common, proximal pathological indicators. Taken together, these three recent developments in the areas of symptom conceptualization and measurement raise important considerations for future studies attempting to identify reliable biomarkers in psychiatry.
|
[
"Behavioral Symptoms",
"Biomarkers",
"Comorbidity",
"Depressive Disorder",
"Female",
"Humans",
"Male",
"Mental Health",
"Prodromal Symptoms",
"Psychopathology",
"Research Design"
] |
other
|
PMC5114292
| null |
132
|
[
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"{'Citation': 'Miller AH, Raison CL. Are Anti-inflammatory Therapies Viable Treatments for Psychiatric Disorders? Where the Rubber Meets the Road. JAMA Psychiatry. 2015;72(6)', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC5542670'}, {'@IdType': 'pubmed', '#text': '25853989'}]}}",
"{'Citation': 'Watkins E, Teasdale JD. Rumination and overgeneral memory in depression: effects of self-focus and analytic thinking. J Abnorm Psychol. 2001;110(2):353.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '11358029'}}}",
"{'Citation': 'Kim S, Thibodeau R, Jorgensen RS. Shame, guilt, and depressive symptoms: a meta-analytic review. Psychol Bull. 2011;137(1):68.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '21219057'}}}"
] |
Proc Jpn Acad Ser B Phys Biol Sci. 2016 Jul 29; 92(7):237-254
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NO-CC CODE
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Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and γ-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-γ-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIα and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIα (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).
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gki190f4c
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2
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61e80e8a209153e96217227d0c6248594fb605c230a5ae762a038589c1e8265d
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gki190f4c.jpg
|
multiple
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multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
480,
359
] |
[{'image_id': 'gki190f2', 'image_file_name': 'gki190f2.jpg', 'image_path': '../data/media_files/PMC546168/gki190f2.jpg', 'caption': 'In situ visualization of GFP-DNA LIGIIIα, GFP-DNA LIGIIIβ and XRCC1 before and after local UV-irradiation. GFP–DNA Ligase IIIα (GFP-LIGIIIα) and GFP–DNA Ligase IIIβ (GFP-LIGIIIβ) were transiently expressed in XPA-UVDE cells before UV-irradiation (20 J/m2). The fluorescence images of the cells stained with anti-XRCC1 (red, second upper row) and the images of GFP (green, uppermost row) are shown. Co-localization appears yellow (third row). GFP-tagged proteins were visualized 2 min after local UV-irradiation (20 J/m2). Column c is for DIQ treated cells. The fluorescent images of GFP were superimposed onto the Nomarsky images in the bottom row.', 'hash': '8485117fbfd83fb303933fc70474d9529570f04ccd3cc5fc90890926fa08aa83'}, {'image_id': 'gki190f5', 'image_file_name': 'gki190f5.jpg', 'image_path': '../data/media_files/PMC546168/gki190f5.jpg', 'caption': 'Distribution of XRCC1 in human cell lines. The upper left panel: fluorescent micrograph of HeLa cell in prophase by immunolabeling for XRCC1. The cells were fixed with paraformaldehyde, and stained with anti-XRCC1 monoclonal antibody. The lower left panel: fluorescent micrograph of AT5BI-VA cell in metaphase by immunolabeling for XRCC1. The cells were fixed with methanol:acetone, and stained with anti-XRCC1 antibody. The DNA was stained with TOPRO-3 and the corresponding images were superimposed onto the fluorescent images in the left columns and shown in the right columns.', 'hash': '71697002b5c408f1f9ab56fbb1feced8f05e1d41b866a7e7ef3123a0576f231c'}, {'image_id': 'gki190f4c', 'image_file_name': 'gki190f4c.jpg', 'image_path': '../data/media_files/PMC546168/gki190f4c.jpg', 'caption': 'Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and γ-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-γ-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIα and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIα (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).', 'hash': '61e80e8a209153e96217227d0c6248594fb605c230a5ae762a038589c1e8265d'}, {'image_id': 'gki190f3', 'image_file_name': 'gki190f3.jpg', 'image_path': '../data/media_files/PMC546168/gki190f3.jpg', 'caption': 'XRCC1 in centrosomes of HeLa cells during interphase. (A) Fluorescent micrographs of HeLa cells in interphase obtained by double immunolabeling for γ-tubulin and XRCC1. Cells were fixed with methanol:acetone and co-stained with anti-XRCC1 antibody (a), and anti-γ-tubulin antibody (b). XRCC1 and γ-tubulin appear red and green, respectively. The corresponding Nomarsky image is shown in (c). Co-localization of both XRCC1 and γ-tubulin appears yellow in overlay (d). (B) Fluorescent micrographs of HeLa cells in interphase expressing XRCC1-GFP. The cells were fixed with paraformaldehyde. DNA was stained with TOPRO-3 (middle panel) and superimposed onto the fluorescent image in the uppermost panel. Arrows indicate the position of centrosomes.', 'hash': 'ef37ac152d2217ce3dbb734d66b997f1901fa87e6fc543c64d5626efcef38231'}, {'image_id': 'gki190f4a', 'image_file_name': 'gki190f4a.jpg', 'image_path': '../data/media_files/PMC546168/gki190f4a.jpg', 'caption': 'Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and γ-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-γ-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIα and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIα (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).', 'hash': '8fb1a142fb5363a14e687d0a40fd15c98e2c67871a0a9f8e97ef245b19955bb4'}, {'image_id': 'gki190f1', 'image_file_name': 'gki190f1.jpg', 'image_path': '../data/media_files/PMC546168/gki190f1.jpg', 'caption': 'Accumulation of XRCC1 and LIGIIIα at SSBs after local UV-irradiation in XPA-UVDE cells. Co-localization of XRCC1 with LIGIIIα was identified by double immunolabeling. Two minutes after local UV-irradiation (20 J/m2) cells were fixed with paraformaldehyde and co-stained with anti-XRCC1 antibody (red, upper row) and anti-LIGIIIα antibody (green, middle row); the column c is for XPA-UVDE cells treated with DIQ, an inhibitor of PARP, before UV-irradiation. Co-localization of XRCC1 with LIGIIIα appears yellow in overlay (bottom row).', 'hash': 'db15c04f75edad43c15a8496b3038d1ffc06c1968afca21ab873a2d93f461cc8'}]
|
{'gki190f1': ['Previously, using local UV-irradiation of human nucleotide excision repair-deficient XPA cells expressing UVDE, we had shown that PAR synthesis occurred only within the UV-irradiated regions and that the recruitment of XRCC1 to these regions was dependent upon PAR synthesis (19). Since there is evidence that XRCC1 functions independently of LIGIIIα under certain circumstances (15,28), we asked whether LIGIIIα was also recruited to the sites of SSBs induced by the action of UVDE in XPA cells after local UV-irradiation. As shown previously (19), XRCC1 accumulated at the irradiated spots 2 min after irradiation (<xref ref-type="fig" rid="gki190f1">Figure 1</xref>, column b, upper panel). LIGIIIα was also recruited to the same sites of DNA damage (, column b, upper panel). LIGIIIα was also recruited to the same sites of DNA damage (<xref ref-type="fig" rid="gki190f1">Figure 1</xref>, column b, middle and bottom panels). As was observed with XRCC1, the recruitment of LIGIIIα to the DNA damage sites was dependent upon the expression of UVDE (, column b, middle and bottom panels). As was observed with XRCC1, the recruitment of LIGIIIα to the DNA damage sites was dependent upon the expression of UVDE (<xref ref-type="fig" rid="gki190f1">Figure 1</xref>, column e) and was prevented by DIQ (100 μM), an inhibitor of PARP (, column e) and was prevented by DIQ (100 μM), an inhibitor of PARP (<xref ref-type="fig" rid="gki190f1">Figure 1</xref> column c). To confirm that the DNA damage-dependent sub-nuclear location of LIGIIIα was not an artifact of the immunocytochemistry, we transiently expressed a GFP-tagged version of LIGIIIα in XPA-UVDE cells. In accord with the results described above, GFP-LIGIIIα was observed in the nucleolus as well as in the nucleoplasm of undamaged cells ( column c). To confirm that the DNA damage-dependent sub-nuclear location of LIGIIIα was not an artifact of the immunocytochemistry, we transiently expressed a GFP-tagged version of LIGIIIα in XPA-UVDE cells. In accord with the results described above, GFP-LIGIIIα was observed in the nucleolus as well as in the nucleoplasm of undamaged cells (<xref ref-type="fig" rid="gki190f2">Figure 2</xref>, column a) and was recruited to the sites of DNA damage induced by irradiation through a membrane filter (, column a) and was recruited to the sites of DNA damage induced by irradiation through a membrane filter (<xref ref-type="fig" rid="gki190f2">Figure 2</xref>, column b). Together, these results demonstrate that LIGIIIα, like its protein partner XRCC1, translocates to the sites of SSBs and that this DNA damage-induced sub-nuclear relocation is dependent upon PAR synthesis. This is consistent with our recent results using local laser irradiation (, column b). Together, these results demonstrate that LIGIIIα, like its protein partner XRCC1, translocates to the sites of SSBs and that this DNA damage-induced sub-nuclear relocation is dependent upon PAR synthesis. This is consistent with our recent results using local laser irradiation (20). To examine whether the recruitment of LIGIIIα to SSBs is dependent upon XRCC1 and not on PARP-1, we transiently expressed a GFP-tagged version of DNA ligase IIIβ (LIGIIIβ), which is normally only expressed in meiotic cells (29). Importantly, LIGIIIβ interacts with poly(ADP-ribosylated) PARP-1 (18) but does not bind to XRCC1. In undamaged cells, LIGIIIβ was present in the nucleolus as well as in the nucleoplasm (<xref ref-type="fig" rid="gki190f2">Figure 2</xref> column d), but, unlike LIGIIIα, LIGIIIβ was not recruited to the sites of DNA damage ( column d), but, unlike LIGIIIα, LIGIIIβ was not recruited to the sites of DNA damage (<xref ref-type="fig" rid="gki190f2">Figure 2</xref>, column e). By a more sensitive assay using UVA laser irradiation and GFP-tagged LIGIIIα, we found a weak accumulation of LIGIIIα at SSBs in XRCC1-deficient cells (L. Lan and A. Yasui, unpublished results), which may be explained by direct interaction between PARP-1 and LIGIIIα. Thus, although LIGIIIα binds to poly(ADP-ribosylated) PARP-1 , column e). By a more sensitive assay using UVA laser irradiation and GFP-tagged LIGIIIα, we found a weak accumulation of LIGIIIα at SSBs in XRCC1-deficient cells (L. Lan and A. Yasui, unpublished results), which may be explained by direct interaction between PARP-1 and LIGIIIα. Thus, although LIGIIIα binds to poly(ADP-ribosylated) PARP-1 in vitro, the in vivo recruitment of LIGIIIα to SSBs is mainly dependent upon its C-terminal BRCT domain, which is required for the interaction with XRCC1 (29,30).'], 'gki190f3': ['When exponentially growing HeLa cells were stained with an XRCC1 antibody, a unique staining pattern that appeared to correspond to the centrosomes was observed in both interphase and mitotic cells. To confirm the co-localization of XRCC1 with the centrosomes, we conducted a series of double-staining experiments using antibodies specific for XRCC1 and γ-tubulin, which is a component of pericentriolar matrix of the centrosome (31). As reported previously (19), the majority of XRCC1 in interphase cells is in the nucleoli, but a small fraction co-localized precisely with the two closely spaced dots of γ-tubulin located near the nuclear envelope (<xref ref-type="fig" rid="gki190f3">Figure 3A</xref>), which correspond to the duplicated centrosomes. The co-localization of XRCC1 with unduplicated centrosomes in interphase cells was also observed (data not shown). As described later, the presence of PAR in the centrosomes is crucial for the centrosomal localization of XRCC1. Detection of XRCC1 in the fraction of the centrosomes by antibodies failed, possibly because the amount of PAR polymers present in the centrosomes is limited partially due to the presence of PAR glycohydrolase and, therefore, the amount of XRCC1 molecules is too low to be detected by western blotting analysis. Therefore, to confirm the centrosomal location of XRCC1, we transiently expressed a GFP-tagged XRCC1 in HeLa cells. Indeed, in interphase cells, GFP-tagged XRCC1 was observed in the centrosomes (), which correspond to the duplicated centrosomes. The co-localization of XRCC1 with unduplicated centrosomes in interphase cells was also observed (data not shown). As described later, the presence of PAR in the centrosomes is crucial for the centrosomal localization of XRCC1. Detection of XRCC1 in the fraction of the centrosomes by antibodies failed, possibly because the amount of PAR polymers present in the centrosomes is limited partially due to the presence of PAR glycohydrolase and, therefore, the amount of XRCC1 molecules is too low to be detected by western blotting analysis. Therefore, to confirm the centrosomal location of XRCC1, we transiently expressed a GFP-tagged XRCC1 in HeLa cells. Indeed, in interphase cells, GFP-tagged XRCC1 was observed in the centrosomes (<xref ref-type="fig" rid="gki190f3">Figure 3B</xref>).).'], 'gki190f4a': ['The association of a small fraction of XRCC1 with the centrosomes in interphase cells prompted us to examine the behavior of XRCC1 during mitosis. Using TOPRO-3 to stain cellular DNA, we determined the distribution of XRCC1 and γ-tubulin (<xref ref-type="fig" rid="gki190f4a">Figure 4A</xref>), XRCC1 and LIGIIIα (), XRCC1 and LIGIIIα (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>), and XRCC1 and PAR (), and XRCC1 and PAR (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>) at different stages of mitosis. As shown in these figures, in metaphase cells the majority of XRCC1 co-localized with γ-tubulin in the centrosomes. XRCC1 was also detected at the periphery of the centrosomes in metaphase. XRCC1 accumulated at mitotic centrosome and its immediate vicinity in the cells from prophase to metaphase () at different stages of mitosis. As shown in these figures, in metaphase cells the majority of XRCC1 co-localized with γ-tubulin in the centrosomes. XRCC1 was also detected at the periphery of the centrosomes in metaphase. XRCC1 accumulated at mitotic centrosome and its immediate vicinity in the cells from prophase to metaphase (<xref ref-type="fig" rid="gki190f5">Figures 5</xref> and and <xref ref-type="fig" rid="gki190f4a">4C</xref>, upper panels). However, the pericentriolar localization of XRCC1 disappeared during anaphase (, upper panels). However, the pericentriolar localization of XRCC1 disappeared during anaphase (<xref ref-type="fig" rid="gki190f4a">Figure 4A</xref>). A similar XRCC1 staining pattern was observed by using paraformaldehyde fixation (). A similar XRCC1 staining pattern was observed by using paraformaldehyde fixation (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>), and by using an XRCC1 monoclonal antibody (), and by using an XRCC1 monoclonal antibody (<xref ref-type="fig" rid="gki190f5">Figure 5</xref>) and in several human fibroblast cell lines including ATM-deficient (AT5BI-VA) cells () and in several human fibroblast cell lines including ATM-deficient (AT5BI-VA) cells (<xref ref-type="fig" rid="gki190f5">Figure 5</xref>). The staining pattern of LIGIIIα detected with two different monoclonal antibodies was indistinguishable from that of XRCC1 (). The staining pattern of LIGIIIα detected with two different monoclonal antibodies was indistinguishable from that of XRCC1 (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>). Thus, we conclude that XRCC1 and LIGIIIα are integral components of centrosomes, and that, during the early stages of mitosis, they also concentrate around the pericentriolar matrix.). Thus, we conclude that XRCC1 and LIGIIIα are integral components of centrosomes, and that, during the early stages of mitosis, they also concentrate around the pericentriolar matrix.', 'To examine whether poly(ADP-ribosyl)ation is involved in the centrosomal localization of XRCC1, the cells were incubated with antibodies for PAR and XRCC1. PAR is reported to be present in the centrosomes in both interphase and mitosis (22). During mitosis, PAR co-localized with XRCC1 in the centrosomes and their immediate vicinity (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>). The change in the amount of PAR in the centrosomes and their immediate vicinity was completely correlated with that of XRCC1 (). The change in the amount of PAR in the centrosomes and their immediate vicinity was completely correlated with that of XRCC1 (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>), suggesting the presence of yet unknown DNA damage-independent mechanisms of PARP activation and the recruitment of XRCC1 to the centrosomal region during mitosis. When cells were incubated for 1 h in the presence of 3-AB (8 mM), a potent inhibitor of PARP, neither PAR nor XRCC1 was observed at or near centrosomes in metaphase cells (), suggesting the presence of yet unknown DNA damage-independent mechanisms of PARP activation and the recruitment of XRCC1 to the centrosomal region during mitosis. When cells were incubated for 1 h in the presence of 3-AB (8 mM), a potent inhibitor of PARP, neither PAR nor XRCC1 was observed at or near centrosomes in metaphase cells (<xref ref-type="fig" rid="gki190f4a">Figure 4A–C</xref>). Similarly, treatment with 3-AB abolished the localization of LIGIIIα to the centrosomes of metaphase cells (). Similarly, treatment with 3-AB abolished the localization of LIGIIIα to the centrosomes of metaphase cells (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>). Moreover, in interphase cells, the centrosomal localization of XRCC1 was also prevented by 3-AB (data not shown). Together, these results show that the synthesis of PAR at or near the centrosomes is a prerequisite for the localization of both XRCC1 and LIGIIIα.). Moreover, in interphase cells, the centrosomal localization of XRCC1 was also prevented by 3-AB (data not shown). Together, these results show that the synthesis of PAR at or near the centrosomes is a prerequisite for the localization of both XRCC1 and LIGIIIα.', 'Since PARP-1 is present in both centrosomes and chromosomes in mitotic cells (21), we examined whether during mitosis PARP is activated by DNA-damaging agents and whether this activation affects the localization of XRCC1 and LIGIIIα. Following treatment of metaphase cells with H2O2, PAR was synthesized on the metaphase chromosomes (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>). In response to the PAR synthesis, both XRCC1 and LIGIIIα were translocated from the centrosomes to the metaphase chromosomes (). In response to the PAR synthesis, both XRCC1 and LIGIIIα were translocated from the centrosomes to the metaphase chromosomes (<xref ref-type="fig" rid="gki190f4a">Figure 4A–C</xref>). Similar results were obtained by treating the cells with 100 μM of DNA alkylating agent, MMS, for 10 min (data not shown). Thus, it appears that, as in interphase cells (). Similar results were obtained by treating the cells with 100 μM of DNA alkylating agent, MMS, for 10 min (data not shown). Thus, it appears that, as in interphase cells (19), the synthesis of PAR by PARP-1 at DNA strand breaks signals the recruitment of SSBs repair factors in mitotic cells. This suggests that DNA repair of SSBs occurs in the condensed chromatin of metaphase chromosomes.']}
|
Translocation of XRCC1 and DNA ligase IIIα from centrosomes to chromosomes in response to DNA damage in mitotic human cells
| null |
Nucleic Acids Res
|
1105689600
|
DNA single-strand breaks (SSBs) are the most frequent lesions caused by oxidative DNA damage. They disrupt DNA replication, give rise to double-strand breaks and lead to cell death and genomic instability. It has been shown that the XRCC1 protein plays a key role in SSBs repair. We have recently shown in living human cells that XRCC1 accumulates at SSBs in a fully poly(ADP-ribose) (PAR) synthesis-dependent manner and that the accumulation of XRCC1 at SSBs is essential for further repair processes. Here, we show that XRCC1 and its partner protein, DNA ligase IIIalpha, localize at the centrosomes and their vicinity in metaphase cells and disappear during anaphase. Although the function of these proteins in centrosomes during metaphase is unknown, this centrosomal localization is PAR-dependent, because neither of the proteins is observed in the centrosomes in the presence of PAR polymerase inhibitors. On treatment of metaphase cells with H2O2, XRCC1 and DNA ligase IIIalpha translocate immediately from the centrosomes to mitotic chromosomes. These results show for the first time that the repair of SSBs is present in the early mitotic chromosomes and that there is a dynamic response of XRCC1 and DNA ligase IIIalpha to SSBs, in which these proteins are recruited from the centrosomes, where metaphase-dependent activation of PAR polymerase occurs, to mitotic chromosomes, by SSBs-dependent activation of PAR polymerase.
|
[
"Cell Line",
"Centrosome",
"Chromosomes, Human",
"DNA Damage",
"DNA Ligase ATP",
"DNA Ligases",
"DNA-Binding Proteins",
"Humans",
"Hydrogen Peroxide",
"Mitosis",
"Poly Adenosine Diphosphate Ribose",
"Poly-ADP-Ribose Binding Proteins",
"Protein Transport",
"X-ray Repair Cross Complementing Protein 1",
"Xenopus Proteins"
] |
other
|
PMC546168
| null |
34
|
[
"{'Citation': 'Thompson L.H., West M.G. XRCC1 keeps DNA from getting stranded. Mutat. Res. 2000;459:1–18.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '10677679'}}}",
"{'Citation': 'Dominguez I., Daza P., Natarajan A.T., Cortes F. A high yield of translocations parallels the high yield of sister chromatid exchanges in the CHO mutant EM9. Mutat. Res. 1998;398:67–73.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '9626966'}}}",
"{'Citation': 'Tebbs R.S., Flannery M.L., Meneses J.J., Hartmann A., Tucker J.D., Thompson L.H., Cleaver J.E., Pedersen R.A. Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. Dev. Biol. 1999;208:513–529.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '10191063'}}}",
"{'Citation': 'Marsin S., Vidal A.E., Sossou M., Menissier-de Murcia J., Le Page F., Boiteux S., de Murcia G., Radicella J.P. Role of XRCC1 in the coordination and stimulation of oxidative DNA damage repair initiated by the DNA glycosylase hOGG1. J. Biol. Chem. 2003;278:44068–44074.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '12933815'}}}",
"{'Citation': 'Dantzer F., de la Rubia G., Menissier-de Murcia J., Hostomsky Z., de Murcia G., Schreibr V. Base excision repair is impaired in mammalian cells lacking poly(ADP-ribose) polymerase-1. Biochemistry. 2000;39:7559–7569.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '10858306'}}}",
"{'Citation': 'Masson M., Niedergang C., Schreiber V., Muller S., Menissier-de Murcia J., de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. Mol. Cell. Biol. 1998;18:3563–3571.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC108937'}, {'@IdType': 'pubmed', '#text': '9584196'}]}}",
"{'Citation': 'Schreiber V., Ame J.C., Dolle P., Schultz I., Rinaldi B., Fraulob V., Menissier-de Murcia J., de Murcia G. Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1. J. Biol. Chem. 2002;277:23028–23036.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '11948190'}}}",
"{'Citation': 'Caldecott K.W., Aoufouchi S., Johnson P., Shall S. XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly(ADP-ribose) polymerase, and DNA ligase III is a novel molecular ‘nick-sensor’ in vitro. Nucleic Acids Res. 1996;24:4387–4394.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC146288'}, {'@IdType': 'pubmed', '#text': '8948628'}]}}",
"{'Citation': 'Kubota Y., Nash R.A., Klungland A., Schar P., Barnes D.E., Lindahl T. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J. 1996;15:6662–6670.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC452490'}, {'@IdType': 'pubmed', '#text': '8978692'}]}}",
"{'Citation': 'Vidal A.E., Boiteux S., Hickson I.D., Radicella J.P. XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein–protein interactions. EMBO J. 2001;20:6530–6539.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC125722'}, {'@IdType': 'pubmed', '#text': '11707423'}]}}",
"{'Citation': 'Whitehouse C.J., Taylor R.M., Thistlethwaite A., Zhang H., Karimi-Busheri F., Lasko D.D., Weinfeld M., Caldecott K.W. XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. Cell. 2001;104:107–117.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '11163244'}}}",
"{'Citation': 'Caldecott K.W., McKeown C.K., Tucker J.D., Ljungquist S., Thompson L.H. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol. Cell. Biol. 1994;14:68–76.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC358357'}, {'@IdType': 'pubmed', '#text': '8264637'}]}}",
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Nucleic Acids Res. 2005 Jan 14; 33(1):422-429
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NO-CC CODE
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Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and γ-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-γ-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIα and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIα (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).
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8fb1a142fb5363a14e687d0a40fd15c98e2c67871a0a9f8e97ef245b19955bb4
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
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[{'image_id': 'gki190f2', 'image_file_name': 'gki190f2.jpg', 'image_path': '../data/media_files/PMC546168/gki190f2.jpg', 'caption': 'In situ visualization of GFP-DNA LIGIIIα, GFP-DNA LIGIIIβ and XRCC1 before and after local UV-irradiation. GFP–DNA Ligase IIIα (GFP-LIGIIIα) and GFP–DNA Ligase IIIβ (GFP-LIGIIIβ) were transiently expressed in XPA-UVDE cells before UV-irradiation (20 J/m2). The fluorescence images of the cells stained with anti-XRCC1 (red, second upper row) and the images of GFP (green, uppermost row) are shown. Co-localization appears yellow (third row). GFP-tagged proteins were visualized 2 min after local UV-irradiation (20 J/m2). Column c is for DIQ treated cells. The fluorescent images of GFP were superimposed onto the Nomarsky images in the bottom row.', 'hash': '8485117fbfd83fb303933fc70474d9529570f04ccd3cc5fc90890926fa08aa83'}, {'image_id': 'gki190f5', 'image_file_name': 'gki190f5.jpg', 'image_path': '../data/media_files/PMC546168/gki190f5.jpg', 'caption': 'Distribution of XRCC1 in human cell lines. The upper left panel: fluorescent micrograph of HeLa cell in prophase by immunolabeling for XRCC1. The cells were fixed with paraformaldehyde, and stained with anti-XRCC1 monoclonal antibody. The lower left panel: fluorescent micrograph of AT5BI-VA cell in metaphase by immunolabeling for XRCC1. The cells were fixed with methanol:acetone, and stained with anti-XRCC1 antibody. The DNA was stained with TOPRO-3 and the corresponding images were superimposed onto the fluorescent images in the left columns and shown in the right columns.', 'hash': '71697002b5c408f1f9ab56fbb1feced8f05e1d41b866a7e7ef3123a0576f231c'}, {'image_id': 'gki190f4c', 'image_file_name': 'gki190f4c.jpg', 'image_path': '../data/media_files/PMC546168/gki190f4c.jpg', 'caption': 'Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and γ-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-γ-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIα and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIα (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).', 'hash': '61e80e8a209153e96217227d0c6248594fb605c230a5ae762a038589c1e8265d'}, {'image_id': 'gki190f3', 'image_file_name': 'gki190f3.jpg', 'image_path': '../data/media_files/PMC546168/gki190f3.jpg', 'caption': 'XRCC1 in centrosomes of HeLa cells during interphase. (A) Fluorescent micrographs of HeLa cells in interphase obtained by double immunolabeling for γ-tubulin and XRCC1. Cells were fixed with methanol:acetone and co-stained with anti-XRCC1 antibody (a), and anti-γ-tubulin antibody (b). XRCC1 and γ-tubulin appear red and green, respectively. The corresponding Nomarsky image is shown in (c). Co-localization of both XRCC1 and γ-tubulin appears yellow in overlay (d). (B) Fluorescent micrographs of HeLa cells in interphase expressing XRCC1-GFP. The cells were fixed with paraformaldehyde. DNA was stained with TOPRO-3 (middle panel) and superimposed onto the fluorescent image in the uppermost panel. Arrows indicate the position of centrosomes.', 'hash': 'ef37ac152d2217ce3dbb734d66b997f1901fa87e6fc543c64d5626efcef38231'}, {'image_id': 'gki190f4a', 'image_file_name': 'gki190f4a.jpg', 'image_path': '../data/media_files/PMC546168/gki190f4a.jpg', 'caption': 'Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and γ-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-γ-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIα and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIα (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).', 'hash': '8fb1a142fb5363a14e687d0a40fd15c98e2c67871a0a9f8e97ef245b19955bb4'}, {'image_id': 'gki190f1', 'image_file_name': 'gki190f1.jpg', 'image_path': '../data/media_files/PMC546168/gki190f1.jpg', 'caption': 'Accumulation of XRCC1 and LIGIIIα at SSBs after local UV-irradiation in XPA-UVDE cells. Co-localization of XRCC1 with LIGIIIα was identified by double immunolabeling. Two minutes after local UV-irradiation (20 J/m2) cells were fixed with paraformaldehyde and co-stained with anti-XRCC1 antibody (red, upper row) and anti-LIGIIIα antibody (green, middle row); the column c is for XPA-UVDE cells treated with DIQ, an inhibitor of PARP, before UV-irradiation. Co-localization of XRCC1 with LIGIIIα appears yellow in overlay (bottom row).', 'hash': 'db15c04f75edad43c15a8496b3038d1ffc06c1968afca21ab873a2d93f461cc8'}]
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{'gki190f1': ['Previously, using local UV-irradiation of human nucleotide excision repair-deficient XPA cells expressing UVDE, we had shown that PAR synthesis occurred only within the UV-irradiated regions and that the recruitment of XRCC1 to these regions was dependent upon PAR synthesis (19). Since there is evidence that XRCC1 functions independently of LIGIIIα under certain circumstances (15,28), we asked whether LIGIIIα was also recruited to the sites of SSBs induced by the action of UVDE in XPA cells after local UV-irradiation. As shown previously (19), XRCC1 accumulated at the irradiated spots 2 min after irradiation (<xref ref-type="fig" rid="gki190f1">Figure 1</xref>, column b, upper panel). LIGIIIα was also recruited to the same sites of DNA damage (, column b, upper panel). LIGIIIα was also recruited to the same sites of DNA damage (<xref ref-type="fig" rid="gki190f1">Figure 1</xref>, column b, middle and bottom panels). As was observed with XRCC1, the recruitment of LIGIIIα to the DNA damage sites was dependent upon the expression of UVDE (, column b, middle and bottom panels). As was observed with XRCC1, the recruitment of LIGIIIα to the DNA damage sites was dependent upon the expression of UVDE (<xref ref-type="fig" rid="gki190f1">Figure 1</xref>, column e) and was prevented by DIQ (100 μM), an inhibitor of PARP (, column e) and was prevented by DIQ (100 μM), an inhibitor of PARP (<xref ref-type="fig" rid="gki190f1">Figure 1</xref> column c). To confirm that the DNA damage-dependent sub-nuclear location of LIGIIIα was not an artifact of the immunocytochemistry, we transiently expressed a GFP-tagged version of LIGIIIα in XPA-UVDE cells. In accord with the results described above, GFP-LIGIIIα was observed in the nucleolus as well as in the nucleoplasm of undamaged cells ( column c). To confirm that the DNA damage-dependent sub-nuclear location of LIGIIIα was not an artifact of the immunocytochemistry, we transiently expressed a GFP-tagged version of LIGIIIα in XPA-UVDE cells. In accord with the results described above, GFP-LIGIIIα was observed in the nucleolus as well as in the nucleoplasm of undamaged cells (<xref ref-type="fig" rid="gki190f2">Figure 2</xref>, column a) and was recruited to the sites of DNA damage induced by irradiation through a membrane filter (, column a) and was recruited to the sites of DNA damage induced by irradiation through a membrane filter (<xref ref-type="fig" rid="gki190f2">Figure 2</xref>, column b). Together, these results demonstrate that LIGIIIα, like its protein partner XRCC1, translocates to the sites of SSBs and that this DNA damage-induced sub-nuclear relocation is dependent upon PAR synthesis. This is consistent with our recent results using local laser irradiation (, column b). Together, these results demonstrate that LIGIIIα, like its protein partner XRCC1, translocates to the sites of SSBs and that this DNA damage-induced sub-nuclear relocation is dependent upon PAR synthesis. This is consistent with our recent results using local laser irradiation (20). To examine whether the recruitment of LIGIIIα to SSBs is dependent upon XRCC1 and not on PARP-1, we transiently expressed a GFP-tagged version of DNA ligase IIIβ (LIGIIIβ), which is normally only expressed in meiotic cells (29). Importantly, LIGIIIβ interacts with poly(ADP-ribosylated) PARP-1 (18) but does not bind to XRCC1. In undamaged cells, LIGIIIβ was present in the nucleolus as well as in the nucleoplasm (<xref ref-type="fig" rid="gki190f2">Figure 2</xref> column d), but, unlike LIGIIIα, LIGIIIβ was not recruited to the sites of DNA damage ( column d), but, unlike LIGIIIα, LIGIIIβ was not recruited to the sites of DNA damage (<xref ref-type="fig" rid="gki190f2">Figure 2</xref>, column e). By a more sensitive assay using UVA laser irradiation and GFP-tagged LIGIIIα, we found a weak accumulation of LIGIIIα at SSBs in XRCC1-deficient cells (L. Lan and A. Yasui, unpublished results), which may be explained by direct interaction between PARP-1 and LIGIIIα. Thus, although LIGIIIα binds to poly(ADP-ribosylated) PARP-1 , column e). By a more sensitive assay using UVA laser irradiation and GFP-tagged LIGIIIα, we found a weak accumulation of LIGIIIα at SSBs in XRCC1-deficient cells (L. Lan and A. Yasui, unpublished results), which may be explained by direct interaction between PARP-1 and LIGIIIα. Thus, although LIGIIIα binds to poly(ADP-ribosylated) PARP-1 in vitro, the in vivo recruitment of LIGIIIα to SSBs is mainly dependent upon its C-terminal BRCT domain, which is required for the interaction with XRCC1 (29,30).'], 'gki190f3': ['When exponentially growing HeLa cells were stained with an XRCC1 antibody, a unique staining pattern that appeared to correspond to the centrosomes was observed in both interphase and mitotic cells. To confirm the co-localization of XRCC1 with the centrosomes, we conducted a series of double-staining experiments using antibodies specific for XRCC1 and γ-tubulin, which is a component of pericentriolar matrix of the centrosome (31). As reported previously (19), the majority of XRCC1 in interphase cells is in the nucleoli, but a small fraction co-localized precisely with the two closely spaced dots of γ-tubulin located near the nuclear envelope (<xref ref-type="fig" rid="gki190f3">Figure 3A</xref>), which correspond to the duplicated centrosomes. The co-localization of XRCC1 with unduplicated centrosomes in interphase cells was also observed (data not shown). As described later, the presence of PAR in the centrosomes is crucial for the centrosomal localization of XRCC1. Detection of XRCC1 in the fraction of the centrosomes by antibodies failed, possibly because the amount of PAR polymers present in the centrosomes is limited partially due to the presence of PAR glycohydrolase and, therefore, the amount of XRCC1 molecules is too low to be detected by western blotting analysis. Therefore, to confirm the centrosomal location of XRCC1, we transiently expressed a GFP-tagged XRCC1 in HeLa cells. Indeed, in interphase cells, GFP-tagged XRCC1 was observed in the centrosomes (), which correspond to the duplicated centrosomes. The co-localization of XRCC1 with unduplicated centrosomes in interphase cells was also observed (data not shown). As described later, the presence of PAR in the centrosomes is crucial for the centrosomal localization of XRCC1. Detection of XRCC1 in the fraction of the centrosomes by antibodies failed, possibly because the amount of PAR polymers present in the centrosomes is limited partially due to the presence of PAR glycohydrolase and, therefore, the amount of XRCC1 molecules is too low to be detected by western blotting analysis. Therefore, to confirm the centrosomal location of XRCC1, we transiently expressed a GFP-tagged XRCC1 in HeLa cells. Indeed, in interphase cells, GFP-tagged XRCC1 was observed in the centrosomes (<xref ref-type="fig" rid="gki190f3">Figure 3B</xref>).).'], 'gki190f4a': ['The association of a small fraction of XRCC1 with the centrosomes in interphase cells prompted us to examine the behavior of XRCC1 during mitosis. Using TOPRO-3 to stain cellular DNA, we determined the distribution of XRCC1 and γ-tubulin (<xref ref-type="fig" rid="gki190f4a">Figure 4A</xref>), XRCC1 and LIGIIIα (), XRCC1 and LIGIIIα (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>), and XRCC1 and PAR (), and XRCC1 and PAR (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>) at different stages of mitosis. As shown in these figures, in metaphase cells the majority of XRCC1 co-localized with γ-tubulin in the centrosomes. XRCC1 was also detected at the periphery of the centrosomes in metaphase. XRCC1 accumulated at mitotic centrosome and its immediate vicinity in the cells from prophase to metaphase () at different stages of mitosis. As shown in these figures, in metaphase cells the majority of XRCC1 co-localized with γ-tubulin in the centrosomes. XRCC1 was also detected at the periphery of the centrosomes in metaphase. XRCC1 accumulated at mitotic centrosome and its immediate vicinity in the cells from prophase to metaphase (<xref ref-type="fig" rid="gki190f5">Figures 5</xref> and and <xref ref-type="fig" rid="gki190f4a">4C</xref>, upper panels). However, the pericentriolar localization of XRCC1 disappeared during anaphase (, upper panels). However, the pericentriolar localization of XRCC1 disappeared during anaphase (<xref ref-type="fig" rid="gki190f4a">Figure 4A</xref>). A similar XRCC1 staining pattern was observed by using paraformaldehyde fixation (). A similar XRCC1 staining pattern was observed by using paraformaldehyde fixation (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>), and by using an XRCC1 monoclonal antibody (), and by using an XRCC1 monoclonal antibody (<xref ref-type="fig" rid="gki190f5">Figure 5</xref>) and in several human fibroblast cell lines including ATM-deficient (AT5BI-VA) cells () and in several human fibroblast cell lines including ATM-deficient (AT5BI-VA) cells (<xref ref-type="fig" rid="gki190f5">Figure 5</xref>). The staining pattern of LIGIIIα detected with two different monoclonal antibodies was indistinguishable from that of XRCC1 (). The staining pattern of LIGIIIα detected with two different monoclonal antibodies was indistinguishable from that of XRCC1 (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>). Thus, we conclude that XRCC1 and LIGIIIα are integral components of centrosomes, and that, during the early stages of mitosis, they also concentrate around the pericentriolar matrix.). Thus, we conclude that XRCC1 and LIGIIIα are integral components of centrosomes, and that, during the early stages of mitosis, they also concentrate around the pericentriolar matrix.', 'To examine whether poly(ADP-ribosyl)ation is involved in the centrosomal localization of XRCC1, the cells were incubated with antibodies for PAR and XRCC1. PAR is reported to be present in the centrosomes in both interphase and mitosis (22). During mitosis, PAR co-localized with XRCC1 in the centrosomes and their immediate vicinity (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>). The change in the amount of PAR in the centrosomes and their immediate vicinity was completely correlated with that of XRCC1 (). The change in the amount of PAR in the centrosomes and their immediate vicinity was completely correlated with that of XRCC1 (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>), suggesting the presence of yet unknown DNA damage-independent mechanisms of PARP activation and the recruitment of XRCC1 to the centrosomal region during mitosis. When cells were incubated for 1 h in the presence of 3-AB (8 mM), a potent inhibitor of PARP, neither PAR nor XRCC1 was observed at or near centrosomes in metaphase cells (), suggesting the presence of yet unknown DNA damage-independent mechanisms of PARP activation and the recruitment of XRCC1 to the centrosomal region during mitosis. When cells were incubated for 1 h in the presence of 3-AB (8 mM), a potent inhibitor of PARP, neither PAR nor XRCC1 was observed at or near centrosomes in metaphase cells (<xref ref-type="fig" rid="gki190f4a">Figure 4A–C</xref>). Similarly, treatment with 3-AB abolished the localization of LIGIIIα to the centrosomes of metaphase cells (). Similarly, treatment with 3-AB abolished the localization of LIGIIIα to the centrosomes of metaphase cells (<xref ref-type="fig" rid="gki190f4a">Figure 4B</xref>). Moreover, in interphase cells, the centrosomal localization of XRCC1 was also prevented by 3-AB (data not shown). Together, these results show that the synthesis of PAR at or near the centrosomes is a prerequisite for the localization of both XRCC1 and LIGIIIα.). Moreover, in interphase cells, the centrosomal localization of XRCC1 was also prevented by 3-AB (data not shown). Together, these results show that the synthesis of PAR at or near the centrosomes is a prerequisite for the localization of both XRCC1 and LIGIIIα.', 'Since PARP-1 is present in both centrosomes and chromosomes in mitotic cells (21), we examined whether during mitosis PARP is activated by DNA-damaging agents and whether this activation affects the localization of XRCC1 and LIGIIIα. Following treatment of metaphase cells with H2O2, PAR was synthesized on the metaphase chromosomes (<xref ref-type="fig" rid="gki190f4a">Figure 4C</xref>). In response to the PAR synthesis, both XRCC1 and LIGIIIα were translocated from the centrosomes to the metaphase chromosomes (). In response to the PAR synthesis, both XRCC1 and LIGIIIα were translocated from the centrosomes to the metaphase chromosomes (<xref ref-type="fig" rid="gki190f4a">Figure 4A–C</xref>). Similar results were obtained by treating the cells with 100 μM of DNA alkylating agent, MMS, for 10 min (data not shown). Thus, it appears that, as in interphase cells (). Similar results were obtained by treating the cells with 100 μM of DNA alkylating agent, MMS, for 10 min (data not shown). Thus, it appears that, as in interphase cells (19), the synthesis of PAR by PARP-1 at DNA strand breaks signals the recruitment of SSBs repair factors in mitotic cells. This suggests that DNA repair of SSBs occurs in the condensed chromatin of metaphase chromosomes.']}
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Translocation of XRCC1 and DNA ligase IIIα from centrosomes to chromosomes in response to DNA damage in mitotic human cells
| null |
Nucleic Acids Res
|
1105689600
|
DNA single-strand breaks (SSBs) are the most frequent lesions caused by oxidative DNA damage. They disrupt DNA replication, give rise to double-strand breaks and lead to cell death and genomic instability. It has been shown that the XRCC1 protein plays a key role in SSBs repair. We have recently shown in living human cells that XRCC1 accumulates at SSBs in a fully poly(ADP-ribose) (PAR) synthesis-dependent manner and that the accumulation of XRCC1 at SSBs is essential for further repair processes. Here, we show that XRCC1 and its partner protein, DNA ligase IIIalpha, localize at the centrosomes and their vicinity in metaphase cells and disappear during anaphase. Although the function of these proteins in centrosomes during metaphase is unknown, this centrosomal localization is PAR-dependent, because neither of the proteins is observed in the centrosomes in the presence of PAR polymerase inhibitors. On treatment of metaphase cells with H2O2, XRCC1 and DNA ligase IIIalpha translocate immediately from the centrosomes to mitotic chromosomes. These results show for the first time that the repair of SSBs is present in the early mitotic chromosomes and that there is a dynamic response of XRCC1 and DNA ligase IIIalpha to SSBs, in which these proteins are recruited from the centrosomes, where metaphase-dependent activation of PAR polymerase occurs, to mitotic chromosomes, by SSBs-dependent activation of PAR polymerase.
|
[
"Cell Line",
"Centrosome",
"Chromosomes, Human",
"DNA Damage",
"DNA Ligase ATP",
"DNA Ligases",
"DNA-Binding Proteins",
"Humans",
"Hydrogen Peroxide",
"Mitosis",
"Poly Adenosine Diphosphate Ribose",
"Poly-ADP-Ribose Binding Proteins",
"Protein Transport",
"X-ray Repair Cross Complementing Protein 1",
"Xenopus Proteins"
] |
other
|
PMC546168
| null |
34
|
[
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] |
Nucleic Acids Res. 2005 Jan 14; 33(1):422-429
|
NO-CC CODE
|
|
Kitchen paper towel treated with an NaCl only solution shows a notable decrease in penetration of nanoparticles. (A and B) NaCl solution-soaked kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for (A) 10 minutes and (B) 2 hours. (C and D) NaCl solution-soaked kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for (C) 10 minutes and (D) 2 hours. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.
|
gr4_lrg
|
2
|
93d03d203b983a8af848e9e98946d2a1f3327d3e9a0804886828d4b86b714592
|
gr4_lrg.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
714,
598
] |
[{'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC7255289/gr2_lrg.jpg', 'caption': 'Materials treated with NaCl\u202f+\u202fTWEEN20 solution show dramatically decreased penetration of nanoparticles. (A-F) NaCl\u202f+\u202fTWEEN20 solution-soaked materials were treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL (A-C) or 1 mg/mL (D-F) for 10 minutes. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.', 'hash': '4cdff73aa09c5869c4442649f1fe88fbc044180daa069984f998bab4c0d3b1f0'}, {'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC7255289/gr6_lrg.jpg', 'caption': 'Schema showing potential application of the pretreated paper towel. Pretreated paper towel can be applied over a homemade mask, surgical mask, or N95 respirator to increase its filtration ability and lifespan for reuse.', 'hash': 'b33ec3a43e6df37d9ca869ab179c1489a1a9949be6cfce758e610e89249e9d29'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC7255289/gr3_lrg.jpg', 'caption': 'Materials treated with NaCl\u202f+\u202fTWEEN20 solution show dramatically decreased penetration of nanoparticles after an extended treatment time. (A-F) NaCl\u202f+\u202fTWEEN20 solution-soaked materials were treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL (A-C) or 1 mg/mL (D-F) for 2 hours. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.', 'hash': '258dabd600892339295d3a027d8315397edac989cd61ec0f2e8ce05336cb2471'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC7255289/gr1_lrg.jpg', 'caption': 'Untreated filters do not adequately protect providers from the penetration of nanoparticles. (A-F) Control (untreated) materials were treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL (A-C) or 1 mg/mL (D-F) for 10 minutes. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.', 'hash': '2a2a6d984cee6c98439cca32ae83f2d6a2d1223932ddc939ae91fe3ccec680ef'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC7255289/gr5_lrg.jpg', 'caption': 'Analysis of microscopy images captured. Using ImageJ, microscopy images were analyzed. Materials treated with NaCl\u202f+\u202fTWEEN20 and NaCl only solution showed a statistically significant reduced penetration of nanoparticles when compared to the control (untreated) middle filter layer of a standard surgical mask. (A) control (untreated), NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for 10 minutes. (B) control (untreated), NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for 10 minutes. (C) NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for 2 hours. Statistical analysis compares these results to the control (untreated) middle filter layer of surgical mask after 10 minute treatment of nanoparticles at a concentration of 10 mg/mL. (D) NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for 2 hours. Statistical analysis compares these results to the control (untreated) middle filter layer of surgical mask after 10 minute treatment of nanoparticles at a concentration of 1 mg/mL. (E) control (no filter), NaCl\u202f+\u202fTween solution-treated materials, NaCl only solution-treated kitchen paper towel, and untreated filter materials were treated with 50 μL E. coli (1.7\u202f×\u202f107 CFU/mL) for 1.5 hours and relative growth density was measured after 8 hours in incubation in LB media. Bars, mean ± SD; *P < .05, **P < .01.', 'hash': 'a944a3155af46fb352bc1bb1626fdb93d101217e06303d53ae7c14710e9aa2c4'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC7255289/gr4_lrg.jpg', 'caption': 'Kitchen paper towel treated with an NaCl only solution shows a notable decrease in penetration of nanoparticles. (A and B) NaCl solution-soaked kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for (A) 10 minutes and (B) 2 hours. (C and D) NaCl solution-soaked kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for (C) 10 minutes and (D) 2 hours. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.', 'hash': '93d03d203b983a8af848e9e98946d2a1f3327d3e9a0804886828d4b86b714592'}]
|
{'gr1_lrg': ['COVID-19 has been reported to be 70-90 nm in diameter.14 To test the effectiveness of standard surgical masks in protecting against the penetration of COVID-19 and nanoparticles of similar size, OMVs harvested from E. coli were used because of their similarity in size (20-200 nm).15\n,\n16 Additionally, OMVs are also known to deliver virulence factors into host cells therefore it is useful to test their ability to penetrate a standard surgical mask.16 We first tested control (untreated) kitchen paper towel, laboratory paper towel, and the middle filter layer of a standard surgical mask. The purpose of using 2 different paper towels was to test effects of using a thick, highly absorbent paper towel (kitchen paper towel) vs a thin, less absorbent paper towel (laboratory paper towel). Given the nationwide shortage of PPE, hospitals have been urging providers to conserve the supply of N95 masks and instead reuse surgical masks for lengthy shifts. Therefore, we believed including a surgical mask was critical to test its ability to filter out nanoparticles of this size. We found all 3 materials did a poor job at filtering out OMVs when untreated (<xref rid="gr1_lrg" ref-type="fig">Fig\xa01</xref>\n) Most worrisome is the large amount of OMVs which penetrated a surgical masks filter when tested with only a 10 minute treatment (\n) Most worrisome is the large amount of OMVs which penetrated a surgical masks filter when tested with only a 10 minute treatment (<xref rid="gr1_lrg" ref-type="fig">Fig\xa01</xref>C and F).C and F).Fig 1Untreated filters do not adequately protect providers from the penetration of nanoparticles. (A-F) Control (untreated) materials were treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL (A-C) or 1 mg/mL (D-F) for 10 minutes. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.Fig 1'], 'gr2_lrg': ['In 2017, Quan et al. reported soaking the filter layer of surgical masks in a solution comprised of NaCl, TWEEN20, and filtered deionized water prevented penetration of various flu viruses while also deactivating it on the surface.13 Given the time-consuming and lack of practicality for this method to be used by providers themselves, we wanted to include household products in our study, specifically paper towels, to offer an alternative that may be more convenient. Both the kitchen and laboratory paper towels, as well as the surgical mask filter, when presoaked in a similar NaCl\u202f+\u202fTWEEN20 solution and left to dry overnight dramatically blocked the penetration of nanoparticles after 10 minutes of treatment (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\nand \nand <xref rid="gr5_lrg" ref-type="fig">5</xref>). The same effects were seen when the presoaked materials were treated for 2 hours (). The same effects were seen when the presoaked materials were treated for 2 hours (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\nand \nand <xref rid="gr5_lrg" ref-type="fig">5</xref>), which shows the durability and longevity of this method of filtration.), which shows the durability and longevity of this method of filtration.Fig 2Materials treated with NaCl\u202f+\u202fTWEEN20 solution show dramatically decreased penetration of nanoparticles. (A-F) NaCl\u202f+\u202fTWEEN20 solution-soaked materials were treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL (A-C) or 1 mg/mL (D-F) for 10 minutes. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.Fig 2Fig 3Materials treated with NaCl\u202f+\u202fTWEEN20 solution show dramatically decreased penetration of nanoparticles after an extended treatment time. (A-F) NaCl\u202f+\u202fTWEEN20 solution-soaked materials were treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL (A-C) or 1 mg/mL (D-F) for 2 hours. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.Fig 3'], 'gr4_lrg': ['Polysorbate 20, commercially mostly known as TWEEN20, is a polyoxyethylene sorbitol ester and nonionic surfactant commonly found in the laboratory setting which is added to buffers and reagents used in immunohistochemistry. Given the unlikeliness that most healthcare providers have TWEEN20 on hand or readily available, we wanted to test the effectiveness of soaking the filter materials in an NaCl-only solution. We found that both the kitchen and laboratory paper towels, as well as the surgical mask filter, were notably effective at reducing the amount of nanoparticle penetration after 10 minute and 2 hour treatments (<xref rid="gr4_lrg" ref-type="fig">Fig 4</xref>, , <xref rid="gr5_lrg" ref-type="fig">Fig 5</xref>\n) Interestingly, the few nanoparticles that penetrated the filter material in this method were relatively larger in size. These results suggest that providers unable to use TWEEN20 can use just NaCl to make a filter themselves, at home, that will reduce the penetration of nanoparticles. TWEEN20 most likely plays a role in evenly distributing the salt across the entire filter, preventing any gaps in the NaCl lattice that serves to filter out particles. Although it is probable that a filter made with solution of NaCl and TWEEN20 is more effective, an NaCl only solution will still offer providers more protection than a normal surgical mask would.\n) Interestingly, the few nanoparticles that penetrated the filter material in this method were relatively larger in size. These results suggest that providers unable to use TWEEN20 can use just NaCl to make a filter themselves, at home, that will reduce the penetration of nanoparticles. TWEEN20 most likely plays a role in evenly distributing the salt across the entire filter, preventing any gaps in the NaCl lattice that serves to filter out particles. Although it is probable that a filter made with solution of NaCl and TWEEN20 is more effective, an NaCl only solution will still offer providers more protection than a normal surgical mask would.Fig 4Kitchen paper towel treated with an NaCl only solution shows a notable decrease in penetration of nanoparticles. (A and B) NaCl solution-soaked kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for (A) 10 minutes and (B) 2 hours. (C and D) NaCl solution-soaked kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for (C) 10 minutes and (D) 2 hours. An image was captured in the microscope field containing the highest penetration of nanoparticles, and each sample was tested 3 times. The image included in the figure represents the closest level of average penetration determined by immunofluorescence microscopy.Fig 4Fig 5Analysis of microscopy images captured. Using ImageJ, microscopy images were analyzed. Materials treated with NaCl\u202f+\u202fTWEEN20 and NaCl only solution showed a statistically significant reduced penetration of nanoparticles when compared to the control (untreated) middle filter layer of a standard surgical mask. (A) control (untreated), NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for 10 minutes. (B) control (untreated), NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for 10 minutes. (C) NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 10 mg/mL for 2 hours. Statistical analysis compares these results to the control (untreated) middle filter layer of surgical mask after 10 minute treatment of nanoparticles at a concentration of 10 mg/mL. (D) NaCl\u202f+\u202fTween solution-treated materials, and NaCl only solution-treated kitchen paper towel was treated with 20 μL of labeled nanoparticles at a concentration of 1 mg/mL for 2 hours. Statistical analysis compares these results to the control (untreated) middle filter layer of surgical mask after 10 minute treatment of nanoparticles at a concentration of 1 mg/mL. (E) control (no filter), NaCl\u202f+\u202fTween solution-treated materials, NaCl only solution-treated kitchen paper towel, and untreated filter materials were treated with 50 μL E. coli (1.7\u202f×\u202f107 CFU/mL) for 1.5 hours and relative growth density was measured after 8 hours in incubation in LB media. Bars, mean ± SD; *P < .05, **P < .01.Fig 5'], 'gr5_lrg': ['In addition to filtering out nanoparticles in the size range of viruses, it\'s critical for an effective mask to also prevent penetration of bacteria, which are larger in size. We found that after treating each presoaked filter material with a drop of E. coli for 1.5 hours there was an observed decrease in bacterial growth on the filter paper beneath it (<xref rid="gr5_lrg" ref-type="fig">Fig\xa05</xref>E). This was observed after presoaking the filter materials in both the NaCl\u202f+\u202fTWEEN20 solution or the NaCl-only solution. These results suggest presoaking the filter materials in either solution effectively prevents penetration of larger bacteria as well.E). This was observed after presoaking the filter materials in both the NaCl\u202f+\u202fTWEEN20 solution or the NaCl-only solution. These results suggest presoaking the filter materials in either solution effectively prevents penetration of larger bacteria as well.'], 'gr6_lrg': ['The purpose of our study is not to replace surgical or N95 masks. Our goal was to increase the protection of a surgical mask, add an additional layer outside of surgical and N95 masks to increase its lifespan, and give members of the community a convenient and economic option to increase the effectiveness of their home-made masks so surgical masks can be rationed for medical workers. These potential applications are illustrated in <xref rid="gr6_lrg" ref-type="fig">Figure 6</xref>\n.\n.Fig 6Schema showing potential application of the pretreated paper towel. Pretreated paper towel can be applied over a homemade mask, surgical mask, or N95 respirator to increase its filtration ability and lifespan for reuse.Fig 6']}
|
Pretreated household materials carry similar filtration protection against pathogens when compared with surgical masks
|
[
"Medical disposable face mask",
"Reused masks",
"Homemade mask",
"OMV",
"COVID-19"
] |
Am J Infect Control
|
1598338800
|
In response to Voitsidis et al. (2020) published in Psychiatry Research addressing the paucity of research on insomnia during a pandemic, we obtained data from an online cross-sectional survey by documenting the prevalence of clinical insomnia and its contributing factors in a French general public sample. Participants (N = 556) completed the Insomnia Severity Index, UCLA Loneliness scale, and provided information on sociodemographics, antecedents of mental and physical health conditions, and COVID-19-related stressful life events. In our sample, 19.1% met the diagnostic criteria of clinical insomnia, which was twice lower than that reported in the study by Voitsidis et al., but close to those found among Chinese and Italian populations. We confirmed COVID-19-related worries and loneliness to be the major contributing factors to clinical insomnia, in addition to education status, being infected by the virus and pre-existing mental health illness. These findings underscore that sleep-related problems should be an important component of mental health interventions during pandemics.
|
[
"Betacoronavirus",
"COVID-19",
"Coronavirus Infections",
"Cross-Sectional Studies",
"Greece",
"Humans",
"Italy",
"Pandemics",
"Pneumonia, Viral",
"Prevalence",
"Risk Factors",
"SARS-CoV-2",
"Sleep Initiation and Maintenance Disorders"
] |
other
|
PMC7255289
| null |
10
|
[
"{'Citation': 'Brooks S.K., Webster R.K., Smith L.E., Woodland L., Wessely S., Greenberg N., Rubin G.J. The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020;395(10227):912–920.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7158942'}, {'@IdType': 'pubmed', '#text': '32112714'}]}}",
"{'Citation': 'Cénat J.M., Felix N., Blais-Rochette C., Rousseau C., Bukaka J., Derivois D. . Prevalence of mental health problems in populations affected by the Ebola virus disease: a systematic review and meta-analysis. Psychiatry Res. 2020;289 doi: 10.1016/j.psychres.2020.113033.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.psychres.2020.113033'}, {'@IdType': 'pubmed', '#text': '32388176'}]}}",
"{'Citation': 'Griffin S.C., Williams A.B., Mladen S.N., Perrin P.B., Dzierzewski J.M., Rybarczyk B.D. Reciprocal effects between loneliness and sleep disturbance in older Americans. J. Aging Health. 2019 doi: 10.1177/0898264319894486.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1177/0898264319894486'}, {'@IdType': 'pmc', '#text': 'PMC7309370'}, {'@IdType': 'pubmed', '#text': '31868077'}]}}",
"{'Citation': 'Huang Y., Zhao N. Generalized anxiety disorder, depressive symptoms and sleep quality during COVID-19 outbreak in China: a web-based cross-sectional survey. Psychiatry Res. 2020 doi: 10.1016/j.psychres.2020.112954.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.psychres.2020.112954'}, {'@IdType': 'pmc', '#text': 'PMC7152913'}, {'@IdType': 'pubmed', '#text': '32325383'}]}}",
"{'Citation': 'Kalmbach D.A., Cuamatzi-Castelan A.S., Tonnu C.V., Tran K.M., Anderson J.R., Roth T., 2, Drake C.L. Hyperarousal and sleep reactivity in insomnia: current insights. Nat. Sci. Sleep. 2018;10:193–201. doi: 10.2147/NSS.S138823.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.2147/NSS.S138823'}, {'@IdType': 'pmc', '#text': 'PMC6054324'}, {'@IdType': 'pubmed', '#text': '30046255'}]}}",
"{'Citation': 'Kay-Stacey M., Attarian H. Advances in the management of chronic insomnia. BMJ. 2016;354:1–14. doi: 10.1136/bmj.i2123.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1136/bmj.i2123'}, {'@IdType': 'pubmed', '#text': '27383400'}]}}",
"{'Citation': 'Morin C.M. Guilford Press; New York: 1993. Insomnia: Psychological Assessment and Management.'}",
"{'Citation': 'Rossi, R., Socci, V., Talevi, D., Mensi, S., Niolu, C., Pacitti, F., et\\xa0al., 2020. COVID-19 pandemic and lockdown measures impact on mental health among the general population in Italy. An N=18147 web-based survey. medRxiv preprint doi:10.1101/2020.04.09.20057802.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1101/2020.04.09.20057802'}, {'@IdType': 'pmc', '#text': 'PMC7426501'}, {'@IdType': 'pubmed', '#text': '32848952'}]}}",
"{'Citation': 'Russell D.W. UCLA Loneliness Scale (Version 3): reliability, Validity, and Factor Structure. J. Pers. Assess. 1996;66(1):20–40.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '8576833'}}}",
"{'Citation': 'Voitsidis P., Gliatas I., Bairachtari V., Papadopoulou K., Papageorgiou G., Parlapani E. Insomnia during the COVID-19 pandemic in a Greek population. Psychiatry Res. 2020;289', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7217074'}, {'@IdType': 'pubmed', '#text': '32434093'}]}}"
] |
Am J Infect Control. 2020 Aug 25; 48(8):883-889
|
NO-CC CODE
|
|
Testing serial diluted SARS-CoV-2 RNA Reference Sample by rapid digital RT-PCR. (a) & (b) & (c) In the right column, fluorescence images of droplets in the reaction chamber after PCR. Concentration of templates are no template, 10, 50, 100, 1000, 5000 copies/μL for (ORF1ab & N gene in (a) & (b). And the dilution factor of 10−3, 5 × 10−3, 10−2, 10−1, 5 × 10−1 is used to mark the concentration of the reference gene in each sample (c). No positive droplets are observed in negative control samples. Correlation between detected target concentration and actual concentration calculated by the dilution factor. In the left column, R2>0.99 in (a) & (b) and R2 = 0.9996 in (c). Error bars represent the standard deviation based on at least 3 replicates of each experiment. Scale bars are 100 μm.
|
gr3_lrg
|
2
|
af37c7a83428db97fc18884ba12ad18438291860151dc9e740543bbf2d48cc36
|
gr3_lrg.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
694,
655
] |
[{'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC8093165/gr3_lrg.jpg', 'caption': 'Testing serial diluted SARS-CoV-2 RNA Reference Sample by rapid digital RT-PCR. (a) & (b) & (c) In the right column, fluorescence images of droplets in the reaction chamber after PCR. Concentration of templates are no template, 10, 50, 100, 1000, 5000 copies/μL for (ORF1ab & N gene in (a) & (b). And the dilution factor of 10−3, 5\xa0×\xa010−3, 10−2, 10−1, 5\xa0×\xa010−1 is used to mark the concentration of the reference gene in each sample (c). No positive droplets are observed in negative control samples. Correlation between detected target concentration and actual concentration calculated by the dilution factor. In the left column, R2>0.99 in (a) & (b) and R2\xa0=\xa00.9996 in (c). Error bars represent the standard deviation based on at least 3 replicates of each experiment. Scale bars are 100\xa0μm.', 'hash': 'af37c7a83428db97fc18884ba12ad18438291860151dc9e740543bbf2d48cc36'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC8093165/gr2_lrg.jpg', 'caption': 'Optimization of process parameters for rapid digital RT-PCR. (a) Fluorescent signals in microfluidic chip are obtained using three fluorescence excitation channels: excitation wavelengths of 485\xa0nm (FAM), 535\xa0nm (HEX), 640\xa0nm (Cy5) for ORF1ab, N gene, RNase P, respectively. Positive signals of ORF1ab are missing when performing rapid digital RT-PCR for 30 cycles with a cycle time of 3s (b) and 25 cycles with a cycle time of 5s (c). (d) Positive signals of all targets are detected after 28 cycles with a cycle time of 2.8s. All experiments are performed at least 3 times. Scale bars, 100\xa0μm (red) and 250\xa0μm (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)', 'hash': '0749265c02304f959ff9b158c1820c8e41b256861db8df2c234939ecd3f35baf'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC8093165/gr4_lrg.jpg', 'caption': 'Comparing results of detecting SARS-CoV-2 RNA Reference Sample using RT-qPCR and rapid digital RT-PCR. (a) Amplification curves and Ct values from RT-qPCR for serial diluted reference samples with the concentration of targeted N gene at 5, 10, 20, 100, 500, 1000, 5000 copies/test. (b) & (c) Ten detection amplification curves and Ct values for low-copy concentration samples with concentration of (b) 10 copies/test (target: N) and (c) 5 copies/test (target: ORF1ab) by RT-qPCR. (d) Ct values from 10 replicated testing of low-concentration reference samples using RT-qPCR. By definition, Ct scales inversely with the log-scale concentration of the target gene in the sample. (e) Accuracy of testing the low-concentration reference samples by rapid digital RT-PCR (10 replicated tests). To enable direct comparison with the result by RT-qPCR, the detected concentration in plotted in the log scale. Error bars represent the standard deviation based on 10 replicated testing.', 'hash': 'ac9eeca607f2052fde1c1d167f8486053c640e5be01f7d91197fade8c16ad014'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC8093165/gr1_lrg.jpg', 'caption': 'Principles and characteristics of the rapid digital PCR system. (a) Workflow of rapid digital PCR method consists of four key steps, sample preparation, reaction mixture partition, amplification process, and targets quantification. (b) Hydrolysis of different TaqMan probes generates distinct positive fluorescence signals. (c) Average diameter of droplets in each storage chamber and error bars show the standard deviation droplet size distribution. (d) Thermal profile of the heater (blue line) and PCR sample (red dotted line) during an 8-s thermal cycle of 2\xa0s at 95\xa0°C and 6\xa0s at 60\xa0°C. The semi-transparent red zone represents the effective temperature range for DNA denaturation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)', 'hash': 'd1e0e7deefc9ea2e0568bc73b7eba93b2cc91cb81b192fb1261c8ce4878734cb'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC8093165/gr5_lrg.jpg', 'caption': 'Quantitative analysis of ORF1ab, N gene, and RNase P in clinical samples. (a) Detected copy number of three target sequences in 9 nucleic acid samples by rapid digital RT-PCR. (b) Ct values from RT-qPCR are plotted against the target copy number acquired by rapid digital PCR. (c) Fluorescence images showing the test results of nucleic acid samples from rapid digital RT-PCR. Top, 9 clinic samples. Bottom, negative (RNAse-free water, Sangon Biotech) and positive (SARS-CoV-2 genomic RNA reference, National Institute of Metrology, China) control samples. Scale bars, 100\xa0μm.', 'hash': 'f72f0c5479f22618b6491d05020b31331d81a6be7c1276949299ec4a468c9484'}]
|
{'gr1_lrg': ['When the system reaches the thermal equilibrium, the temperature of PCR mixtures inside droplets should be synchronized with that measured by temperature sensors. However, the thin glass separating the sensor and droplet storage chamber causes the delay of temperature change in PCR mixtures during the thermal cycling. To investigate the effect of this delay on the denaturation of PCR, a thin gold film temperature sensor is embedded in the droplet storage chamber to record temperature in the mineral oil filled chamber during the thermal cycling. Owing to the big surface-to-volume ratio of the chambers and the small volumes of PCR mix, the temperatures of the sensor can be seen as identical to that of the PCR mix. The temperature measured by the gold sensor in the storage chamber closely followed that from the platinum sensors next to the heater array with a dynamic offset of less than 0.5\xa0s. In a 2-s denaturing step programmed by the heater control, the duration of the effective denaturation temperature (between 90\xa0°C and 95\xa0°C) is measured for approximately 1.2\xa0s in the storage chamber (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>\nd), which is sufficient for the denaturation of double DNA strands (\nd), which is sufficient for the denaturation of double DNA strands (Wittwer and Garling, 1991). Therefore, the delay in temperature change due to the thin glass slide would not have dramatic effect on the rapid PCR process.Fig. 1Principles and characteristics of the rapid digital PCR system. (a) Workflow of rapid digital PCR method consists of four key steps, sample preparation, reaction mixture partition, amplification process, and targets quantification. (b) Hydrolysis of different TaqMan probes generates distinct positive fluorescence signals. (c) Average diameter of droplets in each storage chamber and error bars show the standard deviation droplet size distribution. (d) Thermal profile of the heater (blue line) and PCR sample (red dotted line) during an 8-s thermal cycle of 2\xa0s at 95\xa0°C and 6\xa0s at 60\xa0°C. The semi-transparent red zone represents the effective temperature range for DNA denaturation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 1', 'Our rapid ddPCR system (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>a) involves two key components: a microfluidic chip to handle droplets for digital PCR and a micro-heater array for rapid PCR. Starting from the droplet generation in the microfluidic chip, the PCR reaction mixture involving extracted nucleic acid samples and PCR reagents is rapidly dispersed into more than 20,000 droplets with mineral oil as the external phase. Droplets are generated using a single PCR sample inlet and oil inlet feeding into T-conjunctions on both sides of the chip (a) involves two key components: a microfluidic chip to handle droplets for digital PCR and a micro-heater array for rapid PCR. Starting from the droplet generation in the microfluidic chip, the PCR reaction mixture involving extracted nucleic acid samples and PCR reagents is rapidly dispersed into more than 20,000 droplets with mineral oil as the external phase. Droplets are generated using a single PCR sample inlet and oil inlet feeding into T-conjunctions on both sides of the chip (Thorsen et al., 2001) (Supplementary Figure. 1a). Parent droplets are formed and then passed through the splitter (Chaudhury et al., 2014), where the parent droplet is split equally to into 16 daughter droplets. The structure of the droplet splitter ensures a similar number of droplets entering each chamber and increases the droplet-generation rate.', 'The storage compartment in the microfluidic device involves eight independent, small chambers to enhance the efficiency of thermal cycling. Each chamber is 2\xa0mm\xa0×\xa02\xa0mm with curved corners to prevent air bubbles during oil filling. In addition, narrow and long channels between droplet splitters provide large flow resistance to prevent droplet flow back. Therefore, droplets enter the storage chamber and remain inside stably. After filling eight chambers with more than 20,000 droplets in less than 2\xa0min, the average diameter of droplets in the chamber is measured to be 35.7\xa0μm with the coefficient of variation (CV) smaller than 3% within and among chambers, respectively (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>c).c).', 'Only droplets with targets sequences undergo PCR cycles, where specific fluorescent probes are cut off by the DNA polymerase and emit fluorescence that increase with PCR cycles. A charge-coupled device (CCD) is used to analyze the end-point fluorescent signal of PCR, where the number fraction of fluorescent droplets is determined and used to calculate the copy number of target sequences via the Poisson distribution formula (Whale et al., 2012). By involving different fluorescent probes targeting different gene sequences, our rapid digital PCR system allows simultaneous detection of multiple targets with a single PCR test (schematic diagram shown in <xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>b).b).'], 'gr2_lrg': ['Based on our experience, the starting point for optimizing the denaturation time at 95\xa0°C and annealing time at 60\xa0°C is 3s and 5s, respectively. And the total number of cycles is selected as 40. By fixing the total cycle number and the annealing time (5s), we gradually reduce the denaturation time to 1s and find this is sufficient for detecting clear positive droplets. Similarly, using the total cycle number (40) and updated denaturation time (1s), we then find 3-s annealing time can yield good results (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\na). This completes optimized parameters of PCR as: 95\xa0°C/1\xa0s for denaturation, 60\xa0°C/3\xa0s for annealing, 72\xa0°C/1\xa0s for extension, and the total number of cycles 40.\na). This completes optimized parameters of PCR as: 95\xa0°C/1\xa0s for denaturation, 60\xa0°C/3\xa0s for annealing, 72\xa0°C/1\xa0s for extension, and the total number of cycles 40.Fig. 2Optimization of process parameters for rapid digital RT-PCR. (a) Fluorescent signals in microfluidic chip are obtained using three fluorescence excitation channels: excitation wavelengths of 485\xa0nm (FAM), 535\xa0nm (HEX), 640\xa0nm (Cy5) for ORF1ab, N gene, RNase P, respectively. Positive signals of ORF1ab are missing when performing rapid digital RT-PCR for 30 cycles with a cycle time of 3s (b) and 25 cycles with a cycle time of 5s (c). (d) Positive signals of all targets are detected after 28 cycles with a cycle time of 2.8s. All experiments are performed at least 3 times. Scale bars, 100\xa0μm (red) and 250\xa0μm (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 2', 'Because the COVID-19 diagnosis yields essentially a “positive” or “negative” result, we postulate that the detection time for positive signals could be reduced by providing sufficient PCR reagents in droplets. The duration of a single PCR cycle and the total number of cycles are further reduced to realize extreme fast diagnosis of COVID-19 within 2\xa0min. While fixing total PCR cycles and the duration for denaturing and extension, we find that the annealing time can be further reduced to 2\xa0s. In addition, positive droplets of ORF1ab only appear clearly after at least 25 PCR cycles (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>b and b and <xref rid="gr2_lrg" ref-type="fig">c</xref>). Moreover, the denaturing time is reduced from 1\xa0s to 0.8\xa0s without losing the detection capability. Finally, the total duration of reverse transcription (43\xa0°C/4s) and pre-denaturation (95\xa0°C/4s) is reduced to 8\xa0s that can still yield reliable end-point detection results. Altogether, our rapid droplet digital PCR system can return “positive” or “negative” diagnosis of COVID-19 within 2\xa0min, involving 28 PCR cycles and each cycle includes 95\xa0°C/0.8\xa0s for denaturation, 60\xa0°C/2\xa0s for annealing, 72\xa0°C/1\xa0s for extension (). Moreover, the denaturing time is reduced from 1\xa0s to 0.8\xa0s without losing the detection capability. Finally, the total duration of reverse transcription (43\xa0°C/4s) and pre-denaturation (95\xa0°C/4s) is reduced to 8\xa0s that can still yield reliable end-point detection results. Altogether, our rapid droplet digital PCR system can return “positive” or “negative” diagnosis of COVID-19 within 2\xa0min, involving 28 PCR cycles and each cycle includes 95\xa0°C/0.8\xa0s for denaturation, 60\xa0°C/2\xa0s for annealing, 72\xa0°C/1\xa0s for extension (Supplementary Figure. 5). This analysis is faster than most present systems (Udugama et al., 2020).'], 'gr3_lrg': ['Positive droplets under separate fluorescence channels distribute uniformly in each chamber, indicating similar PCR efficiency for both ORF1ab and N gene sequences through all droplets. More importantly, the concentration of these two target sequences predicted by rapid ddRT-PCR agrees well with that calculated from the dilution factor (R2\xa0>\xa00.99 for the Pearson correlation, <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\na and \na and <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>b). For the reference gene RNase P, because its absolute concentration in the sample not known, we plot the detected copy number against the dilution factor used in preparing the specific sample, which also indicates good linear correlation (Rb). For the reference gene RNase P, because its absolute concentration in the sample not known, we plot the detected copy number against the dilution factor used in preparing the specific sample, which also indicates good linear correlation (R2\xa0=\xa00.9996, <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>c).c).Fig. 3Testing serial diluted SARS-CoV-2 RNA Reference Sample by rapid digital RT-PCR. (a) & (b) & (c) In the right column, fluorescence images of droplets in the reaction chamber after PCR. Concentration of templates are no template, 10, 50, 100, 1000, 5000 copies/μL for (ORF1ab & N gene in (a) & (b). And the dilution factor of 10−3, 5\xa0×\xa010−3, 10−2, 10−1, 5\xa0×\xa010−1 is used to mark the concentration of the reference gene in each sample (c). No positive droplets are observed in negative control samples. Correlation between detected target concentration and actual concentration calculated by the dilution factor. In the left column, R2>0.99 in (a) & (b) and R2\xa0=\xa00.9996 in (c). Error bars represent the standard deviation based on at least 3 replicates of each experiment. Scale bars are 100\xa0μm.Fig. 3'], 'gr4_lrg': ['The same serial diluted reference samples used for benchmarking rapid ddPCR system are tested with RT-qPCR. The sample concentration is indicted by the Ct value (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\na). RT-qPCR yields accurate results for negative samples and diluted reference solutions with high viral load (>10 copies/test), where Ct linearly correlates with the concentration (copies/test) in the log scale (R\na). RT-qPCR yields accurate results for negative samples and diluted reference solutions with high viral load (>10 copies/test), where Ct linearly correlates with the concentration (copies/test) in the log scale (R2\xa0>\xa00.99, Supplementary Figure. 7).Fig. 4Comparing results of detecting SARS-CoV-2 RNA Reference Sample using RT-qPCR and rapid digital RT-PCR. (a) Amplification curves and Ct values from RT-qPCR for serial diluted reference samples with the concentration of targeted N gene at 5, 10, 20, 100, 500, 1000, 5000 copies/test. (b) & (c) Ten detection amplification curves and Ct values for low-copy concentration samples with concentration of (b) 10 copies/test (target: N) and (c) 5 copies/test (target: ORF1ab) by RT-qPCR. (d) Ct values from 10 replicated testing of low-concentration reference samples using RT-qPCR. By definition, Ct scales inversely with the log-scale concentration of the target gene in the sample. (e) Accuracy of testing the low-concentration reference samples by rapid digital RT-PCR (10 replicated tests). To enable direct comparison with the result by RT-qPCR, the detected concentration in plotted in the log scale. Error bars represent the standard deviation based on 10 replicated testing.Fig. 4', 'We further compare the performance of RT-qPCR and rapid ddRT-PCR in detecting low-viral-load samples. 10 repeated tests of these two methods are performed separately for diluted reference samples, where the concentration of target sequences is 10 copies/test and 5 copies/test (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>b and b and <xref rid="gr4_lrg" ref-type="fig">c</xref>). While both methods yield positive signals for tested low-viral-load samples, detection results from RT-qPCR (Ct values) have more significant variations that are possibly due to inconsistent amplification efficiency. Such variations often lead to false-negative results that are undesired in the diagnosis of COVID-19 (). While both methods yield positive signals for tested low-viral-load samples, detection results from RT-qPCR (Ct values) have more significant variations that are possibly due to inconsistent amplification efficiency. Such variations often lead to false-negative results that are undesired in the diagnosis of COVID-19 (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>d).d).', 'By contrast, rapid digital RT-PCR results correctly predict 3.8 copies/test and 9.4 copies/test for two low-viral-load samples, respectively (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>e). These values are slightly smaller than actual ones (calculated by the dilution factor), which could be caused by the degradation of the RNA templates during serial dilution (e). These values are slightly smaller than actual ones (calculated by the dilution factor), which could be caused by the degradation of the RNA templates during serial dilution (Fleige et al., 2006). And the relative uncertainly of digital PCR would increase as the random distribution of the template molecules number during serial dilution. This could be another reason for the deviation from the calculated concentration (Bhat et al., 2009; Zhu et al., 2014). However, this is unlikely to occur in clinical diagnosis that usually does not involve sample dilution. These results demonstrate that quantitative and un-biased PCR amplification in the rapid ddPCR system enable accurate detection of low-viral-load samples.'], 'gr5_lrg': ['After benchmarking the performance of rapid ddRT-PCR with reference samples, we apply this system to test clinical nucleic acid samples collected for the diagnosis of COVID-19, which involve six positive and three negative cases. The rapid ddPCR system simultaneously targets three specific sequences, ORF1ab, N and RNase P gene, and detect their copy numbers in each sample (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>\na and \na and <xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>b, calculated concentration is listed in b, calculated concentration is listed in Supplementary Table 2). Meanwhile, RT-qPCR is performed for the same clinical samples in the LightCycler 480 (Roche, Switzerland) using an approved RT-qPCR kit (Sansure Biotech, China).Fig. 5Quantitative analysis of ORF1ab, N gene, and RNase P in clinical samples. (a) Detected copy number of three target sequences in 9 nucleic acid samples by rapid digital RT-PCR. (b) Ct values from RT-qPCR are plotted against the target copy number acquired by rapid digital PCR. (c) Fluorescence images showing the test results of nucleic acid samples from rapid digital RT-PCR. Top, 9 clinic samples. Bottom, negative (RNAse-free water, Sangon Biotech) and positive (SARS-CoV-2 genomic RNA reference, National Institute of Metrology, China) control samples. Scale bars, 100\xa0μm.Fig. 5', 'Noticeably, rapid ddRT-PCR correctly diagnose all positive and negative cases of COVID-19 samples. In addition to qualitative results (positive or negative), rapid ddRT-PCR also provide the copy number of target sequences in each sample, which correlate linearly with the corresponding Ct value from RT-qPCR (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>b). Furthermore, rapid ddRT-PCR yields these accurate results within 10\xa0min, whereas the standard RT-qPCR analysis takes at least 2\xa0h.b). Furthermore, rapid ddRT-PCR yields these accurate results within 10\xa0min, whereas the standard RT-qPCR analysis takes at least 2\xa0h.']}
|
Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system
|
[
"Nucleic testing",
"Rapid digital PCR system",
"SARS-CoV-2 detection",
"Microfluidic technology"
] |
Biosens Bioelectron
|
1631689200
|
We report the first combination of droplet digital and rapid PCR techniques for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. The presented rapid digital PCR system simultaneously detects two specific targets (ORF1ab and N genes) and one reference gene (RNase P) with a single PCR thermal cycling period around 7 s and the total running time less than 5 min. A clear positive signal could be identified within 115 s via the rapid digital RT-PCR, suggesting its efficiency for the end-point detection. In addition, benchmark tests with serial diluted reference samples of SARS-CoV-2 RNA reveal the excellent accuracy of our system (R>0.99). More importantly, the rapid digital PCR system gives consistent and accurate detection of low-concentration reference samples, whereas qPCR yields Ct values with significant variations that could lead to false-negative results. Finally, we apply the rapid digital PCR system to analyze clinical samples with both positive and control cases, where results are consistent with qPCR test outcomes. By providing similar accuracy with qPCR while minimizing the detection time-consuming and the false-negative tendency, the presented rapid digital PCR system represents a promising improvement on the rapid diagnosis of COVID-19.
|
[
"Biosensing Techniques",
"COVID-19",
"COVID-19 Nucleic Acid Testing",
"Humans",
"RNA, Viral",
"SARS-CoV-2",
"Sensitivity and Specificity"
] |
other
|
PMC8093165
| null |
31
|
[
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] |
Biosens Bioelectron. 2021 Sep 15; 188:113282
|
NO-CC CODE
|
|
FACS analysis using E17 FVB/N-Tg (GFAPGFP)14Mes/J transgenic micea, Cortical neurons were isolated from E17 FVB/N-Tg
(GFAPGFP)14Mes/J transgenic mice. Immunocytochemistry showed that cultured
neurons did not express either GFP or GFAP protein following oxygen-glucose
deprivation, suggesting that stroke-like stress may not likely lead to
“leakiness” in this astrocyte-specific GFP mouse.
b, Brain cell suspension was prepared from FVB/N-Tg
(GFAPGFP)14Mes/J mice subjected to transient ischemia, then FACS analysis
was performed. c, Representative image before cell sorting.
d, Purity after cell sorting. e, Either
MAP2+/GFP- or MAP2+/GFP+ population was positive for
DAPI as 92.5% or 85.9%, respectively. f,
Western blot analysis demonstrated that both GFP-positive and negative
neurons expressed mature neuron marker (neurofilament) but not neuronal stem
cell marker (nestin). These data exclude the possibility that GFAP-positive
cells included subsets of neuronal precursor cells that are known to also
express GFAP.
|
nihms795847f11
|
2
|
dbda3ab4dd8eb5358cadf480d18ff86f194c7d83c71beb2f677e2aaa8775fb3d
|
nihms795847f11.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
764,
442
] |
[{'image_id': 'nihms795847f11', 'image_file_name': 'nihms795847f11.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f11.jpg', 'caption': 'FACS analysis using E17 FVB/N-Tg (GFAPGFP)14Mes/J transgenic micea, Cortical neurons were isolated from E17 FVB/N-Tg\n(GFAPGFP)14Mes/J transgenic mice. Immunocytochemistry showed that cultured\nneurons did not express either GFP or GFAP protein following oxygen-glucose\ndeprivation, suggesting that stroke-like stress may not likely lead to\n“leakiness” in this astrocyte-specific GFP mouse.\nb, Brain cell suspension was prepared from FVB/N-Tg\n(GFAPGFP)14Mes/J mice subjected to transient ischemia, then FACS analysis\nwas performed. c, Representative image before cell sorting.\nd, Purity after cell sorting. e, Either\nMAP2+/GFP- or MAP2+/GFP+ population was positive for\nDAPI as 92.5% or 85.9%, respectively. f,\nWestern blot analysis demonstrated that both GFP-positive and negative\nneurons expressed mature neuron marker (neurofilament) but not neuronal stem\ncell marker (nestin). These data exclude the possibility that GFAP-positive\ncells included subsets of neuronal precursor cells that are known to also\nexpress GFAP.', 'hash': 'dbda3ab4dd8eb5358cadf480d18ff86f194c7d83c71beb2f677e2aaa8775fb3d'}, {'image_id': 'nihms795847f9', 'image_file_name': 'nihms795847f9.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f9.jpg', 'caption': 'Role of astrocytic CD38 in mitochondria transfer during starvation in\nvitroa, Immunocytochemistry in neuron-astrocyte co-cultures\ndemonstrated that CD38 was primarily expressed within astrocytes.\nb, Extracellular ATP levels were higher in media collected\nfrom neurons co-cultured with astrocytes compared to neuron-alone cultures\nalone (n=9 or 11). c, After serum/glucose starvation,\nneurons were significantly damaged, as expected. But neurons co-cultured\nwith astrocytes were protected (n=6 or 4). d, CD38\nsuppression with siRNA significantly decreased extracellular ATP levels in\nneuron-astrocyte co-culture, but CD38 suppression did not affect\nextracellular ATP level in neuron-alone cultures (n=9 or 6).\ne, Blockade of astrocytic CD38 with siRNA significantly\nincreased LDH release (indicative of cell damage) in the co-culture,\nsuggesting that CD38 may be important to maintain neuroglial homeostasis\n(n=6). f, Rat primary neurons were co-cultured with rat\nastrocytes. Immunocytochemistry showed that CD38 suppression with siRNA\nreduced astrocytic mitochondria (red) transfer into neurons compared to\ncontrol. g, h, Western blot analysis indicated that CD38\nsuppression with siRNA can be successfully performed in astrocyte culture\nwithout affecting cell viability (n=4 or 3). All values are mean\n+/− SEM.', 'hash': '2367d0605bb1542020caef44c598f271ec5f4b7bba4df62507a474d8cbac9257'}, {'image_id': 'nihms795847f7', 'image_file_name': 'nihms795847f7.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f7.jpg', 'caption': 'Production of astrocytic mitochondria particle in a\nCa2+-dependent mechanisma, The known CD38 downstream signal, cADPR increased\nintracellular calcium shown in Fluo-4 intensity in a concentration-dependent\nmanner (n=3). b, Intracellular ATP in astrocytes was\nupregulated by cADPR stimulation (n=4). **P<0.01\nvs cADPR 0 μM. c, To measure ATP levels in\nextracellular particles, astrocyte-conditioned media were collected and\nlarge debris were excluded by centrifugation and filtration using 1.2\nμm filter. Following another centrifugation at 20,000g for 30 min,\neach 100 μl from top or bottom fractions were used for ATP assay.\nd, The bottom fraction had higher ATP content, and cADPR (1\nμM) increased ATP content in this bottom fraction (n= 6 or\n8). e, cADPR-induced extracellular ATP levels within\nextracellular particles was diminished by intracellular calcium blocker,\nBAPTA-AM (n= 4 or 6). All values are mean +/−\nSEM.', 'hash': 'eb75cac8d41d77339e338f0b3526100b6b45d32956b127b8b97870ec1a822ce2'}, {'image_id': 'nihms795847f1', 'image_file_name': 'nihms795847f1.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f1.jpg', 'caption': 'Astrocytic CD38 and extracellular mitochondriaa, Transmission electron microscopy (TEM) of extracellular\nmitochondria in astrocyte-conditioned medium (ACM). Scale: 500 nm.\nb, Rat cortical astrocytes were labeled by Mitotracker Red\nCMXRos. FACS showed that 0.2 μm filter depleted extracellular\nmitochondria in ACM (mdACM). c–e, 0.2 μm filters\nreduced markers of extracellular mitochondrial function in ACM - c,\nextracellular ATP (n=4), d, membrane potential\n(n=4), e, oxygen consumption (n= 9 or 6).\nf, Western blot confirmed higher CD38 in rat cortical\nastrocytes compared to neurons. g, High and low levels of CD38\ncyclase activity in astrocytes and neurons respectively (n= 8 or 5).\nh,. Experimental schematic for testing CRISPR/Cas9-mediated\nCD38 activation. i, Twenty four hours after transfection, CD38\ncyclase activity was upregulated by CD38 activation plasmid (n=4).\nj, k, Extracellular ATP production (j) and oxygen\nconsumption (k) were significantly increased by CD38 activation\n(n=5). l, FACS showed that extracellular mitochondria were\nincreased by cADPR (1 μM) stimulation in astrocytes (n=3).\nm, cADPR (1 μM) increased extracellular mitochondria\nmembrane potential at 24 hours (n=7). n, Oxygen consumption\nin extracellular mitochondria was increased by cADPR (n=4).\no, cADPR did not cause astrocyte toxicity (n=4). All\nvalues are mean +/− SEM.', 'hash': '6e663feeb73ec4154736671854b3c3758d432ab4dea895185ef9e3a535117ece'}, {'image_id': 'nihms795847f6', 'image_file_name': 'nihms795847f6.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f6.jpg', 'caption': 'Characteristics of astrocytic mitochondria particle in FACS\nanalysisa, Mitochondrial particles were identified by FACS.\nb, Of these mitochondrial particles, FACS analysis\nidentified that approximately 79% and 43% of particles\nexpress β1-integrin and CD63, respectively (n=4). cADPR (1\nμM) did not appear to affect these distributions (n=4). All\nvalues are mean +/− SEM.', 'hash': '134a395cb87c7849ffbffa3611f29e1a5bf1e599f41d41dce41899a97878b39f'}, {'image_id': 'nihms795847f8', 'image_file_name': 'nihms795847f8.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f8.jpg', 'caption': 'Summary of experiment on Figure\n2ca, We repeated the experiment in Fig. 2c with n=4 independent primary\ncultures per group. Similar results were obtained. The extracellular\nmitochondria-depleted astrocyte media (mdACM) group was significantly\ndifferent compared to the ACM group. Furthermore, in this repeated\nexperiment, there was also statistical significance between controls\n(OGD-damaged neurons alone) versus those treated with\nmitochondria-containing astrocyte media (ACM), and there was no\nstatistically significant worsening when comparing control versus\nmitochondria-depleted groups (mdACM). Taken together, these two separate\nexperiments suggest a modest but statistically significant neuroprotection\ninduced by astrocyte-derived mitochondria. All values are mean\n+/− SEM. b, Mitotracker Red CMXRos (200 nM) was\nincubated without astrocytes to obtain no-cell-derived media (negative\ncontrol). Media was collected and further incubated with neurons following\noxygen-glucose deprivation. After 24 hours, there was no mitochondrial\nsignal observed. Scale: 100 μm.', 'hash': 'b3bcc5a4b4d893e9377b1bbff003b90273252871c5d330d0ef87c82c47a4f4f3'}, {'image_id': 'nihms795847f10', 'image_file_name': 'nihms795847f10.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f10.jpg', 'caption': 'Metabolic inhibition in astrocyte causes neuronal cell death and retards\nneurite outgrowth in vitroa, Astrocytic aconitase was inhibited by fluorocitrate\n(FC) which disrupted astrocyte metabolism that was accompanied by\nSA-β-gal signal. b, Intracellular ATP was decreased in\nthese metabolically-disrupted astrocytes (n=6). *P<0.05,\n**P<0.01 vs FC 0 mM. c, PI staining showed\nthat fluorocitrate (0.5 mM) did not induce cell death in astrocytes.\nd, Metabolically-disrupted astrocytes significantly\ndecreased mitochondrial membrane potential. Red: aggregated JC1, Green:\nmonomer JC1. Scale: 20 μm. e, Rat cortical neurons were\nco-cultured with JC1-labeled astrocytes. After 24 hours co-culture, control\nastrocytes transferred mitochondria which had a high-membrane potential\n(aggregated JC1), but metabolically-disrupted astrocytes released and\ntransferred dysfunctional mitochondria into neurons (n=3).\nf, Metabolically-disrupted astrocytes could not support\nneural viability under starvation in the co-culture (n=4).\ng, Co-culture between astrocytes and neurons was conducted\nfor 48 hours to test neurite outgrowth. Immunocytochemistry showed that\nmetabolically-disrupted astrocytes retarded neurite outgrowth and increased\nneuronal cell death (n=3). h, LDH assay indicated that\nfluorocitrate (0.5 mM) did not affect cell viability in either rat cortical\nastrocytes (n=4) or rat cortical neurons (n=4). All values\nare mean +/− SEM.', 'hash': '63391e5aef4e314faf3d1b1a6c8d1829c46d73c42422d98105d7fadb0ab26819'}, {'image_id': 'nihms795847f5', 'image_file_name': 'nihms795847f5.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f5.jpg', 'caption': 'Astrocytic mitochondria particle detectiona, Electron microscopic analysis demonstrated that\nmitochondria were detected within extracellular astrocyte-derived particles.\nFree mitochondria were also found in astrocyte-conditioned medium.\nb, In FACS analysis, control beads were used to gate\npopulation ranging in size from 500 nm to 900 nm. c, In\nastrocyte-derived conditioned media, approximately 53% of particles\nin the range of size were positive for functional mitochondria\n(n=5). d, After FACS analysis to isolate extracellular\nmitochondria fraction from astrocyte-conditioned media, particle size was\nmeasured with qNano analysis. Consistent with electron microscope analysis,\na range of size distributions were observed (~25%: 300 – 400\nnm, ~75%: 400 – 1100 nm). All values are mean\n+/− SEM.', 'hash': '9156089c7bbb358a62dd8745def2819707406df6caf5df3e940c29ef91cc787e'}, {'image_id': 'nihms795847f2', 'image_file_name': 'nihms795847f2.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f2.jpg', 'caption': 'Astrocytic extracellular mitochondria and neuroprotectiona, Experimental schematic to test neuroprotective effects of\nastrocyte conditioned media (ACM) or mitochondria-depleted astrocyte conditioned\nmedia (mdACM) against oxygen-glucose deprivation (OGD) in rat cortical neurons.\nb, ACM but not mdACM rescued ATP levels in damaged neurons\n(n=4). c, ACM but not mdACM recovered neuronal viability\nafter OGD (n=4). d, Immunostaining confirmed that\nneuroprotective effect of ACM but not mdACM (n=4). Scale: 100\nμm. e, No statistically significant neuroprotection with\nliposomal ATP (1–1000 nM) after OGD. f, Fluorescent\nmicroscopy suggests the presence of astrocyte mitochondria (labeled with\nMitotracker Red CMXRos, 200 nM) within neurons. Scale: 100 μm. All\nvalues are mean +/− SEM.', 'hash': '627d32de68288210473ed6a90b87f14b7a63c17203f7c3addaa4e8ec8b1ec713'}, {'image_id': 'nihms795847f13', 'image_file_name': 'nihms795847f13.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f13.jpg', 'caption': 'Neuronal purity confirmed by FACS analysis in vivoTo be sure about our FACS findings, we used two different standard\napproaches that have been published in the literature (Bi et al, J Neurosci\n2011; Cruz et al, Nat Neurosci Rev 2013) a, By FACS, MAP2\npositive population were gated and further assessed by other markers such as\nIba1 (microglia/macrophage) and GFAP (astrocyte) in brain cell samples\nisolated from C57Bl6 mice. These comparisons confirmed that the\nMAP2+ population did not contain any appreciable amounts of\nmicroglia or astrocyte, whereas another neuron marker (NeuN) was highly\nenriched. b, Similar findings were obtained using an\nalternative gating method to isolate neurons.', 'hash': '8e24a2d4f5d0d5383b6c66d2648f8109688837fa92400f4680c8a1915b1f6714'}, {'image_id': 'nihms795847f14', 'image_file_name': 'nihms795847f14.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f14.jpg', 'caption': 'Involvement of integrin-mediate src/syk mechanisms in astrocytic\nmitochondrial entry into neurons in vitroa and b, Cultured rat cortical astrocytes were\nstimulated by cADPR (1 μM) for 24 hours. Intracellular mitochondria\nlabeled by mitotracker dye was significantly increased in astrocytes\nstimulated with cADPR (1 μM) (n=7).\n**P<0.01 vs 0h. c, Some of\nmitochondria were found outside of cells. d, FACS analysis\nrevealed that approximately 5×105 mitochondria were\ncontained in 1mL of astrocyte-derived conditioned media (n=6). cADPR\n(1 μM) significantly increased the number of mitochondria in the\nmedia (n=6). e, Experimental schedule to quantify the\nmitochondrial entry into neurons following oxygen-glucose deprivation. Rat\ncortical neurons (1×105 cells/well) were prepared in\n24-well culture plate. ACM or cADPR-ACM (each 1 mL) was co-incubated with\nneurons for 18 hours. Mitochondrial entry into neurons were calculated by\nmitochondrial intensity measured before and after washing cells with PBS.\nPhenol red free culture media were used to decrease back ground signal. Back\nground signal was subtracted from fluorescent intensity obtained from each\nsample. f, Oxygen-glucose deprivation for 2 hours decreased\napproximately 50% of mitochondria in neurons after 18 h\nreoxygenation (n=4). g, All data are expressed as\nrelative values, with total neuronal mitochondria after 2 h OGD/18 h\nreoxygenation being 100%. Mitochondrial entry into neurons was\nslightly higher in cADPR-ACM treatment (18%) compared to ACM\ntreatment (11%), although there was no statistically significance\n(n=4). h, There was no difference in the percentage of\nmitochondrial entry between ACM treatment and cADPR-ACM treatment\n(n=4). i, cADPR-ACM treatment supported neuronal\nviability better than ACM treatment (n=4). j,\nCo-culture between rat cortical astrocytes in the upper chamber and rat\ncortical neurons in the lower chamber was performed for 18 hours following\noxygen-glucose deprivation for 2 hours in neurons. Then, mitochondrial entry\ninto neurons was measured. k, Immediately after oxygen-glucose\ndeprivation, dynasore (5 μM), RGDS peptide (50 μg/ml), or\nMNS (1 μM) was initially added in neurons for 30 min, then astrocyte\nco-culture was performed for 18 hours. The data are expressed as relative\nvalues, with astrocytic extracellular mitochondria plus entered mitochondria\ninto neurons being 100%. RGDS peptide and MNS significantly\ndecreased mitochondrial entry into neurons, but dynasore did not inhibit the\nentry. l, MNS treatment significantly decreased\nastrocyte-mediated neuroprotection (n=4). m, Dynasore\n(5 μM), RGDS peptide (50 μg/ml), or MNS (1 μM) did\nnot affect neuronal viability after 2 h oxygen-glucose deprivation\n(n=4). All values are mean +/− SEM. These data\nsuggest that astrocyte into neuron mitochondrial particle entry may involve\nintegrin-mediate src/syk mechanisms. However, we acknowledge that these\npathways may be multifactorial and deeper analyses are warranted to dissect\nentry mechanisms under various physiologic and pathologic conditions.', 'hash': '98a72737177c42ec422053419d991d22a13b01a72cff67216f286053f688be3f'}, {'image_id': 'nihms795847f12', 'image_file_name': 'nihms795847f12.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f12.jpg', 'caption': 'Effects of CD38 suppression with siRNA in vivo and in vitroa, Western blot showed that CD38 expression was\nincreased in peri-infarct cortex at days 1 to 7 after stroke.\nb, CD38 siRNA or a scrambled control was injected into\nlateral ventricles at 5 days after stroke. Western blot analysis confirmed\nthat CD38 expression was successfully decreased in peri-infarct cortex at 7\ndays. c, In peri-infarct cortex, CD8 T cell and Iba1 positive\nmicroglia/macrophage were detected by immunohistochemistry. d,\nQuantification of the number of CD8 positive cells or Iba1 positive cells\nindicated that there was no difference between control siRNA and CD38 siRNA\n(n=6). All values are mean +/− SEM. e,\nCultured rat cortical astrocytes were subjected to oxygen-glucose\ndeprivation for 2 hours followed by treating with control siRNA or CD38\nsiRNA. Astrocyte cell morphology or GFAP expression was assessed by\nimmunocytochemistry or western blot after 22-h reoxygenation.\nf, Morphology change was not clearly observed in cultured\nastrocytes suppressed CD38 with siRNA compared to control siRNA.\ng, Western blot analysis showed that CD38 was successfully\ndecreased by siRNA transfection but GFAP expression was not clearly\nchanged.', 'hash': 'dd16e8fe7f7f7631fde320089221e28b7e1313035510649abdc20456268b3a15'}, {'image_id': 'nihms795847f3', 'image_file_name': 'nihms795847f3.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f3.jpg', 'caption': 'Astrocytic mitochondria and neuroplasticity after ischemic stressa, Confocal microscopy revealed that astrocytic mitochondria (red,\nMitotracker Red CMXRos) may be transferred into neural soma (a) and axon (b),\nand some may fuse with neuronal mitochondria (c, green, Cell-light Mito-GFP).\nb, Experimental schematic for co-culture studies.\nc, Soma size was unchanged but astrocytic mitochondrial density\nin neuronal soma was significantly decreased when CD38 was suppressed in\nastrocytes (n=54 or 41 soma were counted). Scale: 20 μm.\nd, Quantification of dendrite elongation (MAP2 staining)\n(n=5 or 6). e, Male C57Bl6 mice were subjected to 60 min\ntransient focal ischemia. Three days later, astrocyte mitochondria particles\n(1,000 particles/2μl, MitoTracker Red CMXRos) were infused into cerebral\ncortex. Confocal images showed transplanted astrocytic mitochondria (red) within\nperi-infarct neurons at 24 hrs. f, FVB/N-Tg (GFAPGFP)14Mes/J\ntransgenic mice with fluorescently labeled astrocytes were subjected to 30 min\ntransient focal ischemia. Immunohistochemistry at 24 hrs suggested that GFP\n(GFAP)-positive particles co-stained with mitochondrial TOM40 were present in\nMAP2-positive neurons in peri-infarct cortex. g, Western blot\nindicated that GFP-positive neurons upregulated cell survival-related proteins\n(phospho-Akt, Bcl-xl) but not apoptosis-related proteins (caspase 3, AIF) along\nwith an increase of mitochondrial TOM40 (n=3). Isolated neurons\nexpressed mature (neurofilament) but not neural stem cell markers (nestin)\n(Extended Data Fig. 7f). All values\nare mean +/− SEM.', 'hash': '574db8a6c1e9e8489d8f0e67be4519a1f720bfaa48eda51e3fd900fe83d45185'}, {'image_id': 'nihms795847f4', 'image_file_name': 'nihms795847f4.jpg', 'image_path': '../data/media_files/PMC4968589/nihms795847f4.jpg', 'caption': 'Effects of CD38 siRNA in focal cerebral ischemiaa, Male C57Bl6 mice were subjected to transient 60 min focal\nischemia and control siRNA or CD38 siRNA was injected into lateral ventricles at\n5 days post-stroke. Immunostaining showed that CD38 siRNA decreased HMGB1\nastrocytes in peri-infarct cortex. b, Nissl staining showed no\ndifference in infarct size (n=8 or 10). c, Immunostaining\ndemonstrated that astrocytic CD38 was diminished by CD38 siRNA. d,\nAstrocytic CD38 suppression with siRNA reduced GFAP-positive mitochondria in CSF\nat 7 days (n=6). e, Neuronal mitochondria were decreased by\nCD38 siRNA (n= 8 or 5). f, CD38 siRNA attenuated\nperi-infarct GAP43 immunostaining. g, Western blot confirmed a\nreduction of peri-infarct GAP43 protein within CD38 siRNA-treated brains\n(n=5). h, i, Suppression of CD38 signaling worsened\nneurological outcomes in neuroscore (h) and grid walking test\n(i) (n=7 or 9).*P<0.05 vs\nday 3 control siRNA, #P<0.05 vs day 7\nCD38 siRNA. j, CD38 suppression decreased oxygen consumption in CSF\nmitochondria (n=7 or 9). k, l, Mitochondrial function in\nCSF was negatively correlated with neurological outcomes. All values are mean\n+/− SEM. m, Schematic of CD38 regulation of\nmitochondria release/transfer hypothesis between astrocytes and neurons.', 'hash': '2b3accb57cf69fc37c9bfac8e296e17e227e0c7dff2262ea562d2d7abeb1f288'}]
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{'nihms795847f1': ['In this study, we asked whether astrocytes can produce functional extracellular\nmitochondria to support neuronal viability after ischemic stroke. Electron microscopy\nconfirmed the presence of extracellular particles containing mitochondria in conditioned\nmedia from rat cortical astrocytes (<xref rid="nihms795847f1" ref-type="fig">Fig. 1a</xref>, , <xref rid="nihms795847f5" ref-type="fig">Extended Data Fig. 1a</xref>). qNano analysis revealed that\nastrocyte-derived mitochondria particles following FACS isolation spanned a range of\nsizes from 300 to 1100 nm (). qNano analysis revealed that\nastrocyte-derived mitochondria particles following FACS isolation spanned a range of\nsizes from 300 to 1100 nm (<xref rid="nihms795847f5" ref-type="fig">Extended Data Fig.\n1b–d</xref>), and included populations that were positive for\nβ1-integrin (79%) and CD63 (43%) (), and included populations that were positive for\nβ1-integrin (79%) and CD63 (43%) (<xref rid="nihms795847f6" ref-type="fig">Extended Data Fig. 2</xref>). Mitotracker-labeling suggested that these\nextracellular mitochondria may still be functional (). Mitotracker-labeling suggested that these\nextracellular mitochondria may still be functional (<xref rid="nihms795847f1" ref-type="fig">Fig.\n1b</xref>), and filtration of astrocyte conditioned media through 0.2 μm\nfilters depleted the amounts of functional mitochondria and reduced measurements of\nmitochondrial ATP, membrane potential and oxygen consumption (), and filtration of astrocyte conditioned media through 0.2 μm\nfilters depleted the amounts of functional mitochondria and reduced measurements of\nmitochondrial ATP, membrane potential and oxygen consumption (<xref rid="nihms795847f1" ref-type="fig">Fig. 1b–e</xref>).).', 'An important question at this point is whether extracellular mitochondria\nrepresent active signals or merely cellular debris. To address this question, we asked\nwhether stimulated astrocytes could actively produce extracellular mitochondria. CD38\ncatalyzes the synthesis of a calcium messenger, cyclic ADP-ribose (cADPR) in\nmitochondrial membranes 14,15. In brain, CD38 is mainly expressed in glial\ncells, and may have a role in neuroglial crosstalk since astrocytes increase CD38\nexpression in response to glutamate release from neurons 16. Based on this background literature and the\nfact that most actively secreted cellular events involve calcium regulation, we decided\nto assess CD38-cADPR-calcium signaling as a candidate mechanism for the astrocytic\nproduction of extracellular mitochondria. First, we confirmed that rat cortical\nastrocytes expressed CD38 protein and CD38/cADPR cyclase activity (<xref rid="nihms795847f1" ref-type="fig">Fig. 1f, g</xref>). Then, we tried two methods to modify this\npathway. When astrocytic CD38 was upregulated using CRISPR/Cas9 activation plasmids,\nfunctional endpoints of extracellular mitochondria were significantly increased in\nconditioned media (). Then, we tried two methods to modify this\npathway. When astrocytic CD38 was upregulated using CRISPR/Cas9 activation plasmids,\nfunctional endpoints of extracellular mitochondria were significantly increased in\nconditioned media (<xref rid="nihms795847f1" ref-type="fig">Fig. 1h–k</xref>). When\nastrocytes were stimulated by cADPR to activate CD38 signaling, extracellular\nmitochondria were increased in conditioned media along with enhancement of functional\nendpoints in a calcium-dependent manner (). When\nastrocytes were stimulated by cADPR to activate CD38 signaling, extracellular\nmitochondria were increased in conditioned media along with enhancement of functional\nendpoints in a calcium-dependent manner (<xref rid="nihms795847f1" ref-type="fig">Fig.\n1l–n</xref>, , <xref rid="nihms795847f7" ref-type="fig">Extended Data Fig. 3</xref>).\nStimulation with cADPR did not appear to damage astrocyte viability ().\nStimulation with cADPR did not appear to damage astrocyte viability (<xref rid="nihms795847f1" ref-type="fig">Fig. 1o</xref>), suggesting that this release of extracellular\nmitochondria was not due to nonspecific cytotoxicity.), suggesting that this release of extracellular\nmitochondria was not due to nonspecific cytotoxicity.'], 'nihms795847f2': ['If astrocytes can produce functional extracellular mitochondria, then is it\npossible that these signals may affect adjacent neurons? When rat cortical neurons were\nsubjected to oxygen-glucose deprivation, intracellular ATP levels fell and neuronal\nviability decreased, as expected (<xref rid="nihms795847f2" ref-type="fig">Fig.\n2a–c</xref>, , <xref rid="nihms795847f8" ref-type="fig">Extended Data Fig. 4</xref>). When\nastrocyte-conditioned media containing extracellular mitochondrial particles was added\nto neurons, ATP levels were increased and neuronal viability was recovered (). When\nastrocyte-conditioned media containing extracellular mitochondrial particles was added\nto neurons, ATP levels were increased and neuronal viability was recovered (<xref rid="nihms795847f2" ref-type="fig">Fig. 2a–c</xref>, , <xref rid="nihms795847f8" ref-type="fig">Extended Data Fig. 4</xref>). But when extracellular mitochondria were removed from\nthe astrocyte-conditioned media, neuroprotection was no longer observed (). But when extracellular mitochondria were removed from\nthe astrocyte-conditioned media, neuroprotection was no longer observed (<xref rid="nihms795847f2" ref-type="fig">Fig. 2a–c</xref>, , <xref rid="nihms795847f8" ref-type="fig">Extended Data\nFig. 4</xref>). Similar results were obtained with immunostaining-based cell counts\n(). Similar results were obtained with immunostaining-based cell counts\n(<xref rid="nihms795847f2" ref-type="fig">Fig. 2d</xref>). As a control, ATP-liposomes were not\nsignificantly protective (). As a control, ATP-liposomes were not\nsignificantly protective (<xref rid="nihms795847f2" ref-type="fig">Fig. 2e</xref>), suggesting that\nthe astrocytic mitochondria entry into neurons may generate additional benefits beyond\nATP energetics per se. Fluorescent microscopy confirmed that astrocyte-derived\nmitochondria appeared to be present within treated neurons (), suggesting that\nthe astrocytic mitochondria entry into neurons may generate additional benefits beyond\nATP energetics per se. Fluorescent microscopy confirmed that astrocyte-derived\nmitochondria appeared to be present within treated neurons (<xref rid="nihms795847f2" ref-type="fig">Fig. 2f</xref>).).', 'a, We repeated the experiment in <xref rid="nihms795847f2" ref-type="fig">Fig. 2c</xref> with n=4 independent primary\ncultures per group. Similar results were obtained. The extracellular\nmitochondria-depleted astrocyte media (mdACM) group was significantly\ndifferent compared to the ACM group. Furthermore, in this repeated\nexperiment, there was also statistical significance between controls\n(OGD-damaged neurons alone) versus those treated with\nmitochondria-containing astrocyte media (ACM), and there was no\nstatistically significant worsening when comparing control versus\nmitochondria-depleted groups (mdACM). Taken together, these two separate\nexperiments suggest a modest but statistically significant neuroprotection\ninduced by astrocyte-derived mitochondria. All values are mean\n+/− SEM. with n=4 independent primary\ncultures per group. Similar results were obtained. The extracellular\nmitochondria-depleted astrocyte media (mdACM) group was significantly\ndifferent compared to the ACM group. Furthermore, in this repeated\nexperiment, there was also statistical significance between controls\n(OGD-damaged neurons alone) versus those treated with\nmitochondria-containing astrocyte media (ACM), and there was no\nstatistically significant worsening when comparing control versus\nmitochondria-depleted groups (mdACM). Taken together, these two separate\nexperiments suggest a modest but statistically significant neuroprotection\ninduced by astrocyte-derived mitochondria. All values are mean\n+/− SEM. b, Mitotracker Red CMXRos (200 nM) was\nincubated without astrocytes to obtain no-cell-derived media (negative\ncontrol). Media was collected and further incubated with neurons following\noxygen-glucose deprivation. After 24 hours, there was no mitochondrial\nsignal observed. Scale: 100 μm.'], 'nihms795847f3': ['Beyond the prevention of acute neuronal death, delayed neuroplasticity is also\nimportant for stroke outcomes. CD38 may be important for brain plasticity because\nCD38-deficient mice show worsened recovery after brain injury 17 and CD38 mutations may comprise risk factors for\nbehavioral dysfunction 18. Hence, we\nasked whether CD38-mediated astrocyte-into-neuron mitochondrial transfer may also\ninfluence neuroplasticity. Neurons were labeled with CellLight Mitochondria-GFP and\nastrocytes were separately labeled with Mitotracker Red CMXRos, and then the two cell\ntypes were co-cultured together for 24 hours. Confocal microscopy indicated that\nastrocyte-derived mitochondria were detected within soma and axon (<xref rid="nihms795847f3" ref-type="fig">Fig. 3a</xref>), and in these co-culture conditions, astrocytes\nsupported neuronal survival after serum/glucose starvation in a CD38-dependent manner\n(), and in these co-culture conditions, astrocytes\nsupported neuronal survival after serum/glucose starvation in a CD38-dependent manner\n(<xref rid="nihms795847f9" ref-type="fig">Extended Data Fig. 5</xref>). When astrocytic\nmitochondria were made dysfunctional via inhibition of mitochondrial aconitase,\ncADPR-stimulated astrocytes no longer supported neuronal survival and axonal extension\n(). When astrocytic\nmitochondria were made dysfunctional via inhibition of mitochondrial aconitase,\ncADPR-stimulated astrocytes no longer supported neuronal survival and axonal extension\n(<xref rid="nihms795847f10" ref-type="fig">Extended Data Fig. 6</xref>). To further assess our\nhypothesis, we asked whether this ability of astrocytes to transfer mitochondria could\nin fact enhance neuroplasticity under pathological conditions. Control or CD38-silenced\nastrocytes were co-cultured with surviving neurons after oxygen-glucose deprivation\n(). To further assess our\nhypothesis, we asked whether this ability of astrocytes to transfer mitochondria could\nin fact enhance neuroplasticity under pathological conditions. Control or CD38-silenced\nastrocytes were co-cultured with surviving neurons after oxygen-glucose deprivation\n(<xref rid="nihms795847f3" ref-type="fig">Fig. 3b</xref>). siRNA suppression of CD38 in\nastrocytes reduced mitochondria transfer (). siRNA suppression of CD38 in\nastrocytes reduced mitochondria transfer (<xref rid="nihms795847f3" ref-type="fig">Fig. 3c</xref>)\nand dendrite regrowth after injury ()\nand dendrite regrowth after injury (<xref rid="nihms795847f3" ref-type="fig">Fig. 3d</xref>).).', 'Taken together, these cellular findings appear consistent with the overall\nhypothesis that CD38 signaling may help astrocytes transfer mitochondria into neurons\nand promote survival and plasticity after injury. But does this mechanism work in vivo?\nTo answer this question, we turned to a mouse model of focal cerebral ischemia. First,\nprimary mouse cortical astrocyte cultures were labeled with MitoTracker Red CMXRos and\nextracellular mitochondria particles were collected. Then mice were subjected to focal\ncerebral ischemia, and 3 days later, extracellular mitochondria particles were directly\ninjected into peri-infarct cortex. After 24 hrs, immunostaining suggested that\ntransplanted astrocytic mitochondria were indeed present in neurons (<xref rid="nihms795847f3" ref-type="fig">Fig. 3e</xref>). Next, we turned to FVB/N-Tg (GFAPGFP)14Mes/J\ntransgenic mice where astrocytes are fluorescently labeled. When these mice were\nsubjected to focal cerebral ischemia, fluorescent mitochondrial particle signals\nappeared within adjacent neurons at 24 hours post-stroke (). Next, we turned to FVB/N-Tg (GFAPGFP)14Mes/J\ntransgenic mice where astrocytes are fluorescently labeled. When these mice were\nsubjected to focal cerebral ischemia, fluorescent mitochondrial particle signals\nappeared within adjacent neurons at 24 hours post-stroke (<xref rid="nihms795847f3" ref-type="fig">Fig. 3f</xref>). Neurons that were collected from ischemic peri-infarct cortex via\nflow cytometry showed a general upregulation of cell survival-related signals such as\nphosphorylated Akt and Bcl-xl along with an increase of the mitochondria marker TOM4\n(). Neurons that were collected from ischemic peri-infarct cortex via\nflow cytometry showed a general upregulation of cell survival-related signals such as\nphosphorylated Akt and Bcl-xl along with an increase of the mitochondria marker TOM4\n(<xref rid="nihms795847f3" ref-type="fig">Fig. 3g</xref>, , <xref rid="nihms795847f11" ref-type="fig">Extended Data Fig. 7</xref>).).'], 'nihms795847f12': ['Finally, we attempted loss-of-function experiments to ask whether blocking CD38\nsignaling results in worsened outcomes after stroke. In our mouse models of focal\ncerebral ischemia, CD38 was upregulated in the peri-infarct cortex (<xref rid="nihms795847f12" ref-type="fig">Extended Data Fig. 8a</xref>). At 5 days post-stroke, CD38 siRNA or\ncontrol siRNA were injected into cerebral ventricles (). At 5 days post-stroke, CD38 siRNA or\ncontrol siRNA were injected into cerebral ventricles (<xref rid="nihms795847f4" ref-type="fig">Fig.\n4a</xref>). By 2 days after siRNA injections, total CD38 expression in the\nperi-infarct cortex was successfully downregulated (). By 2 days after siRNA injections, total CD38 expression in the\nperi-infarct cortex was successfully downregulated (<xref rid="nihms795847f12" ref-type="fig">Extended Data Fig. 8b</xref>). There were no clear differences in infarct area nor\nthe total levels of GFAP-positive reactive astrocytes (). There were no clear differences in infarct area nor\nthe total levels of GFAP-positive reactive astrocytes (<xref rid="nihms795847f4" ref-type="fig">Fig. 4b, c</xref>), but astrocyte subsets that expressed CD38 were significantly\ndecreased without affecting the number of other CD38 expressing cells such as CD8 T\ncells and microglia/macrophages ), but astrocyte subsets that expressed CD38 were significantly\ndecreased without affecting the number of other CD38 expressing cells such as CD8 T\ncells and microglia/macrophages 19\n(<xref rid="nihms795847f4" ref-type="fig">Fig. 4c</xref>, , <xref rid="nihms795847f12" ref-type="fig">Extended Data Fig. 8c–g</xref>). To assess the levels of extracellular\nmitochondrial particles in this in vivo model, flow cytometry was used to analyze\ncerebrospinal fluid (CSF). GFAP-positive mitochondria were detected in CSF, and CD38\nsiRNA injections appeared to reduce this extracellular population of astrocyte-derived\nmitochondria (). To assess the levels of extracellular\nmitochondrial particles in this in vivo model, flow cytometry was used to analyze\ncerebrospinal fluid (CSF). GFAP-positive mitochondria were detected in CSF, and CD38\nsiRNA injections appeared to reduce this extracellular population of astrocyte-derived\nmitochondria (<xref rid="nihms795847f4" ref-type="fig">Fig. 4d</xref>). At the same time, flow\ncytometry was used to quantify levels of MAP2 neuronal mitochondria (). At the same time, flow\ncytometry was used to quantify levels of MAP2 neuronal mitochondria (<xref rid="nihms795847f13" ref-type="fig">Extended Data Fig. 9</xref>). Brains treated with CD38 siRNA showed a\nsignificant reduction in neuronal mitochondria (). Brains treated with CD38 siRNA showed a\nsignificant reduction in neuronal mitochondria (<xref rid="nihms795847f4" ref-type="fig">Fig.\n4e</xref>), suggesting that interfering with CD38 signaling may have suppressed\nendogenous astrocyte-to-neuron mitochondrial transfer. These effects were accompanied by\na reduction in peri-infarct GAP43 (a surrogate marker of neuroplasticity, ), suggesting that interfering with CD38 signaling may have suppressed\nendogenous astrocyte-to-neuron mitochondrial transfer. These effects were accompanied by\na reduction in peri-infarct GAP43 (a surrogate marker of neuroplasticity, <xref rid="nihms795847f4" ref-type="fig">Fig. 4f, g</xref>) as well as worsened neurologic outcomes () as well as worsened neurologic outcomes (<xref rid="nihms795847f4" ref-type="fig">Fig. 4h, i</xref>). Furthermore, CD38 suppression\nsignificantly decreased oxygen consumption measurements in CSF-derived extracellular\nmitochondrial particles (). Furthermore, CD38 suppression\nsignificantly decreased oxygen consumption measurements in CSF-derived extracellular\nmitochondrial particles (<xref rid="nihms795847f4" ref-type="fig">Fig. 4j</xref>), and neurologic\noutcomes seemed to be negatively correlated with these functional endpoints (), and neurologic\noutcomes seemed to be negatively correlated with these functional endpoints (<xref rid="nihms795847f4" ref-type="fig">Fig. 4k, l</xref>), suggesting that CSF mitochondrial\nfunction may be a potential biomarker of neuroglial signaling after stroke.), suggesting that CSF mitochondrial\nfunction may be a potential biomarker of neuroglial signaling after stroke.'], 'nihms795847f4': ['Taken together, these findings suggest that astrocytes may release extracellular\nmitochondrial particles via CD38-mediated mechanisms that enter into neurons after\nstroke (<xref rid="nihms795847f4" ref-type="fig">Fig. 4m</xref>). But there a few caveats and the\ndetailed mechanisms and generalizability of these proof-of-concept findings should\nwarrant further investigation. First, the dynamics of extracellular mitochondria release\nand entry into neurons as well as quantitative thresholds for functional benefit remain\nto be fully defined (). But there a few caveats and the\ndetailed mechanisms and generalizability of these proof-of-concept findings should\nwarrant further investigation. First, the dynamics of extracellular mitochondria release\nand entry into neurons as well as quantitative thresholds for functional benefit remain\nto be fully defined (<xref rid="nihms795847f14" ref-type="fig">Extended Data Fig.\n10a–i</xref>). A second caveat relates to mitochondrial entry mechanisms. In\nneurons, endocytosis may be regulated by dynamin/clathrin ). A second caveat relates to mitochondrial entry mechanisms. In\nneurons, endocytosis may be regulated by dynamin/clathrin 20 or integrin pathways 21. In our models, integrin-mediated src/syk\nsignaling may be involved (<xref rid="nihms795847f14" ref-type="fig">Extended Data Fig.\n10j–m</xref>). How integrin-mediated mitochondrial transfer is modulated\nunder different disease conditions requires further study. Third, CD38 is also expressed\nin immune cells. In this study, CD38 suppression with siRNA in vivo did not appear to\naffect T cells or microglia/macrophages, but the balance between potentially beneficial\nCD38 signals in astrocytes versus deleterious CD38 signals in immune cells should be\ncarefully considered. A fourth caveat is whether other glial cells may participate.\nMicroglia, oligodendrocytes and pericytes are activated after stroke ). How integrin-mediated mitochondrial transfer is modulated\nunder different disease conditions requires further study. Third, CD38 is also expressed\nin immune cells. In this study, CD38 suppression with siRNA in vivo did not appear to\naffect T cells or microglia/macrophages, but the balance between potentially beneficial\nCD38 signals in astrocytes versus deleterious CD38 signals in immune cells should be\ncarefully considered. A fourth caveat is whether other glial cells may participate.\nMicroglia, oligodendrocytes and pericytes are activated after stroke 22,23, so their potential roles in mitochondrial exchange warrants\nfurther investigation. Finally, astrocytes can produce many factors for protecting and\nrestoring neurons, including tPA, high-mobility group box 1 (HMGB1), extracellular\nmicrovesicles containing VEGF and FGF-2, and various microRNAs 24–27. How mitochondrial particles may interact with these other\nextracellular signals should be explored.'], 'nihms795847f11': ['a, Confocal microscopy revealed that astrocytic mitochondria (red,\nMitotracker Red CMXRos) may be transferred into neural soma (a) and axon (b),\nand some may fuse with neuronal mitochondria (c, green, Cell-light Mito-GFP).\nb, Experimental schematic for co-culture studies.\nc, Soma size was unchanged but astrocytic mitochondrial density\nin neuronal soma was significantly decreased when CD38 was suppressed in\nastrocytes (n=54 or 41 soma were counted). Scale: 20 μm.\nd, Quantification of dendrite elongation (MAP2 staining)\n(n=5 or 6). e, Male C57Bl6 mice were subjected to 60 min\ntransient focal ischemia. Three days later, astrocyte mitochondria particles\n(1,000 particles/2μl, MitoTracker Red CMXRos) were infused into cerebral\ncortex. Confocal images showed transplanted astrocytic mitochondria (red) within\nperi-infarct neurons at 24 hrs. f, FVB/N-Tg (GFAPGFP)14Mes/J\ntransgenic mice with fluorescently labeled astrocytes were subjected to 30 min\ntransient focal ischemia. Immunohistochemistry at 24 hrs suggested that GFP\n(GFAP)-positive particles co-stained with mitochondrial TOM40 were present in\nMAP2-positive neurons in peri-infarct cortex. g, Western blot\nindicated that GFP-positive neurons upregulated cell survival-related proteins\n(phospho-Akt, Bcl-xl) but not apoptosis-related proteins (caspase 3, AIF) along\nwith an increase of mitochondrial TOM40 (n=3). Isolated neurons\nexpressed mature (neurofilament) but not neural stem cell markers (nestin)\n(<xref rid="nihms795847f11" ref-type="fig">Extended Data Fig. 7f</xref>). All values\nare mean +/− SEM.). All values\nare mean +/− SEM.']}
|
Transfer of mitochondria from astrocytes to neurons after stroke
| null |
Nature
|
1469602800
|
Neurons can release damaged mitochondria and transfer them to astrocytes for disposal and recycling. This ability to exchange mitochondria may represent a potential mode of cell-to-cell signalling in the central nervous system. Here we show that astrocytes in mice can also release functional mitochondria that enter neurons. Astrocytic release of extracellular mitochondrial particles was mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signalling. Transient focal cerebral ischaemia in mice induced entry of astrocytic mitochondria into adjacent neurons, and this entry amplified cell survival signals. Suppression of CD38 signalling by short interfering RNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke.
|
[
"ADP-ribosyl Cyclase 1",
"Animals",
"Astrocytes",
"Brain Ischemia",
"Calcium",
"Cell Survival",
"Cyclic ADP-Ribose",
"Male",
"Membrane Glycoproteins",
"Mice",
"Mice, Inbred C57BL",
"Mitochondria",
"Neuronal Plasticity",
"Neurons",
"Protective Factors",
"RNA, Small Interfering",
"Signal Transduction",
"Stress, Physiological",
"Stroke"
] |
other
|
PMC4968589
| null |
37
|
[
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] |
Nature. 2016 Jul 27; 535(7613):551-555
|
NO-CC CODE
|
|
ActA–RFP topology and localization by immunofluorescence.Diagram of the topology of ActA–RFP association with the bacterial cell wall.Immunofluorescence of wild-type ActA using an ActA antibody. Top panel shows the ActA distribution on strain DPL-4087 (constitutively expressing ActA) and bottom panel shows ActA in strain DPL-4077 induced to stage IV of polarization.Surface distributions of strains expressing ActA–RFP. Left panels show the ActA–RFP distribution by immunofluorescence as in B. Right panels show RFP fluorescence signal on the inside of bacteria. Top panel shows strain JAT-396 (constitutively expressing ActA–RFP) and bottom panel shows ActA–RFP in strain JAT-395 induced to stage IV of polarization. All strains exhibit the same normal polarized surface distribution of ActA with most protein at the poles and least at the septation zone. The ActA–RFP distribution is the same when visualized by immunofluorescence using an ActA antibody to protein accessible on the outside of bacteria and by the RFP signal on the inside of bacteria. A slight shift of RFP signal further inside bacteria can be seen. Bar = 2 µm.
|
mmi_5025_f1
|
2
|
a1d9b8e41ec833ce6ec17749845eb6f02f4beebdbd88c9971a13fe2824aabdc3
|
mmi_5025_f1.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
400,
264
] |
[{'image_id': 'mmi_5025_f4', 'image_file_name': 'mmi_5025_f4.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f4.jpg', 'caption': 'Time-course of InlA induction and polarization in vitro. InlA was induced identically to ActA in strains JAT-395 and DPL-3078 (Δ-actA in 10403S background) and visualized by immunofluorescence at different times during induction. Stages of polarization were identical in both strains and DPL-3078 is shown here.InlA polarization stages I–IV. InlA exhibited stages of polarization similar to those observed for ActA (Fig. 2) over the course of several (three to four) hours.', 'hash': '1c4889e3c454280d26e604646050322f9dc67dee28dbad77102a5b73fdf3f4c5'}, {'image_id': 'mmi_5025_f3', 'image_file_name': 'mmi_5025_f3.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f3.jpg', 'caption': 'Sudden increase in overall ActA–RFP levels corresponds to an increase in ActA–RFP signal along cylindrical body.Representative images from stages I–IV during an induction time-course. Top panels show ActA–RFP signal, middle panels show phase-contrast and bottom panels show an overlay of ActA–RFP onto the phase-contrast image. Bar = 2 µm.Relative levels of RFP signal per bacterium at each stage in the induction time-course shown in A. Amounts shown as a percentage of the maximum. The sudden increase in RFP levels on bacteria occurred between stages II and III of polarization (approximately eightfold increase) during which ActA–RFP accumulated along the cylindrical body of bacteria but not directly at the poles. Accumulation of ActA–RFP at the poles occurred later in induction, at stage IV, when protein levels had increased approximately another twofold.', 'hash': 'bc3b136ec6eb7f893f0e74c9c86e0efe12793b44be7f0cf56673117ee73bd202'}, {'image_id': 'mmi_5025_f10', 'image_file_name': 'mmi_5025_f10.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f10.jpg', 'caption': 'Multi-step model for passive polarization of ActA.A.3-D representations of possible mechanisms leading to the observed polarization stages I–III. Darkest green lines represent newest cell wall growth, and successively lighter dashed green lines represent successively older cell wall material twisting and changing pitch as new material is incorporated into the cell wall (similar to Carballido-Lopez and Errington, 2003). Darkest red shading represents the greatest concentration of ActA protein over a specific surface area. (i) ActA protein is secreted in several distinct spots along the cylindrical body away from sites of new cell wall synthesis. (ii and iii) ActA is spread over the cylindrical body through two non-exclusive mechanisms: redistribution and sequential secretion. In redistribution spots of ActA already on the surface are stretched into patches through the movement of older cell wall as new cell wall is inserted, effectively pushing protein around. ActA initially present in (i) is diluted, represented by the lighter shades of red. In sequential secretion, ActA continues to be secreted in spots at a site that remains stationary in space (circled in black) as the cell wall moves around it. In (ii), the spot of ActA secreted in (i) has moved because of new cell wall growth. (iv) Eventually, as more ActA accumulates and is spread along the entire bacterial length, the combination of both mechanisms leads to an ActA surface distribution reminiscent of helical cell wall synthesis but localized in a non-overlapping pattern.2-D cross-section representations of observed polarization stages III–IV. (i) Bacterium at the same polarization stage as in (A, iv). Red rectangles represent the helical ActA surface distribution as seen for a 2-D longitudinal cross-section through the centre of the bacterium. (ii) During the transition between stages III and IV, more protein accumulates until the distribution of ActA is more uniform along the cylindrical cell body, represented by the solid darkest red lines. ActA gradually accumulates directly at the poles. The poles of the oldest generation are indicated as ‘a’ and the poles of the youngest generation, created from the septation zone of the previous generation, are indicated as ‘c’. Protein accumulates most efficiently at ‘c’ and least efficiently at ‘a’, as indicated by sequentially lighter shades of red and thinner lines. (iii) A possible mechanism for ActA accumulation at the poles. Slow incorporation of cylindrical cell wall material and ActA protein is shown in red, and indicated by red arrows. Grey arrows indicate continual shedding of cell wall material from the poles.', 'hash': '3c507596aff1d4242b5c81801cd6f518c39517e620f041a11cbfec508b9c9cc0'}, {'image_id': 'mmi_5025_f2', 'image_file_name': 'mmi_5025_f2.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f2.jpg', 'caption': 'Time-course of ActA–RFP induction and polarization in vitro. All ActA–RFP inductions were performed on strain JAT-395 (Rafelski and Theriot, 2005).Relative amounts of ActA–RFP per bacterium during an induction time-course as determined by Western blot analysis. Left panel shows two exposures of a representative immunoblot. Right graph shows quantitation of Western blot with amounts shown as a percentage of the maximum.Polarization stage I. Bacteria displaying the earliest visible ActA–RFP signal, 20–30 min after induction. Initial ActA–RFP accumulation occurred in one to four spots along the sides of bacteria.Polarization stage II. A little later in induction (30 min−1 h later) bacteria displayed an ActA–RFP distribution pattern of irregular patches covering distinct regions of the surface. New ActA–RFP accumulation in spots continued to be seen (top panel). Patches occurred along the sides of bacteria, not at the poles, nor the septation zone.Polarization stage III. ActA–RFP signal continued to increase and protein was seen to cover greater areas of the surface but still remained absent from the poles. Patterns were often reminiscent of a helical surface distribution, as indicated by the arrows. Surface distribution patterns varied greatly among bacteria within a population.Polarization stage IV. Bacteria display a fully polarized ActA–RFP distribution 3–6 h after initial induction. Protein continued to fill in along the sides of bacteria creating a continuous distribution. Additionally, protein was now seen at the poles and remained absent from the septation zone (arrow) as previously described (Kocks et al., 1993; Rafelski and Theriot, 2005).Bar = 2 µm. Far left panels show ActA–RFP signal, centre panels show phase-contrast and far right panels show an overlay of ActA–RFP onto the phase-contrast image to visualize the localization of protein on the bacterial surface.', 'hash': 'bb19dab7c5d9a2144392f5ebb99ca59cf7a36500cb3763f56cb9ad22ac0fc106'}, {'image_id': 'mmi_5025_f5', 'image_file_name': 'mmi_5025_f5.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f5.jpg', 'caption': 'ActA–RFP and vanc-FL have a non-overlapping surface distribution.L. monocytogenes stained with fluorescently labelled vancomycin (vanc-FL). Vanc-FL patterns were identical to those previously described on B. subtilis and the pairs of dots along the sides of the cylindrical body are typical of a helical distribution (Daniel and Errington, 2003).Immunofluorescence of earliest ActA–RFP signal detectable at polarization stage I (∼20 min after induction). Left panels show ActA, the centre panels show vanc-FL staining and the far right panels show an overlay, with ActA in red and vanc-FL in green. Initial spots of ActA localize to regions on the bacterium with less vanc-FL staining. Inset in bottom example shows ActA–RFP signal for that bacterium.Bacteria at polarization stage III. The top two panels on far left show ActA–RFP signal on live bacteria. The bottom panel on far left shows ActA–RFP on a bacterium at the same stage of induction by immunofluorescence. Left, centre and right panels are as in B. ActA signal localizes to regions of the bacterium with less vanc-FL signal. Inset in bottom example shows ActA–RFP signal for that bacterium.Bar = 2 µm.', 'hash': '2dd98cc9cc2d7bf95b8e4dd2702e75a3a80cbe11f799d025616943ec1530e741'}, {'image_id': 'mmi_5025_f8', 'image_file_name': 'mmi_5025_f8.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f8.jpg', 'caption': 'ActA–RFP is retained at the inert poles.Bacteria were induced for 2 h after ActA–RFP was at polarization stage IV then labelled with FAM-SE. The left panel shows uniform FAM-SE labelling as in 6A. The right panel shows that ActA–RFP on the surface of these bacteria is uniform as well, because of continual accumulation of protein.Bacteria from A grown for several generations without induction. Left panels show that fluorescent signal is retained at those poles that were initially labelled while absent from other poles. Right panels show brightest ActA–RFP signal at the same poles that are labelled with FAM-SE, indicating that protein was retained at those poles.Bar = 2 µm.', 'hash': '06500d3e7e73e057f9e730dd63fc7543d2877b59f98dda01be92e4c6a31adecf'}, {'image_id': 'mmi_5025_f6', 'image_file_name': 'mmi_5025_f6.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f6.jpg', 'caption': 'Differential growth along bacterium visualized by cell wall labelling.Bacteria labelled with FAM-SE show a uniform surface distribution with staining at the septation zones. Diagram on the right indicates uniform FAM-SE distribution at the time of labelling.Bacteria from A 2 h of growth later. The most fluorescent signal is seen at the poles, the least at the septation zones and intermediate amounts along the sides of bacteria, also diagrammed on the right. Regions with most growth show least FAM-SE labelling. Left panel is FAM-SE labelling. Bar = 2 µm.Two ways of considering the age of the bacterial poles. (i) Traditionally a single bacterial division is considered. The poles of the original bacterium are considered the old poles while the poles created from the septation zone upon division are considered the new poles. (ii) Over several generations, the poles that are created in each generation can be assigned a generational age. Poles of the first generation (marked with a 1) are considered the oldest poles while poles of the last generation (here marked as 3) are the youngest.', 'hash': '3c4e59df46bf50d5559edd2459dc6274f232edf405dd0346fb18208dcca31a2e'}, {'image_id': 'mmi_5025_f1', 'image_file_name': 'mmi_5025_f1.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f1.jpg', 'caption': 'ActA–RFP topology and localization by immunofluorescence.Diagram of the topology of ActA–RFP association with the bacterial cell wall.Immunofluorescence of wild-type ActA using an ActA antibody. Top panel shows the ActA distribution on strain DPL-4087 (constitutively expressing ActA) and bottom panel shows ActA in strain DPL-4077 induced to stage IV of polarization.Surface distributions of strains expressing ActA–RFP. Left panels show the ActA–RFP distribution by immunofluorescence as in B. Right panels show RFP fluorescence signal on the inside of bacteria. Top panel shows strain JAT-396 (constitutively expressing ActA–RFP) and bottom panel shows ActA–RFP in strain JAT-395 induced to stage IV of polarization. All strains exhibit the same normal polarized surface distribution of ActA with most protein at the poles and least at the septation zone. The ActA–RFP distribution is the same when visualized by immunofluorescence using an ActA antibody to protein accessible on the outside of bacteria and by the RFP signal on the inside of bacteria. A slight shift of RFP signal further inside bacteria can be seen. Bar = 2 µm.', 'hash': 'a1d9b8e41ec833ce6ec17749845eb6f02f4beebdbd88c9971a13fe2824aabdc3'}, {'image_id': 'mmi_5025_f7', 'image_file_name': 'mmi_5025_f7.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f7.jpg', 'caption': 'ActA–RFP preferentially localizes to younger poles.Schematic of the experimental design. Diagram along the bottom indicate the type of staining on poles of the first, second and later (> 2) generations (white numbers). The poles already present at the time of induction are considered the first generation. Bacteria were labelled with FAM-SE (uniformly green) and ActA–RFP was induced. One generation later, bacteria were labelled with Marina Blue-SE (older poles now labelled green and blue, younger poles labelled only in blue) and grown until ActA–RFP began to polarize (late stage III–early stage IV). Several categories of labelled bacteria are now present in the population, as represented by the schematic bacteria in the diagram. Unlabelled poles can be from any generation younger than the second generation.Images from experiment described in A. Top panel shows ActA–RFP signal on bacteria and the second and third panels show FAM-SE and Marina Blue-SE staining. The fourth and fifth panels from the top show the overlays of ActA–RFP (red) with FAM-SE (green) and Marina Blue-SE (blue), respectively. Poles of the first generation are labelled with both green and blue (green arrows) while poles of the second generation are labelled only with blue (blue circles). ActA–RFP signal is markedly absent from older generation poles (see green arrows in top panel) while younger generation poles are associated with ActA–RFP (blue circles in top panel). Bar = 2 µm.Larger images of the two bacteria marked with asterisks in A. Left panel shows ActA–RFP, right panel shows overlays with ActA–RFP in red and either FAM-SE (top, in green) or Marina-SE (bottom, blue). Bar = 1 µm.Quantitation of a parallel set of experiments in which three sequential generations were labelled. The experiment was quantitated at two time points, 1 h apart (3.5 and 4.5 h after induction) when the protein was beginning to accumulate at the poles (late stage III–early stage IV). The percentage of poles associated with ActA–RFP was greatest for the youngest, second generation poles and least for the oldest, pre-first generation poles. An increase in the percentage of poles associated with ActA–RFP was seen from 3.5 to 4.5 h for all generations. Between 188 and 381 bacteria are represented by the percentage values. All values were significantly different (Z-test P < 0.05). Error bars show 95% confidence interval.', 'hash': '4f8f9b3c4ac6a345a1c26540a015536bb3f50a7bc65cfbee2d8fd900175ad295'}, {'image_id': 'mmi_5025_f9', 'image_file_name': 'mmi_5025_f9.jpg', 'image_path': '../data/media_files/PMC1413586/mmi_5025_f9.jpg', 'caption': 'Tracking of polar generations from a single bacterium in an infected Ptk2 cell.The generational lineage originating from the single bacterium within the cell seen in Movie 1. Coloured numbers indicate the generational age of the poles. First generation poles were already existent at time of infection, analogous to the first generation poles in Figs 5 and 6, already existent at time of ActA–RFP induction. Circles indicate the generation that began moving. Single asterisk (*) refers to first generation poles that moved very slowly, and, in the absence of a circled number, stopped moving for a period of time. Double asterisk (**) refers to bacteria that had already begun moving, but continued movement after the subsequent division, and are not diagrammed in B. Light grey, outlined bacteria and their progeny were not trackable in the time-lapse movie because they had moved out of the field of view.Table of bacteria shown in A with the times at which they began moving. Within each generation row, bacteria from the top to bottom in the table are shown from left to right in A. Times shown were rounded to the closest 30 s. Single asterisks (*) indicate the first generation poles marked in A. Double asterisks (**) indicate the poles that continued moving over several generations in A but are shown only for the generation at which the first began moving.', 'hash': '895916f51f8481d0f75990045829f5faac61089f19fd24426f30969477394a5e'}]
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{'mmi_5025_f1': ['In order to directly visualize the process of ActA polarization on the L. monocytogenes surface, we induced the de novo expression of an ActA–RFP fusion (<xref ref-type="fig" rid="mmi_5025_f1">Fig. 1A</xref>; ; Rafelski and Theriot, 2005) in vitro and imaged the RFP signal on the surface of live bacteria at sequential time points over several hours using epifluorescence microscopy. The expression of the ActA–RFP protein was regulated by the endogenous actA promoter in a wild-type (10403S) background (JAT-395; Rafelski and Theriot, 2005). To induce ActA–RFP expression de novo, we began with bacteria grown in rich medium at room temperature, conditions in which no ActA–RFP signal was detectable on the surface of bacteria. The actA promoter is regulated by the PrfA transcription factor, a master regulator for many of the L. monocytogenes virulence genes (Chakraborty et al., 1992; Milohanic et al., 2003). We induced ActA–RFP using a combination of three conditions known to upregulate PrfA activity and therefore virulence gene expression: growth at 37°C (Leimeister-Wachter et al., 1992; Johansson et al., 2002), supplementing media with charcoal to bind an inhibitor of PrfA (Ermolaeva et al., 2004) and providing glucose-1-phosphate (G1P) as the main sugar source both to prevent repression by other sugars (Ripio et al., 1997) and to imitate the metabolic pathway used by L. monocytogenes to promote its efficient replication within the host cell cytosol (Chico-Calero et al., 2002). After several hours of growth in these conditions, a fully polarized ActA–RFP distribution was seen on the surface of bacteria. This distribution was indistinguishable from the normal polarized distribution seen by immunofluorescence on bacteria expressing wild-type ActA inside infected cells (Kocks et al., 1993), the distribution of wild-type ActA induced in the identical background (DPL-4077; Lauer et al., 2002; <xref ref-type="fig" rid="mmi_5025_f1">Fig. 1B</xref>), and the distribution of ActA in ), and the distribution of ActA in L. monocytogenes strains constitutively expressing both wild-type ActA (DPL-4087; Lauer et al., 2002) and ActA–RFP (JAT-396; Rafelski and Theriot, 2005) in broth (<xref ref-type="fig" rid="mmi_5025_f1">Fig. 1B and C</xref>). Further, the distribution of ActA–RFP was the same when imaged directly (RFP fluorescence signal) and indirectly (by immunofluorescence of ActA–RFP present on the outer surface using a polyclonal ActA antibody). During the induction time-course both strains DPL-4077 and JAT-395 exhibited identical intermediate distributions of wild-type ActA and ActA–RFP (Fig. S1) as described in ). Further, the distribution of ActA–RFP was the same when imaged directly (RFP fluorescence signal) and indirectly (by immunofluorescence of ActA–RFP present on the outer surface using a polyclonal ActA antibody). During the induction time-course both strains DPL-4077 and JAT-395 exhibited identical intermediate distributions of wild-type ActA and ActA–RFP (Fig. S1) as described in <xref ref-type="fig" rid="mmi_5025_f2">Fig. 2</xref>..'], 'mmi_5025_f2': ['The relative amounts of total ActA–RFP over the course of a specific induction time-course were determined by Western analysis (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2A</xref>). This analysis showed that relative protein levels increased non-linearly over time with the greatest increase occurring several hours after induction. These results were fully consistent with our microscopic observations of ActA–RFP signal on the bacterial surface during the polarization process.). This analysis showed that relative protein levels increased non-linearly over time with the greatest increase occurring several hours after induction. These results were fully consistent with our microscopic observations of ActA–RFP signal on the bacterial surface during the polarization process.', 'The polarization of ActA–RFP occurred in four ordered stages. During stage I, the first detectable ActA–RFP signal on the surface of L. monocytogenes appeared in one to four distinct spots predominantly along the sides of bacteria (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2B</xref>). This signal could be detected as early as 20 min after induction. Stage II occurred a little later in induction (30–90 min after stage I). Spots could still be seen on some bacteria but many displayed a more irregular accumulation of signal in larger patches (). This signal could be detected as early as 20 min after induction. Stage II occurred a little later in induction (30–90 min after stage I). Spots could still be seen on some bacteria but many displayed a more irregular accumulation of signal in larger patches (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2C</xref>). These patches still localized along the sides of bacteria, and were not found at the poles or at the septation zone. ActA–RFP often seemed to cover a continuous irregular region of the surface and its distribution varied greatly among individual bacteria. As the induction progressed to the next stages, significantly more protein accumulated on the surface and the overall intensity of ActA–RFP signal increased (). These patches still localized along the sides of bacteria, and were not found at the poles or at the septation zone. ActA–RFP often seemed to cover a continuous irregular region of the surface and its distribution varied greatly among individual bacteria. As the induction progressed to the next stages, significantly more protein accumulated on the surface and the overall intensity of ActA–RFP signal increased (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2D</xref>), consistent with the dramatic increase in protein levels seen in the Western analysis. At stage III, ActA–RFP covered greater areas of the surface, but was still confined to the cylindrical body of the bacterium (), consistent with the dramatic increase in protein levels seen in the Western analysis. At stage III, ActA–RFP covered greater areas of the surface, but was still confined to the cylindrical body of the bacterium (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2D</xref>). While there was now some signal along most of the length of the cylindrical body, there were still regions that were brighter in intensity and similar to the initial spots and patches seen in earlier stages (). While there was now some signal along most of the length of the cylindrical body, there were still regions that were brighter in intensity and similar to the initial spots and patches seen in earlier stages (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2D</xref>). The ActA–RFP signal continued to increase until bacteria achieved a fully polarized ActA–RFP distribution at stage IV (). The ActA–RFP signal continued to increase until bacteria achieved a fully polarized ActA–RFP distribution at stage IV (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2E</xref>), in which ActA–RFP was localized at the poles, absent from the septation zone (arrow) and distributed in a continuous fashion along the sides. Between stages II and IV, the amount of ActA–RFP on the surface and its distribution varied greatly among individuals within a population as some accumulated ActA–RFP at their poles earlier than others. The overall time for ActA–RFP to be fully polarized on bacteria varied, taking between 3 and 6 h and on average two to three generations, depending on the particular culture induction and on the bacterial growth rate.), in which ActA–RFP was localized at the poles, absent from the septation zone (arrow) and distributed in a continuous fashion along the sides. Between stages II and IV, the amount of ActA–RFP on the surface and its distribution varied greatly among individuals within a population as some accumulated ActA–RFP at their poles earlier than others. The overall time for ActA–RFP to be fully polarized on bacteria varied, taking between 3 and 6 h and on average two to three generations, depending on the particular culture induction and on the bacterial growth rate.', 'InlA polarization stages I–IV. InlA exhibited stages of polarization similar to those observed for ActA (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2</xref>) over the course of several (three to four) hours.) over the course of several (three to four) hours.', 'As the ActA–RFP signal initially appeared in distinct spots, we wondered how it could then become distributed over the bacterial surface. Because the ActA–RFP protein spans both the cell membrane and the entire cell wall, its distribution could potentially be affected by cell wall growth. It has been established that fluorescently labelled vancomycin (vanc-FL) can be used to probe sites of new cell wall synthesis and that the distribution of vanc-FL along the cylindrical body is helical in B. subtilis (Daniel and Errington, 2003). At stage III of polarization, the ActA–RFP distribution was often semi-regular and reminiscent of a partial helical pattern (arrows in <xref ref-type="fig" rid="mmi_5025_f2">Fig. 2D</xref>). To observe the ActA–RFP distribution pattern relative to the pattern of new cell wall growth, we first incubated growing ). To observe the ActA–RFP distribution pattern relative to the pattern of new cell wall growth, we first incubated growing L. monocytogenes with a mixture of Bodipy FL vancomycin (subsequently referred to as vanc-FL) and unlabelled vancomycin (vanc) and found that the distribution of vanc-FL on the L. monocytogenes surface was similar to that reported on B. subtilis (Daniel and Errington, 2003). Labelling was most concentrated at the septation zone and distributed in a helical fashion along the cylindrical cell body but absent from the poles (<xref ref-type="fig" rid="mmi_5025_f5">Fig. 5A</xref>). We achieved optimal labelling with mixtures containing over 50% vanc (versus vanc-FL) at concentrations near the minimum inhibitory concentration (MIC) for our strains (data not shown), conditions similar to those reported as optimal for labelling of ). We achieved optimal labelling with mixtures containing over 50% vanc (versus vanc-FL) at concentrations near the minimum inhibitory concentration (MIC) for our strains (data not shown), conditions similar to those reported as optimal for labelling of B. subtilis using this technique (Daniel and Errington, 2003).', 'During the earlier steps in the polarization of ActA–RFP, protein accumulated along the sides but remained absent from the poles, a process that took one to two bacterial generations and appeared to be directed by cylindrical cell wall growth. However, it took more than two generations before ActA–RFP began to accumulate directly at the poles to lead to the fully polarized surface distribution (<xref ref-type="fig" rid="mmi_5025_f2">Fig. 2E</xref>), suggesting a second, slower mechanism for polar accumulation of ActA–RFP.), suggesting a second, slower mechanism for polar accumulation of ActA–RFP.'], 'mmi_5025_f3': ['Quantitation of the amount of RFP signal per bacterium during an induction confirmed our visual observations that the greatest increase in ActA–RFP levels on the surface occurred as the ActA–RFP distribution progressed from stage II to stage III (<xref ref-type="fig" rid="mmi_5025_f3">Fig. 3</xref>), precluding the possibility that the sudden increase in the levels of ActA–RFP protein production led to the appearance of protein directly at the poles. Instead we observed a delay between ActA–RFP accumulation along the cylindrical body (stage III) and eventual accumulation directly at the poles (stage IV).), precluding the possibility that the sudden increase in the levels of ActA–RFP protein production led to the appearance of protein directly at the poles. Instead we observed a delay between ActA–RFP accumulation along the cylindrical body (stage III) and eventual accumulation directly at the poles (stage IV).'], 'mmi_5025_f4': ['We examined the de novo polarization of InlA by inducing its expression in vitro identically to ActA induction and then visualizing InlA distribution by immunofluorescence with a monoclonal InlA antibody. We found that InlA protein was polarized on the surface of L. monocytogenes in a manner very similar to ActA (<xref ref-type="fig" rid="mmi_5025_f4">Fig. 4</xref>), first appearing at distinct sites along the sides of bacteria (), first appearing at distinct sites along the sides of bacteria (<xref ref-type="fig" rid="mmi_5025_f4">Fig. 4A</xref>), then being distributed along the entire cylindrical body (), then being distributed along the entire cylindrical body (<xref ref-type="fig" rid="mmi_5025_f4">Fig. 4B and C</xref>), and eventually accumulating at the poles (), and eventually accumulating at the poles (<xref ref-type="fig" rid="mmi_5025_f4">Fig. 4D</xref>). The timing of this process was also very similar to the polarization of ActA, taking several (three to four) hours from initial induction to final polarization.). The timing of this process was also very similar to the polarization of ActA, taking several (three to four) hours from initial induction to final polarization.'], 'mmi_5025_f5': ['We then concurrently imaged both the ActA–RFP and the vanc-FL surface distribution patterns at various stages of polarization. ActA–RFP distributions could be observed both by ActA–RFP signal on live bacteria and by immunofluorescence of ActA–RFP present on the outer surface of fixed bacteria using a polyclonal ActA antibody. The distributions of ActA–RFP and ActA antibody were the same at all polarization stages (see insets in <xref ref-type="fig" rid="mmi_5025_f5">Fig. 5B and C</xref> for examples of stages I and III and Fig. S1). The main difference in the precise localization of ActA–RFP versus antibody signal was due to the presence of RFP on the inside and antibody on the outside of the bacterium (compare inset in for examples of stages I and III and Fig. S1). The main difference in the precise localization of ActA–RFP versus antibody signal was due to the presence of RFP on the inside and antibody on the outside of the bacterium (compare inset in <xref ref-type="fig" rid="mmi_5025_f5">Fig. 5C</xref>). The colocalization of the RFP and the antibody signals at the earliest stage of ActA induction indicated that the distribution of ActA visualized by the ActA–RFP signal was not obscured because of the folding rate of mRFP1 on the ActA–RFP fusion protein. Additionally, immunofluorescence allowed for an enhancement of the earliest, weakly detectable ActA–RFP signal on the surface of bacteria during concurrent imaging with vanc-FL staining (). The colocalization of the RFP and the antibody signals at the earliest stage of ActA induction indicated that the distribution of ActA visualized by the ActA–RFP signal was not obscured because of the folding rate of mRFP1 on the ActA–RFP fusion protein. Additionally, immunofluorescence allowed for an enhancement of the earliest, weakly detectable ActA–RFP signal on the surface of bacteria during concurrent imaging with vanc-FL staining (<xref ref-type="fig" rid="mmi_5025_f5">Fig. 5B</xref>).).', 'At polarization stage I, distinct spots of ActA were detected by immunofluorescence and were seen in regions of the bacterial surface with relatively little vanc-FL staining (<xref ref-type="fig" rid="mmi_5025_f5">Fig. 5B</xref>). Later in polarization (stage III) ActA–RFP and vanc-FL continued to be distributed in a non-overlapping surface distribution on both live and fixed bacteria (). Later in polarization (stage III) ActA–RFP and vanc-FL continued to be distributed in a non-overlapping surface distribution on both live and fixed bacteria (<xref ref-type="fig" rid="mmi_5025_f5">Fig. 5C</xref>). It seemed conceivable that cell wall-associated proteins, such as ActA, might be more easily incorporated into the highly cross-linked cell wall by being secreted directly at sites of new peptidoglycan synthesis suggesting a possible colocalization of earliest ActA–RFP signal with vanc-FL staining. Instead, the observed anti-localization between newest ActA–RFP at the surface and vanc-FL indicates secretion of ActA–RFP occurred at sites distinct from new cell wall synthesis. Further, the helical nature of ActA–RFP distributions and the anti-localized nature of both ActA–RFP and vanc-FL distributions at the later stage III suggest that protein could be spread from discrete spots over the bacterial surface via new cell wall growth. To test this possible requirement of growth for ActA–RFP redistribution we induced ActA–RFP under conditions inhibiting bacterial growth. We found that bacterial growth was required for ActA–RFP accumulation and therefore also for its subsequent polarization (Figs S2 and S3, ). It seemed conceivable that cell wall-associated proteins, such as ActA, might be more easily incorporated into the highly cross-linked cell wall by being secreted directly at sites of new peptidoglycan synthesis suggesting a possible colocalization of earliest ActA–RFP signal with vanc-FL staining. Instead, the observed anti-localization between newest ActA–RFP at the surface and vanc-FL indicates secretion of ActA–RFP occurred at sites distinct from new cell wall synthesis. Further, the helical nature of ActA–RFP distributions and the anti-localized nature of both ActA–RFP and vanc-FL distributions at the later stage III suggest that protein could be spread from discrete spots over the bacterial surface via new cell wall growth. To test this possible requirement of growth for ActA–RFP redistribution we induced ActA–RFP under conditions inhibiting bacterial growth. We found that bacterial growth was required for ActA–RFP accumulation and therefore also for its subsequent polarization (Figs S2 and S3, Supplementary material).', 'The generational lineage originating from the single bacterium within the cell seen in Movie 1. Coloured numbers indicate the generational age of the poles. First generation poles were already existent at time of infection, analogous to the first generation poles in <xref ref-type="fig" rid="mmi_5025_f5">Figs 5</xref> and and <xref ref-type="fig" rid="mmi_5025_f6">6</xref>, already existent at time of ActA–RFP induction. Circles indicate the generation that began moving. Single asterisk (*) refers to first generation poles that moved very slowly, and, in the absence of a circled number, stopped moving for a period of time. Double asterisk (**) refers to bacteria that had already begun moving, but continued movement after the subsequent division, and are not diagrammed in B. Light grey, outlined bacteria and their progeny were not trackable in the time-lapse movie because they had moved out of the field of view., already existent at time of ActA–RFP induction. Circles indicate the generation that began moving. Single asterisk (*) refers to first generation poles that moved very slowly, and, in the absence of a circled number, stopped moving for a period of time. Double asterisk (**) refers to bacteria that had already begun moving, but continued movement after the subsequent division, and are not diagrammed in B. Light grey, outlined bacteria and their progeny were not trackable in the time-lapse movie because they had moved out of the field of view.'], 'mmi_5025_f6': ['Recently, fluorescent succimidyl esters (SEs) have been used to covalently label outer membrane proteins in E. coli (de Pedro et al., 2004). The Gram-positive cell wall contains many covalently attached surface proteins that would likely be labelled by this method (Navarre and Schneewind, 1999; Cabanes et al., 2002). We successfully used this method to label the surface of L. monocytogenes and visualize the differential growth rates along the bacterial surface. Bacteria were labelled with 5(6)-carboxyfluorescein succimidyl ester (FAM-SE) and initially showed uniform labelling of the entire bacterial surface including the septation zones (<xref ref-type="fig" rid="mmi_5025_f6">Fig. 6A</xref>). Bacteria were then grown for 2 h and a clear segregation of fluorescent signal was seen (). Bacteria were then grown for 2 h and a clear segregation of fluorescent signal was seen (<xref ref-type="fig" rid="mmi_5025_f6">Fig. 6B</xref>), with least signal at the septation zone (arrows), most at the poles, and intermediate signal along the cylindrical body as seen previously for cell wall segregation in ), with least signal at the septation zone (arrows), most at the poles, and intermediate signal along the cylindrical body as seen previously for cell wall segregation in E. coli (de Pedro et al., 1997). The FAM-SE signal at the poles after growth was overall less than at the time of initial labelling, indicating that some shedding of cell wall material occurred, even at the relatively inert poles. This labelling pattern is consistent with the greatest dilution of signal occurring at the most rapidly growing septation zone, an intermediate dilution of signal through cylindrical growth, where signal would uniformly decrease as the helical pattern of growth continuously changed, and the least dilution due to little cell wall growth or turnover at the poles. As opposed to vanc-FL, which labels newest cell wall growth at one moment in time, FAM-SE signal effectively labels the oldest cell wall and the signal can persist as bacteria grow over many generations.', 'Within a single generation a bacterium grows, forms a septation zone, and then divides such that each of the two progeny has two bacterial poles. The one that was a pole since the beginning of that cell cycle is considered the old pole, while the one that originated from the septation zone is considered the new pole (<xref ref-type="fig" rid="mmi_5025_f6">Fig. 6C, i</xref>). However, poles can also be assigned a generational age if a single bacterium is tracked over several division cycles (). However, poles can also be assigned a generational age if a single bacterium is tracked over several division cycles (<xref ref-type="fig" rid="mmi_5025_f6">Fig. 6C, ii</xref>). While each pole is created from the septation zone of the previous generation, its generational age is determined by the division cycle during which it was created.). While each pole is created from the septation zone of the previous generation, its generational age is determined by the division cycle during which it was created.'], 'mmi_5025_f7': ['In an example of a sequential labelling experiment (<xref ref-type="fig" rid="mmi_5025_f7">Fig. 7</xref>), bacteria were first labelled with FAM-SE (all labelled poles referred to as the first generation) and ActA–RFP was induced (), bacteria were first labelled with FAM-SE (all labelled poles referred to as the first generation) and ActA–RFP was induced (<xref ref-type="fig" rid="mmi_5025_f7">Fig. 7A</xref>). One generation later, bacteria were labelled with Marina Blue-SE (newly labelled poles referred to as the second generation; ). One generation later, bacteria were labelled with Marina Blue-SE (newly labelled poles referred to as the second generation; <xref ref-type="fig" rid="mmi_5025_f6">Fig. 6A</xref>). Several generations of growth (2.5 generations) later ActA–RFP was beginning to accumulate at the pole, between late stage III and early stage IV (). Several generations of growth (2.5 generations) later ActA–RFP was beginning to accumulate at the pole, between late stage III and early stage IV (<xref ref-type="fig" rid="mmi_5025_f7">Fig. 7A and B</xref>). Those poles that retained both FAM-SE and Marina Blue-SE signal corresponded to older, first generation poles that were already poles at the time of ActA–RFP induction (green arrows in ). Those poles that retained both FAM-SE and Marina Blue-SE signal corresponded to older, first generation poles that were already poles at the time of ActA–RFP induction (green arrows in <xref ref-type="fig" rid="mmi_5025_f7">Fig. 7B</xref>). Younger, second generation poles, non-existent at the time of the first labelling reaction (with FAM-SE) and ActA–RFP induction could only retain Marina Blue-SE signal as they were labelled one generation later (blue circles in ). Younger, second generation poles, non-existent at the time of the first labelling reaction (with FAM-SE) and ActA–RFP induction could only retain Marina Blue-SE signal as they were labelled one generation later (blue circles in <xref ref-type="fig" rid="mmi_5025_f7">Fig. 7B</xref>). Correlating the presence of ActA–RFP at the poles with the generational age of those poles showed that often ActA–RFP signal was markedly absent from the older, first generation poles (green arrows and asterisks in ). Correlating the presence of ActA–RFP at the poles with the generational age of those poles showed that often ActA–RFP signal was markedly absent from the older, first generation poles (green arrows and asterisks in <xref ref-type="fig" rid="mmi_5025_f7">Fig. 7B and C</xref>) while younger, second generation poles (blue circles and asterisks in ) while younger, second generation poles (blue circles and asterisks in <xref ref-type="fig" rid="mmi_5025_f7">Fig. 7B and C</xref>) were associated with polarized ActA–RFP (61% of second generation poles associated with ActA–RFP compared with 21% of first generation poles in this experiment). We also noticed that often younger, second generation poles were labelled less strongly than those of the older, first generation poles () were associated with polarized ActA–RFP (61% of second generation poles associated with ActA–RFP compared with 21% of first generation poles in this experiment). We also noticed that often younger, second generation poles were labelled less strongly than those of the older, first generation poles (<xref ref-type="fig" rid="mmi_5025_f7">Fig. 7B</xref>; compare intensity of labelling on bacteria indicated with circles in Marina-SE panel with those indicated by arrows in FAM-SE panel), consistent with the hypothesis that younger poles remain more dynamic over at least one bacterial generation.; compare intensity of labelling on bacteria indicated with circles in Marina-SE panel with those indicated by arrows in FAM-SE panel), consistent with the hypothesis that younger poles remain more dynamic over at least one bacterial generation.', 'We calculated the frequency of poles associated with ActA–RFP for poles of increasing generational age over time to further quantitate our observations. We performed a parallel set of experiments such that three sequential generations of poles were labelled by labelling the middle generation in both experiments (see Experimental procedures). Bacteria were labelled one generation prior to ActA–RFP induction (pre-first generation), at the time of ActA–RFP induction (first generation) and one generation post induction (second generation). The percentage of poles associated with ActA–RFP was determined for each labelled polar generation at 3.5 and 4.5 h after induction, between late stage III and early stage IV, when ActA–RFP was beginning to accumulate at the poles (<xref ref-type="fig" rid="mmi_5025_f7">Fig. 7D</xref>). At both time points examined, the poles from the youngest generation (second) most efficiently accumulated ActA–RFP while the poles from the oldest generation (pre-first) accumulated protein the least efficiently. There was also an increase in the frequency of poles associated with ActA–RFP within each generation between the two time points (all pairwise comparisons were statistically significantly different; ). At both time points examined, the poles from the youngest generation (second) most efficiently accumulated ActA–RFP while the poles from the oldest generation (pre-first) accumulated protein the least efficiently. There was also an increase in the frequency of poles associated with ActA–RFP within each generation between the two time points (all pairwise comparisons were statistically significantly different; P < 0.05). The preference for ActA–RFP to be associated with the younger poles rather than with the older poles, and the increase in the frequency of polar ActA–RFP over time was found in five other sequential-generation labelling experiments, although the percentages and time points after induction varied depending on the growth and induction of each specific experiment. Thus, the gradual accumulation of ActA–RFP at the pole over several generations occurred preferentially at poles of younger generational age, suggesting that these poles may be more dynamic than poles of older generations, possibly due to their more recent creation from the rapidly growing septation zone.'], 'mmi_5025_f8': ['To investigate the fate of ActA protein that had accumulated at the poles, we continued to induce ActA–RFP for 2 h after it had reached stage IV. L. monocytogenes continued to accumulate ActA–RFP on the surface until the ActA–RFP distribution eventually became uniform (<xref ref-type="fig" rid="mmi_5025_f8">Fig. 8A</xref>). At this time we covalently labelled the surface of these bacteria with FAM-SE (). At this time we covalently labelled the surface of these bacteria with FAM-SE (<xref ref-type="fig" rid="mmi_5025_f8">Fig. 8A</xref>) and allowed them to grow in non-inducing conditions (rich BHI media at room temperature). After two generations of growth, the oldest, most inert bacterial poles, initially labelled with FAM-SE, could be clearly identified () and allowed them to grow in non-inducing conditions (rich BHI media at room temperature). After two generations of growth, the oldest, most inert bacterial poles, initially labelled with FAM-SE, could be clearly identified (<xref ref-type="fig" rid="mmi_5025_f8">Fig. 8B</xref>). ActA–RFP signal was uniformly diluted over the cylindrical surface (compare ). ActA–RFP signal was uniformly diluted over the cylindrical surface (compare <xref ref-type="fig" rid="mmi_5025_f8">Fig. 8A and B</xref>) but bright signal was specifically retained at the poles that remained labelled with FAM-SE. These results indicate a similar lack of protein mobility as seen at poles in ) but bright signal was specifically retained at the poles that remained labelled with FAM-SE. These results indicate a similar lack of protein mobility as seen at poles in E. coli (de Pedro et al., 2004). Thus, once ActA–RFP had accumulated at the pole, it could be retained there over several generations, allowing the pole to act as a trap and enhance the polarization of ActA–RFP.'], 'mmi_5025_f9': ['The polar distribution of ActA on the surface of L. monocytogenes is required for motility within an infected host cell (Smith et al., 1995), where actin tails form on the bacterial poles associated with the higher density of ActA protein (Kocks et al., 1993; Rafelski and Theriot, 2005). Thus, the ability of bacterial poles to support actin-based motility in vivo should indicate the presence of ActA at those poles. Analogous to our ActA–RFP induction in vitro, a bacterium that initially infects a host cell must begin expression and polarization of the ActA protein de novo. We investigated whether the generational age of the pole could affect the ability of L. monocytogenes to move by actin-based motility within an infected host cell. A single bacterium inside an infected Potoroo tridactylis kidney epithelial (PtK2) cell was imaged for five generations beginning 40 min after infection (Supplementary material Movie 1). By following the bacterium through multiple division cycles, we correlated the generational age of a pole with the time at which that pole formed an actin tail and bacteria began moving (<xref ref-type="fig" rid="mmi_5025_f9">Fig. 9A and B</xref>).).', 'We are grateful to Matt Footer for providing polyclonal ActA antibody and to Matt Footer and Jarrett Wrenn for assistance in creating cell wall labelling protocols. We thank Dan Portnoy for his contribution to the original video data used for <xref ref-type="fig" rid="mmi_5025_f9">Fig. 9</xref> and for providing bacterial strains. We thank Eddie Johnson at Cedarlane Laboratories for providing InlA monoclonal antibody. We are grateful to Aretha Fiebig and Zach Pincus for advice on visual representations of polarization. We thank the Theriot lab for valuable and stimulating discussions, especially Cyrus Wilson, Catherine Lacayo and Natalie Dye. We also thank Bill Burkholder, Natalie Dye, Catherine Lacayo and Mike Ruvolo for critical reading of the manuscript. This work was supported by NIH RO1 AI36929 and the American Heart Association. S.M.R. was supported by a NSF Predoctoral Fellowship. and for providing bacterial strains. We thank Eddie Johnson at Cedarlane Laboratories for providing InlA monoclonal antibody. We are grateful to Aretha Fiebig and Zach Pincus for advice on visual representations of polarization. We thank the Theriot lab for valuable and stimulating discussions, especially Cyrus Wilson, Catherine Lacayo and Natalie Dye. We also thank Bill Burkholder, Natalie Dye, Catherine Lacayo and Mike Ruvolo for critical reading of the manuscript. This work was supported by NIH RO1 AI36929 and the American Heart Association. S.M.R. was supported by a NSF Predoctoral Fellowship.'], 'mmi_5025_f10': ['Here we have shown a new mechanism for the polarization of surface proteins on bacteria, distinct from experimentally observed results for other proteins (Steinhauer et al., 1999; Robbins et al., 2001; Rudner et al., 2002) and previously proposed general models (Shapiro et al., 2002; Pugsley and Buddelmeijer, 2004). Our results indicate that the ActA protein is neither initially targeted directly to the poles nor secreted uniformly, but instead is secreted at several specific sites along the cylindrical body. This observation requires modifications of the simplest passive polarization model. Here we discuss a more detailed multi-step model for the passive polarization of ActA: (i) non-uniform secretion and incorporation into the cell wall at distinct sites along the cylindrical body; (ii) spread of ActA over the cylindrical surface due to helical cell wall growth; and (iii) gradual accumulation of ActA at the hemispherical pole through a slow incorporation of cylindrical wall material at a rate proportional to the generational age of the pole (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10</xref>).).', 'Initially, ActA–RFP appeared at distinct sites along the cylindrical body (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10A, i</xref>). At these sites both the ActA–RFP signal on the inside and the immunofluorescence signal of ActA–RFP accessible to the outside were the same. Contrary to SpoIVFB in ). At these sites both the ActA–RFP signal on the inside and the immunofluorescence signal of ActA–RFP accessible to the outside were the same. Contrary to SpoIVFB in B. subtilis, which is redistributed over the bacterial surface by lateral diffusion within the membrane (Rudner et al., 2002), our results in L. monocytogenes indicate that ActA does not laterally diffuse over such large (> 1 µm) distances within the membrane prior to being incorporated into the cell wall. Further, if the Sec apparatus were directly associated with the cell wall, it would travel along with the cell wall during cylindrical growth and the localization of both Sec and the proteins it secreted (including ActA) would remain in one region on the bacterial surface, preventing the observed spread of ActA–RFP over the entire bacterial surface. Instead, our results prove that the Sec apparatus must be anchored in space as the cell wall grows around it, which could be possible through an association with an internal scaffold, or through a dynamic localization independent of cylindrical cell wall growth. The sites of new cell wall synthesis have been shown to be regulated by one such internal scaffold in B. subtilis, Mbl (Daniel and Errington, 2003). Our results of an anti-localization between ActA–RFP and vanc-FL suggest that the internal scaffold directing cylindrical cell wall growth may be distinct from the localization of the Sec apparatus, consistent with the finding that neither Mbl nor MreB is responsible for the observed Sec distribution in B. subtilis (Campo et al., 2004).', 'The observed steps of ActA–RFP polarization are inconsistent with a mechanism of uniform diffusion of protein within the cell wall away from the sites of initial incorporation. Instead, the continuous incorporation of new material into the rigid cell wall during bacterial growth appears to directly affect the distribution of this protein that spans the cell wall, a result that may also be generally true for proteins such as InlA that are covalently attached to the peptidoglycan. Previous experiments suggest that the synthesis of new cell wall occurs in a helical pattern along the entire length of the cylindrical cell body (Daniel and Errington, 2003). This sort of growth pattern could lead to the spread of protein, independently inserted into the cell wall, into irregularly shaped patches by two non-exclusive mechanisms: protein redistribution and sequential secretion (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10A</xref>). Protein redistribution on the surface would occur when ActA adjacent to sites of new cell wall synthesis is pushed away from those sites, travelling with regions of older cell wall. As new cell wall is inserted helically into the cylindrical body, it would cause a twisting and distortion of older cell wall material (). Protein redistribution on the surface would occur when ActA adjacent to sites of new cell wall synthesis is pushed away from those sites, travelling with regions of older cell wall. As new cell wall is inserted helically into the cylindrical body, it would cause a twisting and distortion of older cell wall material (Mendelson, 1976; Carballido-Lopez and Errington, 2003; Daniel and Errington, 2003) and therefore a redistribution of ActA protein already associated with the older cell wall into irregular patches on the bacterial surface (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10A</xref>, ii and iii). Concurrently, the secretion apparatus, whose localization is not directed by the cell wall, would continue to insert ActA into the cell wall at specific sites on the surface. Movement of the cell wall, directed by new cell wall synthesis, over these sites would lead to the sequential secretion of ActA to the surface in irregular patches of helical nature (, ii and iii). Concurrently, the secretion apparatus, whose localization is not directed by the cell wall, would continue to insert ActA into the cell wall at specific sites on the surface. Movement of the cell wall, directed by new cell wall synthesis, over these sites would lead to the sequential secretion of ActA to the surface in irregular patches of helical nature (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10A, ii–iv</xref>). With persistent secretion and continuous cell wall remodelling, the ActA protein will eventually cover the cylindrical cell body (). With persistent secretion and continuous cell wall remodelling, the ActA protein will eventually cover the cylindrical cell body (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10B, i and ii</xref>).).', 'Although bacterial poles are known to be relatively inert, with very little new cell wall growth, it was previously shown in B. subtilis that new material did accumulate at the poles and seemed to do so in a much slower manner than along the cylindrical body, appearing first at regions of the pole proximal to the cylindrical body and eventually at the tip of the pole (Clarke-Sturman et al., 1989). This accumulation of new material at the pole could be due to a slower rate of new cell wall synthesis at the different regions of the pole or through remodelling of the pole due to gradual incorporation of cylindrical cell wall material from the sides. As we found that ActA–RFP was not secreted at the poles and accumulated along the cylindrical cell body prior to polarization, the fact that ActA–RFP did eventually appear at the poles suggests that ActA redistribution occurred by the second mechanism of cylindrical cell wall incorporation into the poles (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10B, iii</xref>). We propose that the rate at which cylindrical cell wall material, and therefore also ActA, is incorporated into the poles is a function of the generational age of the poles (). We propose that the rate at which cylindrical cell wall material, and therefore also ActA, is incorporated into the poles is a function of the generational age of the poles (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10B</xref>). That is, poles of younger generations are more dynamic than those of older generations because of the dramatic change in growth rates as a rapidly growing septation zone gradually transforms into an inert pole over several bacterial divisions.). That is, poles of younger generations are more dynamic than those of older generations because of the dramatic change in growth rates as a rapidly growing septation zone gradually transforms into an inert pole over several bacterial divisions.', 'Overall, our results suggest that the polarization of the ActA protein on the surface of L. monocytogenes is a direct and inevitable consequence of the pattern of cell wall growth rates and dynamics along the bacterial cell. Furthermore, it seems inevitable that this sort of mechanism should occur for other cell wall-associated proteins on Gram-positive bacteria. Different final surface distributions could be achieved by regulating protein surface half-life via secretion and degradation rates. The topology of ActA is such that the protein spans both the cell membrane and the entire cell wall. This topology could increase the residence time of ActA on the surface as older cell wall would be turned over and shed but ActA could be left behind because of its association with the membrane (<xref ref-type="fig" rid="mmi_5025_f10">Fig. 10B, iii</xref>). A genomic analysis has shown that there are at least 10 other proteins in the ). A genomic analysis has shown that there are at least 10 other proteins in the L. monocytogenes genome that could share this topology (Cabanes et al., 2002). One of these, SvpA, was recently shown to be associated with the cell wall similarly to ActA (Borezee et al., 2001). SvpA additionally showed a polarized surface distribution (Bierne et al., 2004) similar to that of ActA, consistent with our general hypothesis.']}
|
Listeria monocytogenes Mechanism of polarization of surface protein ActA
| null |
Mol Microbiol
|
1139472000
|
The polar distribution of the ActA protein on the surface of the Gram-positive intracellular bacterial pathogen, Listeria monocytogenes, is required for bacterial actin-based motility and successful infection. ActA spans both the bacterial membrane and the peptidoglycan cell wall. We have directly examined the de novo ActA polarization process in vitro by using an ActA-RFP (red fluorescent protein) fusion. After induction of expression, ActA initially appeared at distinct sites along the sides of bacteria and was then redistributed over the entire cylindrical cell body through helical cell wall growth. The accumulation of ActA at the bacterial poles displayed slower kinetics, occurring over several bacterial generations. ActA accumulated more efficiently at younger, less inert poles, and proper polarization required an optimal balance between protein secretion and bacterial growth rates. Within infected host cells, younger generations of L. monocytogenes initiated motility more quickly than older ones, consistent with our in vitro observations of de novo ActA polarization. We propose a model in which the polarization of ActA, and possibly other Gram-positive cell wall-associated proteins, may be a direct consequence of the differential cell wall growth rates along the bacterium and dependent on the relative rates of protein secretion, protein degradation and bacterial growth.
|
[
"Bacterial Proteins",
"Cell Membrane",
"Cell Polarity",
"Cell Wall",
"Listeria monocytogenes",
"Listeriosis",
"Membrane Proteins",
"Models, Biological"
] |
other
|
PMC1413586
| null |
54
|
[
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] |
Mol Microbiol. 2006 Feb 9; 59(4):1262-1279
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NO-CC CODE
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D-MAP induces antibody responses and recruitment of myeloid cells via adaptive immunity.a-c) Measurement of anti-D specific IgG subtype antibodies by ELISA 21 days following wound healing experiments in SKH1 mice treated with indicated hydrogels. d-f) Measurement of anti-L specific IgG subtype antibodies by ELISA 21 days following wound healing experiments in SKH1 mice treated with indicated hydrogels. Each data point represents one animal and all analysis in a-f is by unpaired two-tailed t-test comparing each condition to L only. g-i) Measurement of anti-D specific IgG subtype antibodies in Balb/c or Balb/c.Rag2−/−γc−/− mice given a subcutaneous injection of D-MAP 21 days after injection. Each data point represents one animal and all analysis in g-I is by unpaired two-tailed t-test (** denotes p=0.0022). j-l) Representative examples of confocal immunofluorescent imaging for CD11b, DAPI, and hydrogel from subcutaneous implants of L- or D-MAP hydrogel implants in Balb/c or Balb/c.Rag2−/−γc−/− mice. Scale = 200μm. (j) and quantification of total DAPI+ cells (k) and CD11b+ myeloid cells (l). Data is plotted as a scatter plot showing the mean and standard deviation. Each point represents average of 3 slides for each wound. All analysis is by unpaired two-tailed t-test (* denotes p=0.0455, *** denotes p=0.0006, **** denotes p<0.0001) represent statistical significance by student t-test for the comparison indicated.
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nihms-1632597-f0004
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2
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5e49543a8f0de719927a8b43922605b16e39cd4356879b5e593f5b9b0be7167a
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nihms-1632597-f0004.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
1372,
2055
] |
[{'image_id': 'nihms-1632597-f0001', 'image_file_name': 'nihms-1632597-f0001.jpg', 'image_path': '../data/media_files/PMC8005402/nihms-1632597-f0001.jpg', 'caption': 'D-MAP hydrogel degradation is enhanced in wounds of SKH1 hairless mice.a) Rheological characterization of MAP hydrogels composed of L or D-peptide crosslinked microgels. The r-ratio (ratio of -SH to -VS) used to form the microgels was changed to arrive at the same storage modulus for both L and D MAP scaffolds. NS represents a no statistical significance between the L MAP scaffold to the D-MAP scaffold indicated using a two-tailed student t-test. b) Fabricated L or D hydrogels were tested for in vitro enzymolysis behavior through exposure to a solution of collagenase I (5U/mL). c-f) Representative low power view of H&E sections from healed skin 21 days after splinted excisional wounding from a Sham (c), L-MAP (d), D-MAP (e), and 1:1 mixture of L-MAP and D-MAP treated wound in SKH1 mice (f). g-i) Histologic quantification of dermal thickness including gels (in mm), hair follicles, and sebaceous glands. Each point represents average of 2 sections from 2 separate slides of one wound. Each data point represents one animal and all analysis is by one-way ANOVA (respective F-values (3,12): 4.448, 10.89, 5.074, stars denote statistical significance by Tukey multiple comparisons test: g) *p=0.0460, **p=0.0341, h) *p=0.0220, **p= 0.0133, ***p=0.0007 i) *p=0.0110). j) 28 days after incisional, unsplinted wounds were created, healed wounds that were treated without or with different hydrogels were tested against unwounded skin in the same mouse. Tensile strength was evaluated by tensiometry and reported as a percentage of the tensile strength of the scar tissue when compared to the normal skin of the same mouse. Each data point represents average of two measurement from one wound, separate from wounds used in b-i with analysis by one-way ANOVA (F-value (3, 20): 5.400, *p=0.0273, **p=0.0131). Data is plotted as a scatter plot showing the mean and standard deviation.', 'hash': 'e82ece9eb290968dd80d6b54b2893822460d97fc4f3315fe5f828011342ef73f'}, {'image_id': 'nihms-1632597-f0006', 'image_file_name': 'nihms-1632597-f0006.jpg', 'image_path': '../data/media_files/PMC8005402/nihms-1632597-f0006.jpg', 'caption': 'D-MAP changes the wound fate from scar formation to regeneration by type 2 immune activation.a) Representation of amino acid chirality within the cross-linking peptides, microfluidic formation of the hydrogel microbeads incorporating L- or D- chirality peptides. b) The use of L- or D-MAP in a wound healing model demonstrates that both L- or D-MAP hydrogel fill the wound defect. While wounds that heal in the absence of hydrogel heal with an atrophic scar with loss of tissue, the epidermis forms over the scaffold in both cases and allows for increased dermal thickness. However, in the case of D-MAP, the hydrogel activates the adaptive immune system over time, resulting in tissue remodeling and skin regeneration as the adaptive immune system degrades the D-MAP scaffold.', 'hash': 'c3ad0555e17304ce2f21519d49def6a11a44f796d59a47533100aadb83dd3102'}, {'image_id': 'nihms-1632597-f0003', 'image_file_name': 'nihms-1632597-f0003.jpg', 'image_path': '../data/media_files/PMC8005402/nihms-1632597-f0003.jpg', 'caption': 'Peptide recognition by pattern recognition receptors is not required for myeloid cell recruitment.a) Representative confocal immunofluorescent images staining myeloid cells (CD11b+) within healed wounds of B6 mice in the absence or presence of the indicated hydrogel. Scale = 100μm. b) Quantification of CD11b+ cellular infiltrate in healed tissue 21 days after wounding in the presence or absence of hydrogel. Each point represents average of 3 slides for each wound. All analysis is by one-way ANOVA (F-value (3,21): 41.10; **** denotes p<0.0001). c-d) Representative high-resolution confocal immunofluorescence imaging for CD11b, F4/80, DAPI, and IL-33 from subcutaneous implants of L- or D-MAP hydrogel implants (c) and quantification of IL-33 producing macrophages and other myeloid cells at hydrogel edge and core. n=5 B6 mice, mean +/− SEM (d), multiple t-tests adjusted for multiple comparisons using Holm-Sidak method (** denotes p=0.00014). Scale = 100μm. e-h) Murine bone marrow derived macrophages (BMDMs) from B6 mice were stimulated with 500 μg/ml of full-length L- or D- crosslinker peptide in the presence or absence of lipopolysaccharide (10ng/ml) for 6 hours. Shown are qPCR results of 4 inflammatory genes from two separate experiment performed with n = 6. All analysis is by one-way ANOVA (Respective F-values (5, 30): 15.66, 17.62, 107.1, and 8.229, ** denotes p=0.009 and **** denotes p<0.0001) i-l) BMDMs were stimulated with LPS (10ng/ml) or cleaved D-crosslinker peptide (500μg/ml) that possessed an N-terminal D-amino acid. Experiment was performed in triplicate. All analysis is by one-way ANOVA (Respective F-values (2,6): 20.28, 30.86, 2.178, and 22.72). Data is plotted as a scatter plot showing the mean and standard deviation.', 'hash': '1ebae3b7346bbddc5fa4b6302a7cdc927c984b56a4295f17247a76cc0934ff13'}, {'image_id': 'nihms-1632597-f0004', 'image_file_name': 'nihms-1632597-f0004.jpg', 'image_path': '../data/media_files/PMC8005402/nihms-1632597-f0004.jpg', 'caption': 'D-MAP induces antibody responses and recruitment of myeloid cells via adaptive immunity.a-c) Measurement of anti-D specific IgG subtype antibodies by ELISA 21 days following wound healing experiments in SKH1 mice treated with indicated hydrogels. d-f) Measurement of anti-L specific IgG subtype antibodies by ELISA 21 days following wound healing experiments in SKH1 mice treated with indicated hydrogels. Each data point represents one animal and all analysis in a-f is by unpaired two-tailed t-test comparing each condition to L only. g-i) Measurement of anti-D specific IgG subtype antibodies in Balb/c or Balb/c.Rag2−/−γc−/− mice given a subcutaneous injection of D-MAP 21 days after injection. Each data point represents one animal and all analysis in g-I is by unpaired two-tailed t-test (** denotes p=0.0022). j-l) Representative examples of confocal immunofluorescent imaging for CD11b, DAPI, and hydrogel from subcutaneous implants of L- or D-MAP hydrogel implants in Balb/c or Balb/c.Rag2−/−γc−/− mice. Scale = 200μm. (j) and quantification of total DAPI+ cells (k) and CD11b+ myeloid cells (l). Data is plotted as a scatter plot showing the mean and standard deviation. Each point represents average of 3 slides for each wound. All analysis is by unpaired two-tailed t-test (* denotes p=0.0455, *** denotes p=0.0006, **** denotes p<0.0001) represent statistical significance by student t-test for the comparison indicated.', 'hash': '5e49543a8f0de719927a8b43922605b16e39cd4356879b5e593f5b9b0be7167a'}, {'image_id': 'nihms-1632597-f0005', 'image_file_name': 'nihms-1632597-f0005.jpg', 'image_path': '../data/media_files/PMC8005402/nihms-1632597-f0005.jpg', 'caption': 'D-MAP requires an intact adaptive immunity to induce hair follicle neogenesis.a) Representative examples of gross clinical images of healed splinted excisional wounds in B6 or B6.Rag1−/− mice by DSLR camera 17 days later treated as sham (no hydrogel) or 1:1 L/D-MAP treatment. Scale = 5mm. b) Histologic sections of healed tissue from B6 or B6.Rag1−/− mice. Scale = 200μm. White dashed lines denotes wounded area. Quantification of the average numbers of c) hair follicles and d) sebaceous glands from 3 histological sections per sample from B6 mice and B6.Rag1−/− mice. Data is plotted as a scatter plot showing the mean and SEM. * denotes two-tailed p=0.002 by Mann Whitney test, for inter-strain/identical treatment comparison and ** denotes p=0.0039 by Wilcoxon test for intra-strain/different treatment comparison.', 'hash': 'cc4b08f569f7ee83511e4c4af0f312273faf9749c2615dab75190b75c0fd9424'}, {'image_id': 'nihms-1632597-f0002', 'image_file_name': 'nihms-1632597-f0002.jpg', 'image_path': '../data/media_files/PMC8005402/nihms-1632597-f0002.jpg', 'caption': 'D-MAP hydrogel induces neogenesis of hair follicles in full-thickness skin wounds in B6 mice.a-f) H&E (a, c, d) and Trichrome staining (b, e, f) of healed 4-mm full-thickness splinted skin wound on day 18. Control (sham-treated) wounds heal with scarring (a, c, e), while D-MAP gel treated wounds form numerous epidermal cysts (asterisks) and, prominently, regenerate de novo hair follicles (green arrowheads) (b, d, f). In some instances, neogenic hair follicles form in close association with epidermal cysts. As compared to normal, pre-existing anagen hair follicles at the wound edges, neogenic hair follicles display early anagen stage morphology (Wound edges in b-d are outlined and D-MAP hydrogel remnants in b are marked with red arrowheads). g-h) Immunostaining for epithelial marker KRT5 (green) and adipocyte marker PLIN (red), reveals normal KRT5+ anagen hair follicles and many mature PLIN+ dermal adipocytes (left panels in g and h). Regeneration of new KRT5+ hair follicles (arrowheads in h) along with KRT5+ epidermal cysts is observed only in D-MAP hydrogel-treated wounds (right panel g vs. h). No neogenic adipocytes are observed in hair-forming D-MAP-treated wounds. i-j) Immunostaining for SOX9 (green) and SMA (red), reveals many SOX9+ epithelial cells within the bulge region of neogenic hair follicles in day 18 DMAP-treated wounds (arrowheads in j). In contrast, in control (sham-treated) wounds that undergo scarring, dermal wound portion contains many Sox9+ cells, many of which also co-express contractile marker SMA (i). Expression of SMA is also seen in both control and D-MAP-treated samples in blood vessels. Scales in a-j = 100 μm. The images are representative of slides from 4 animals per group.', 'hash': '1e6ad764d695d9dcd1d7bc2f8d73a1bbdc461e57210a8a6aad09cdd949f48475'}]
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{'nihms-1632597-f0001': ['We first used enantiomeric chemistry to change degradation rate without changing the initial material properties (e.g. hydrophobicity, mesh size, and charge) of the hydrogel4. All amino acids at the site of enzymatic cleavage for the MMP-degradable peptide were changed to D-amino acids (Ac-GCRDGPQDGIDWDGQDRCG-NH2, D-peptide). We matched the stiffness (i.e. storage modulus) by rheology of both the D-peptide MAP (D-MAP) and L-peptide (L-MAP) formulations to that used in our previous MAP-based cutaneous application (~500Pa; <xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1a</xref>). After formulation optimization, we generated the microsphere particles using a previously published microfluidic technique). After formulation optimization, we generated the microsphere particles using a previously published microfluidic technique1. Following application of Collagenase I to L-MAP, D-MAP or a 50% mixture of D-MAP and L-MAP (1:1 L/D-MAP), the L-MAP hydrogel degraded within minutes, while the degradation of D-MAP by itself or within a mixture with L-MAP was minimal even after an 1 hour (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1b</xref> and and Supplementary Figure 1).', 'Since no differences in wound closure results were noted, we next examined whether the degradation of hydrogels containing D amino acid cross-linkers was slowed in vivo by examining excised tissue 21 days after the wound was completely healed. Unexpectedly, histological sections of wounds treated with D-MAP or a 1:1 L/D-MAP hydrogel mixture displayed minimal to no hydrogel persistence 21 days after wounding, nearing levels seen in mice not treated with hydrogel (Sham), whereas wounds treated with L-MAP hydrogel displayed large amounts of hydrogel remaining (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1c</xref>––<xref rid="nihms-1632597-f0001" ref-type="fig">f</xref>).).', 'Of note, initial examination of histologic sections of D-MAP and 1:1 L/D-MAP displayed a much different overall appearance than that of healed sham- or L-MAP-treated wounds. Previous reports suggest that, unlike large excisional wounds in adult mice (wounds larger than 1×1 cm) that result in significant regenerative healing with wound induced hair neogenesis (WIHN)11–13, wounds smaller than 1×1cm in mice, like the punch biopsies performed in our studies, typically heal without regeneration of new hair and fat and, instead, form scars12,14,15. Despite these reports, when the correct regenerative cues are provided from wound fibroblasts, through transgenic activation of specific Hedgehog signals, small wounds can regenerate16. Consistent with these results, histological examination of 4-mm excisional splinted wounds in mice that did not receive hydrogel (sham) displayed the typical appearance of scar tissue with a flattened epidermis, a thinned dermis with horizontally-oriented collagen bundles, vertically-oriented blood vessels, and lack of hair follicles and sebaceous glands (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1c</xref>, , <xref rid="nihms-1632597-f0001" ref-type="fig">g</xref>––<xref rid="nihms-1632597-f0001" ref-type="fig">i</xref>). Tissue from mice treated with L-MAP hydrogel displayed a similar appearance, but with thicker overall tissue compared to sham wounds, due to the substantial residual L-MAP hydrogels (). Tissue from mice treated with L-MAP hydrogel displayed a similar appearance, but with thicker overall tissue compared to sham wounds, due to the substantial residual L-MAP hydrogels (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1d</xref>, , <xref rid="nihms-1632597-f0001" ref-type="fig">g</xref>). Within the dermis surrounding the hydrogel, fibroblasts secreting collagen/extracellular matrix and blood vessels formed between the hydrogel microparticles (). Within the dermis surrounding the hydrogel, fibroblasts secreting collagen/extracellular matrix and blood vessels formed between the hydrogel microparticles (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1d</xref>). Only rare hair follicles and associated sebaceous glands were observed in the wound areas (). Only rare hair follicles and associated sebaceous glands were observed in the wound areas (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1d</xref>, , <xref rid="nihms-1632597-f0001" ref-type="fig">h</xref>––<xref rid="nihms-1632597-f0001" ref-type="fig">i</xref>). Remarkably, examination of histological sections of the D-MAP- or 1:1 L/D-MAP-treated tissue revealed a ). Remarkably, examination of histological sections of the D-MAP- or 1:1 L/D-MAP-treated tissue revealed a de novo regenerated appearance. The overlying epidermis often displayed physiological undulation, while numerous immature-appearing hair follicles were seen spanning the length of the healed full thickness injury (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1e</xref>––<xref rid="nihms-1632597-f0001" ref-type="fig">i</xref>). Samples treated with D-MAP or 1:1 L/D-MAP also displayed increased skin thickness despite less hydrogel remaining in these samples (). Samples treated with D-MAP or 1:1 L/D-MAP also displayed increased skin thickness despite less hydrogel remaining in these samples (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1f</xref>). Many samples also displayed epidermal cyst formation. In samples that displayed residual hydrogel, hair follicles were apparent directly overlying the degrading MAP hydrogel particles (). Many samples also displayed epidermal cyst formation. In samples that displayed residual hydrogel, hair follicles were apparent directly overlying the degrading MAP hydrogel particles (Supplementary Figure 2c). The presence of hair follicles in SKH1 mice was suggestive of embryonic-like tissue regeneration, a phenomenon not often observed in the murine small wound model.', 'To further quantify tissue regeneration, we next performed tensile strength testing on unsplinted incisional wounds in SKH1 mice using a modified literature protocol17. We found that scar tissue from sham wounds revealed tensile strength that was approximately 15% of unwounded skin from the same animal (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1i</xref>). While treatment of wounds with L-MAP hydrogel did not result in a significant increase in tissue tensile strength, treatment with either D or L/D MAP resulted in an ~80% improvement in tensile strength (). While treatment of wounds with L-MAP hydrogel did not result in a significant increase in tissue tensile strength, treatment with either D or L/D MAP resulted in an ~80% improvement in tensile strength (<xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1j</xref>).).', 'For each tissue sample, stress/strain curves were calculated from force/elongation curves (provided from the Instron Bluehill software) using the known cross-sectional dimensions of the “dog bone” samples (each measured with calipers prior to placement on the Instron), and by measuring the starting distance between pneumatic grips with a caliper. The starting distance was standardized by preloading the sample to 0.5N, followed by measurement and then running of the tensile test to failure. This analysis enabled calculation of Yield Stress, which are reporting in <xref rid="nihms-1632597-f0001" ref-type="fig">Figure 1j</xref>..'], 'nihms-1632597-f0002': ['We next repeated wound healing experiments in B6 mice to investigate if the regenerative phenomenon observed in D-MAP treated wounds was similar to WIHN. We chose sham as control and D-MAP as a treatment method that showed evidence of regeneration in SKH1 mice. Similar to the sham and L-MAP treated wounds in SKH1 mice, the B6 mice wounds without hydrogel (sham) displayed the typical scar appearance by H&E and Masson Trichrome staining (<xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2a</xref>, , <xref rid="nihms-1632597-f0002" ref-type="fig">c</xref>, , <xref rid="nihms-1632597-f0002" ref-type="fig">e</xref>). In contrast, histological sections of the D-MAP treated tissue revealed clear signs of WIHN. As in SKH1 mice, D-MAP treated B6 mice wounds displayed undulations and numerous epidermal cysts under the epidermis, while the dermis was thicker. Importantly, many neogenic hair follicles developed in the wound (). In contrast, histological sections of the D-MAP treated tissue revealed clear signs of WIHN. As in SKH1 mice, D-MAP treated B6 mice wounds displayed undulations and numerous epidermal cysts under the epidermis, while the dermis was thicker. Importantly, many neogenic hair follicles developed in the wound (<xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2b</xref>, , <xref rid="nihms-1632597-f0002" ref-type="fig">d</xref>, , <xref rid="nihms-1632597-f0002" ref-type="fig">f</xref>). The neogenic hair follicles were in early anagen phases with immature appearance, yet many of them already had formed new sebaceous glands (). The neogenic hair follicles were in early anagen phases with immature appearance, yet many of them already had formed new sebaceous glands (<xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2b</xref>) and featured prominent SOX9) and featured prominent SOX9+ bulge stem cell region (<xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2j</xref>). In several instances, neogenic hair follicles were physically connected to epidermal cysts (morphology not expected from pre-existing follicles). This suggests that in D-MAP treated wounds, epidermal cysts can be the initiation sites for ). In several instances, neogenic hair follicles were physically connected to epidermal cysts (morphology not expected from pre-existing follicles). This suggests that in D-MAP treated wounds, epidermal cysts can be the initiation sites for de novo morphogenesis for at least some of the neogenic hair follicles (<xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2h</xref>). Masson trichrome staining confirmed the presence of neogenic hair follicles within the collagen matrix of the wound bed (). Masson trichrome staining confirmed the presence of neogenic hair follicles within the collagen matrix of the wound bed (<xref rid="nihms-1632597-f0002" ref-type="fig">Fig 2b</xref>, , <xref rid="nihms-1632597-f0002" ref-type="fig">f</xref>). Furthermore, regenerating day 18 D-MAP treated wounds with neogenic hair follicles lacked PLIN). Furthermore, regenerating day 18 D-MAP treated wounds with neogenic hair follicles lacked PLIN+ dermal adipocytes (<xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2h</xref>), which is consistent with slower regeneration of neogenic adipocytes that occurs four weeks after wounding in the large wound-induced WIHN), which is consistent with slower regeneration of neogenic adipocytes that occurs four weeks after wounding in the large wound-induced WIHN18,19. Thus, addition of D-MAP to normally non-regenerating 4-mm excisional wounds activates hair follicle neogenesis.', 'The evaluation of hair neogenesis in B6 mice Control vs D-MAP for <xref rid="nihms-1632597-f0002" ref-type="fig">Figure 2</xref> was performed on samples from n= 4 for each group. The wound healing studies comparing WT to Rag1 was performed on samples from n= 4 for each group. The wound healing studies comparing WT to Rag1−/− mice were repeated three times (n=4 each group). In the first experiment, all Rag1−/− mice were euthanized due to the development of severe and worsening wound infections, and thus were not included in the final analysis. In addition, wounds/scars that showed more than 50% contraction of the wound area from the underlying fascia from any group were removed from the final data set or if the histological processing failed to identify the wound/scar bed (i.e. sample was cut through). For the histological analysis, Sham vs 1:1 L/D in B6 mice from 3 separate experiments was used (n=9 histological samples available out of an available n=12 wounds performed), while samples in the B6.Rag1−/− mice were obtained from the latter two experiments performed in B6 vs B6.Rag1−/− mice performed at the same time (n=6 histological samples available out of an n=8 wounds. The findings within this manuscript were observed in two different mouse strains (CRL-SKH and C57Bl/6) that have different adnexal structures (vellus hair only and mature/terminal follicles, respectively).'], 'nihms-1632597-f0003': ['To determine whether an enhanced immune response was leading to enhanced D-MAP or 1:1 L:D MAP degradation in the wound microenvironment, we utilized a subcutaneous implantation model which also allows for larger amounts of hydrogel to be implanted, and thus remain present longer, than in the small excisional wound model. To test whether the subcutaneous implants of D-MAP hydrogel resulted in enhanced immune cell recruitment, we utilized immunofluorescent microscopy with AlexaFluor488 labeled MAP hydrogel. We found that implants containing just L-MAP display a background level of CD11b cells within the hydrogel, as previously observed1, while D-MAP or L/D-MAP resulted in the robust accumulation of CD11b-expressing myeloid cells within and around the scaffold (<xref rid="nihms-1632597-f0003" ref-type="fig">Figure 3a</xref>––<xref rid="nihms-1632597-f0003" ref-type="fig">b</xref>). Standard histological analysis of a repeat experiment of different formulations of subcutaneously implanted MAP hydrogel confirmed the activation of type 2 immunity with an atypical, type 2 granulomatous response dominated by the accumulation of individual macrophages within and around D-MAP hydrogel implants, but not L-MAP hydrogel implants (see ). Standard histological analysis of a repeat experiment of different formulations of subcutaneously implanted MAP hydrogel confirmed the activation of type 2 immunity with an atypical, type 2 granulomatous response dominated by the accumulation of individual macrophages within and around D-MAP hydrogel implants, but not L-MAP hydrogel implants (see Supplementary Figure 3a and Supplementary Discussion). Immunofluorescent staining for F4/80 and CD11b confirms the enhanced recruitment of macrophages, without giant cell formation, in D-MAP implants (see Supplementary Figure 3b and 3c and Supplementary Discussion). These results confirm that D-MAP elicit a more robust immune response, and degradation by the accumulated immune cells likely contributes to the enhanced degradation of D-MAP in our previous wound experiments.', 'Allergic responses and parasites can elicit a type 2 immune response including atypical type 2 granulomatous responses at least partially through interleukin (IL)-33 production by epithelial cells, recruited myeloid cells and resident macrophages20–23. Implanted, non-degradable microparticle-based materials elicit an IL-33-dependent type 2 innate immune response by circulating CD11b+ myeloid cells and macrophages24. It is possible that MAP particles could activate this same program, especially given the atypical type 2 foreign body responses observed in D-MAP samples. Indeed, 21 days after implantation, we found similar numbers of IL-33-expressing F4/80+CD11b+ macrophages in the center/core of both L- and D-MAP implants (<xref rid="nihms-1632597-f0003" ref-type="fig">Figure 3c</xref> and and <xref rid="nihms-1632597-f0003" ref-type="fig">3d</xref>), consistent with both L- and D-MAP samples activating this type 2 pathway. However, there was a dramatic increase in IL-33 producing IL-33), consistent with both L- and D-MAP samples activating this type 2 pathway. However, there was a dramatic increase in IL-33 producing IL-33+F4/80+ macrophages at the edges of only D-MAP implants (<xref rid="nihms-1632597-f0003" ref-type="fig">Figure 3c</xref> and and <xref rid="nihms-1632597-f0003" ref-type="fig">3d</xref>). These results confirm that the hydrogel possesses a type 2 innate “adjuvant” effect, that may activate the adaptive immune system and contribute to enhanced immune activation with D-MAP hydrogel. When L-MAP scaffolds are used, the immune response remains mild as the hydrogel degrades slowly over time). These results confirm that the hydrogel possesses a type 2 innate “adjuvant” effect, that may activate the adaptive immune system and contribute to enhanced immune activation with D-MAP hydrogel. When L-MAP scaffolds are used, the immune response remains mild as the hydrogel degrades slowly over time25, but the presence of D-peptide accelerates immune mediated degradation.', 'We next tested whether D-peptides could directly activate innate immunity through traditional PRR-induced transcriptional response. We stimulated murine bone marrow derived macrophages (BMDMs) with L-peptide or D-peptide in the presence or absence of bacterial lipopolysaccharide (LPS), the Toll-like receptor 4 agonist that results in rapid macrophage transcriptional responses. We chose to examine genes reliably and potently induced downstream of the major signaling pathways downstream of a variety of cellular insult (AP-1, MAPK, NF-κB, and type I IFN) to simultaneously interrogate multiple PRR pathways26–28. To our surprise, neither L- nor D-amino acid containing crosslinking peptides alone at high doses (1mg/ml) induced the expression of pro-inflammatory genes Tnf (NF-κB dependent), Il1b (NF-κB and MAPK dependent), Cxcl2 (AP-1 dependent early response), or Mx1 (type I IFN dependent) in murine BMDMs at 6 hours (tmax of gene induction; <xref rid="nihms-1632597-f0003" ref-type="fig">Figure 3e</xref>––<xref rid="nihms-1632597-f0003" ref-type="fig">l</xref>). Additionally, neither L- nor D-peptides enhanced the ability of LPS to induce the expression of these same genes (). Additionally, neither L- nor D-peptides enhanced the ability of LPS to induce the expression of these same genes (<xref rid="nihms-1632597-f0003" ref-type="fig">Figure 3e</xref>––<xref rid="nihms-1632597-f0003" ref-type="fig">h</xref>).).', 'Previous studies have shown that peptides containing an N-terminal D-methionine can activate the innate immune receptor formyl peptide receptor 2 and formyl peptide like receptor 229–31. Since cleavage of D-amino acid peptide can result in shorter peptides that contain a D-amino acid at the N-terminus, we next wished to examine whether a peptide corresponding to the cleaved D-peptide could activate inflammatory responses in BMDMs. Similar to the results with intact D-peptide, high concentrations of cleaved D-peptide (1mg/ml) did not induce the transcription of Tnf, Il1b, Cxcl2, or Mx1 at 6 hours (<xref rid="nihms-1632597-f0003" ref-type="fig">Figure 3i</xref>––<xref rid="nihms-1632597-f0003" ref-type="fig">l</xref>). Since there is a very low likelihood that cleaved D-peptide will be present at such high local concentrations within the implanted hydrogel while it is being degraded ). Since there is a very low likelihood that cleaved D-peptide will be present at such high local concentrations within the implanted hydrogel while it is being degraded in vivo, these show that D-chiral peptides are poor activators of a traditional PRR-mediated inflammatory response in macrophages and suggest that D-peptides may be acting as antigens to enhance immunity, leading to enhanced degradation of D-MAP.'], 'nihms-1632597-f0004': ['Indeed, regardless of whether D-containing MAP hydrogel was applied to wounded tissue or given via subcutaneous implants, mice developed a T cell dependent IgG1 and IgG2a response against the D-amino acid containing peptide, but not a T cell independent IgG3 response. These results are more consistent with a T cell-dependent immune response against D-peptides (<xref rid="nihms-1632597-f0004" ref-type="fig">Figure 4a</xref>––<xref rid="nihms-1632597-f0004" ref-type="fig">b</xref>). IgG1 is typically associated with a Th2 “tissue repair” type response, while IgG2a is typically associated with a Th1 “foreign body” response that typically requires strong adjuvants to develop, depending on the strain of mice). IgG1 is typically associated with a Th2 “tissue repair” type response, while IgG2a is typically associated with a Th1 “foreign body” response that typically requires strong adjuvants to develop, depending on the strain of mice37,38. The fact that anti-D peptide-specific IgG2a induced when the hydrogel was given to mice in a wound environment but not when the hydrogel was given in the subcutaneous implant model suggests that, by itself, the hydrogel does not possess sufficient adjuvant effects to induce robust Th1 responses. However, the inflammation present in the wound environment may result in a mixed Th2/Th1 response to the D-MAP (<xref rid="nihms-1632597-f0004" ref-type="fig">Figure 4b</xref> and and <xref rid="nihms-1632597-f0004" ref-type="fig">3e</xref>). Mice that were treated with L-MAP alone did not develop antibody responses to L-peptide.). Mice that were treated with L-MAP alone did not develop antibody responses to L-peptide.', 'Our data suggest that the activation of adaptive immune responses to D-MAP contributes to immune infiltration and degradation of D-MAP. To test this hypothesis further, we examined whether Balb/c.Rag2−/−γc−/− mice, which are devoid of an adaptive immune system, innate lymphoid cells, and IL-2/IL-15 signaling, but possess a fully functional myeloid system, will exhibit reduced immune infiltration39. Indeed, total cellularity and the specific recruitment of CD11b+ myeloid cells to D-MAP hydrogel in Balb/c.Rag2−/−γc−/− mice were decreased to comparable levels to those seen in L-MAP in WT mice (<xref rid="nihms-1632597-f0004" ref-type="fig">Figure 4k</xref>––<xref rid="nihms-1632597-f0004" ref-type="fig">l</xref>).).'], 'nihms-1632597-f0005': ['Sham wounds in B6 mice demonstrated an obvious depigmented, irregularly-shaped scar, while scars in B6 mice treated with 1:1 L/D-MAP gel were difficult to identify visually as they displayed hair growth over the wounds and less atrophy/surface changes typically seen in scars (representative example <xref rid="nihms-1632597-f0005" ref-type="fig">Figure 5a</xref>, all wound images , all wound images Supplementary Figure 4). Scars in sham-treated or 1:1 L/D-MAP-treated B6.Rag1−/− mice were smaller than those in sham-treated B6 mice, but were identifiable in B6.Rag1−/− mice regardless of whether wounds were sham treated or hydrogel treated (<xref rid="nihms-1632597-f0005" ref-type="fig">Figure 5a</xref>). All wound (including 1:1 L/D-MAP-treated B6 wound areas) injuries were confirmed by examining the defect on the fascial side of the tissue after excision of skin. Histological sections of the healed skin of mice displayed significant neogenic hairs and sebaceous glands only in wounds of wildtype mice treated with 1:1 L/D-MAP (). All wound (including 1:1 L/D-MAP-treated B6 wound areas) injuries were confirmed by examining the defect on the fascial side of the tissue after excision of skin. Histological sections of the healed skin of mice displayed significant neogenic hairs and sebaceous glands only in wounds of wildtype mice treated with 1:1 L/D-MAP (<xref rid="nihms-1632597-f0005" ref-type="fig">Figure 5b</xref>––<xref rid="nihms-1632597-f0005" ref-type="fig">d</xref>, and , and Supplementary Figure 5). Sham wounds in B6 and Rag−/− mice, and in the 1:1 L/D-MAP treated B6.Rag1−/− mice displayed prominent scars, without hairs or sebaceous glands, confirming the requirement of adaptive immune system in skin regeneration by D-peptide containing MAP gel (<xref rid="nihms-1632597-f0005" ref-type="fig">Figure 5b</xref>––<xref rid="nihms-1632597-f0005" ref-type="fig">d</xref>). These studies highlight that hair follicle structures can be regenerated through adaptive immune activation from MAP hydrogel scaffolds.). These studies highlight that hair follicle structures can be regenerated through adaptive immune activation from MAP hydrogel scaffolds.'], 'nihms-1632597-f0006': ['In most mammals, the natural process of scar formation and tissue fibrosis is highly evolved and is a tissue-scale attempt to restore critical barrier functions for survival. This process, however, is ultimately a biological ‘triage’ that favors the rapid deposition of a fibrotic matrix to restore the barrier at the expense of a loss of function of complex tissue. In the skin, this fibrotic response results not only in a loss of functioning adnexal structures, but skin tissue that is more fragile and prone to re-injury. A major goal when engineering skin regeneration is to allow for the rapid restoration of barrier function while providing increased tissue tensile strength and higher tissue function. Many biomaterial-based approaches including addition of growth factors and decellularized extracellular matrix constructs display limited success in restoring function in wounds. We previously showed that the MAP scaffold can accelerate wound closure in murine wounds1. Our findings reported here further highlight that incorporation of a modest adaptation to MAP that enhanced a type 2 innate and adaptive immune response, induced skin regeneration: hair neogenesis and improved tensile strength (<xref rid="nihms-1632597-f0006" ref-type="fig">Figure 6</xref>). This response was dependent upon the generation of an adaptive immune response to D-enantiomeric peptides, and occurred without addition of stem cells, growth factors, or adjuvants. Importantly, this regenerative response was decoupled from wound closure that begins immediately, consistent with the time needed to generate an antigen specific immune response.). This response was dependent upon the generation of an adaptive immune response to D-enantiomeric peptides, and occurred without addition of stem cells, growth factors, or adjuvants. Importantly, this regenerative response was decoupled from wound closure that begins immediately, consistent with the time needed to generate an antigen specific immune response.']}
|
Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing.
| null |
Nat Mater
|
1617951600
|
A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.
|
[
"Amino Acid Sequence",
"Coronavirus",
"Coronavirus 229E, Human",
"Coronavirus Papain-Like Proteases",
"Humans",
"Models, Molecular",
"Molecular Sequence Data",
"Papain",
"Protein Structure, Tertiary",
"Viral Proteins",
"Zinc Fingers"
] |
other
|
PMC8005402
| null |
86
|
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Nat Mater. 2021 Apr 9; 20(4):560-569
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Glucose uptake is decreased in RON3loxP parasites. The glucose analogue 2-NBDG was used to track glucose uptake into v2 cycle 2 DMSO- or rapamycin (Rapa)-treated parasites. (A) Representative images of DMSO- and Rapa-treated parasites after 2 h of incubation with 2-NBDG. Parasites were additionally stained with Hoechst 33342 (DNA marker) and MitoTracker Deep Red (Mito DR), a mitochondrial marker. Bars, 2 μm. (B) Example illustration of strategy for determining change in mean fluorescent intensity (ΔMFI) of 2-NBDG. MFI (green) was measured by first measuring fluorescent intensity attributed to the parasite (red circle as determined by localization with Hoechst/MitoTracker [blue/red] localization). Background MFI in the blood cell in which the parasite resides (blue circle) was subsequently subtracted to provide ΔMFI. (C) ΔMFI of 2-NBDG in DMSO-treated and rapamycin-treated ring-stage parasites (n = 35/group). (D) ΔMFI of 2-NBDG in DMSO-treated and rapamycin-treated merozoites was measured by subtracting background MFI from merozoite MFI. (E) Representative images of DMSO- and Rapa-treated merozoites stained with 2-NBDG (n = 35/group). Data represent the mean ± standard error of the mean (SEM). Statistical significance was determined by two-tailed unpaired t test where P < 0.05 is considered significant (****, P < 0.0001).
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
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[{'image_id': 'mBio.01460-19-f0002', 'image_file_name': 'mBio.01460-19-f0002.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0002.jpg', 'caption': 'RON3-deficient parasites invade normally but stall at the ring stage. (A and B) Version 1 (v1) clone 7C4 (A) or version 2 (v2) clone B11 (B) parasites were treated with DMSO or rapamycin (Rapa) for 4\u2009h during ring stage and allowed to mature and progress into the next cycle. Microscopic images of Giemsa-stained thin blood smears were taken at 46\u2009h, 52\u2009h, 72\u2009h, and 94\u2009h following the commencement of cycle 1. Images are representative of at least three independent experiments. (C) v1 clones 7C4 and 3E10 and the parental line, 1G5DC, were treated with DMSO or rapamycin to investigate invasive capacity after loss of RON3. Schizonts were purified after treatment and allowed to undergo egress and invasion into fresh RBCs for 4\u2009h before ring-stage parasitemia was determined by flow cytometry. Data were averaged from three biological replicate experiments, using blood samples from different donors. Values are means ± 1 standard deviation (error bars). Statistical significance was determined by a two-tailed t test where P\u2009>\u20090.05 is considered nonsignificant (ns). (D) Differential count of the DMSO- or rapamycin-treated v2 B11 parasite blood stages (rings, trophozoites, or schizonts) at 46\u2009h, 52\u2009h, and 94\u2009h. Percentages of each blood stage (rings, trophozoites, or schizonts) were normalized to total percentage parasitemia at each time point. Data represent two independent experiments, and error bars depict standard deviations (SD).', 'hash': '5d6422b939143cf9345d7890a84d936e993cd6f56d4e66700b39cafba7653e6d'}, {'image_id': 'mBio.01460-19-f0004', 'image_file_name': 'mBio.01460-19-f0004.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0004.jpg', 'caption': 'Glucose uptake is decreased in RON3loxP parasites. The glucose analogue 2-NBDG was used to track glucose uptake into v2 cycle 2 DMSO- or rapamycin (Rapa)-treated parasites. (A) Representative images of DMSO- and Rapa-treated parasites after 2 h of incubation with 2-NBDG. Parasites were additionally stained with Hoechst 33342 (DNA marker) and MitoTracker Deep Red (Mito DR), a mitochondrial marker. Bars, 2\u2009μm. (B) Example illustration of strategy for determining change in mean fluorescent intensity (ΔMFI) of 2-NBDG. MFI (green) was measured by first measuring fluorescent intensity attributed to the parasite (red circle as determined by localization with Hoechst/MitoTracker [blue/red] localization). Background MFI in the blood cell in which the parasite resides (blue circle) was subsequently subtracted to provide ΔMFI. (C) ΔMFI of 2-NBDG in DMSO-treated and rapamycin-treated ring-stage parasites (n\u2009=\u200935/group). (D) ΔMFI of 2-NBDG in DMSO-treated and rapamycin-treated merozoites was measured by subtracting background MFI from merozoite MFI. (E) Representative images of DMSO- and Rapa-treated merozoites stained with 2-NBDG (n\u2009=\u200935/group). Data represent the mean ± standard error of the mean (SEM). Statistical significance was determined by two-tailed unpaired t test where P\u2009<\u20090.05 is considered significant (****, P\u2009<\u20090.0001).', 'hash': 'aaf12af922b05d486babb2af6dc5791a359d453e340673567fee60bb55c8c41b'}, {'image_id': 'mBio.01460-19-f0003', 'image_file_name': 'mBio.01460-19-f0003.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0003.jpg', 'caption': 'RON3 is essential for the export of proteins into the infected RBC. Ring infected RBCs from RON3loxP v1 (A) and v2 (B) parasites were treated with DMSO or rapamycin (Rapa) for 4\u2009h, washed, and cultured in cRPMI for ∼40 to 44\u2009h. The parasites were fixed and stained with antibodies to RON3, KAHRP, RESA, MAHRP1, EXP2, or PTEX150 antibodies. RON3, KAHRP, and EXP2 staining is shown in green, and RESA, MAHRP1, and PTEX150 staining is shown in red. The nuclei staining (4′,6′-diamidino-2-phenylindole [DAPI]) is shown in blue. MAHRP1, KAHRP, or RESA was exported into the DMSO-treated infected RBCs while they appeared to be around the rapamycin-treated parasite. All images are representative of the majority of parasites over several fields and/or of three independent experiments. DIC, differential interference contrast. Bars, 2\u2009μm.', 'hash': '20867e4f79f8ad67196a6a84478fa59418e72b3435aa389a5e0bc286e9b86a9a'}, {'image_id': 'mBio.01460-19-f0001', 'image_file_name': 'mBio.01460-19-f0001.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0001.jpg', 'caption': 'Rapid and efficient conditional disruption of the P. falciparum\nRON3 gene. The native PfRON3 gene on chromosome 12 comprises 8 exons (indicated as gray boxes) and 7 introns. (A) Version 1 (v1). Schematic representation of pre- and postintegration events after CRISPR-Cas9 editing to produce the modified RON3loxP and the expected locus architecture before and after rapamycin treatment (pre- and postexcision). Recodonized exons 2 to 6 are shown as a green box. Red arrows indicate target areas of primers RON3_exon1_F (solid arrow)/RON3_exon7_R (dashed arrow). (B) PCR RON3loxP v1 clones 3E10 and 7C4 were treated with DMSO (D) or rapamycin (R), and schizonts were analyzed ∼44\u2009h after treatment to determine excision. DNA markers (M) ranged from 200 to 10,000 bp. Excision leads to a decrease in amplicon size from 1,615\u2009bp to 422\u2009bp. (C) Version 2 (v2). Schematic representation of pre- and postintegration events after CRISPR-Cas9 editing to produce the modified RON3loxP and the expected locus architecture before and after rapamycin treatment (pre- and postexcision). Recodonized exons 6 to 7 are shown as a yellow box. Blue arrows indicate target areas of primers RON3_exon4_F (solid arrow)/RON3_exon8_R (dashed arrow). (D) PCR RON3loxP v2 clone B11 was treated with DMSO (D) or rapamycin (R); excision leads to s decrease in amplicon size from 2,364\u2009bp to 757\u2009bp. DNA markers (M) ranged from 250 to 10,000 bp. Samples were run in duplicate on the same gel, with the white line indicating splicing of the image to show only one replicate.', 'hash': '95eecc550e8fa1a1ec3aa16a91218c917bd530043c98e7cf9bee07b338670fbc'}]
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{'mBio.01460-19-f0001': ['To investigate the role of RON3 in the Plasmodium falciparum life cycle, we conditionally disrupted the gene in the 3D7 parasite clone 1G5DC using the dimerizable P1 bacteriophage cyclization recombination (DiCre) recombinase system (14). Two forms of RON3loxP were created by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 (Cas9)-mediated gene editing, whereby the locus of X-over P1 (loxP) sites were inserted into different segments of the RON3 gene. The RON3 gene contains eight exons with the signal sequence located in exon 1. For version 1 (v1), the RON3 gene was modified such that exons 2 to 6 were recodonized without introns between them. This was flanked by synthetic introns containing loxP sites (<xref ref-type="fig" rid="mBio.01460-19-f0001">Fig.\xa01A</xref>). In version 2 (v2), ). In version 2 (v2), RON3 was edited further toward the 3′ end of the gene by replacing exons 6 to 7 with a recodonized version containing no introns. The endogenous introns on either side of exons 6 to 7 were replaced with synthetic loxP-containing introns (<xref ref-type="fig" rid="mBio.01460-19-f0001">Fig.\xa01C</xref>).).', 'Disruption of the RON3 gene was induced in ring-stage parasites; excision of the gene is mediated by brief rapamycin treatment, allowing the activation of the Cre recombinase that recognizes loxP sequences by dimerization of the DiCre components (14). Rapamycin treatment was predicted to lead to the excision of the recodonized exons. In v1, this would eliminate the first two transmembrane domains, while in v2, this would lead to excision of the third transmembrane domain. Diagnostic PCR, using DNA extracted from dimethyl sulfoxide (DMSO)- or rapamycin-treated v1 (clones 3E10 and 7C4) and v2 (clone B11), showed the expected loss of the intervening recodonized sequence (<xref ref-type="fig" rid="mBio.01460-19-f0001">Fig.\xa01B</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0001">D</xref>); v2 parasites were further sequenced, confirming the excision after rapamycin treatment and demonstrating efficient excision of the floxed sequences (data not shown).); v2 parasites were further sequenced, confirming the excision after rapamycin treatment and demonstrating efficient excision of the floxed sequences (data not shown).'], 'mBio.01460-19-f0002': ['Rapamycin-treated v1 or v2 parasites were observed to progress normally through the cycle in which they were treated (cycle 1; <xref ref-type="fig" rid="mBio.01460-19-f0002">Fig.\xa02A</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0002">B</xref>). However, following invasion into cycle 2, rapamycin-treated parasites did not develop beyond the ring stage (). However, following invasion into cycle 2, rapamycin-treated parasites did not develop beyond the ring stage (<xref ref-type="fig" rid="mBio.01460-19-f0002">Fig. 2A</xref>, , <xref ref-type="fig" rid="mBio.01460-19-f0002">B</xref>, and , and <xref ref-type="fig" rid="mBio.01460-19-f0002">D</xref>). Comparison of ring-stage parasites at 94\u2009h was found to be significantly different between treatment conditions (). Comparison of ring-stage parasites at 94\u2009h was found to be significantly different between treatment conditions (P\u2009<\u20090.001). Examination of the invasion capacity of v1 parasites (clones 3E10 and 7C4) found that rapamycin-treated parasites invaded new RBCs at a similar rate to DMSO-treated parasites of the same clone (<xref ref-type="fig" rid="mBio.01460-19-f0002">Fig.\xa02C</xref>). The parental parasite clone, 1G5DC, was also included in these experiments to assess whether rapamycin treatment itself could affect invasion; this confirmed that there were no significant differences between DMSO and rapamycin treatment as previously shown (). The parental parasite clone, 1G5DC, was also included in these experiments to assess whether rapamycin treatment itself could affect invasion; this confirmed that there were no significant differences between DMSO and rapamycin treatment as previously shown (14). To rule out any effects of loxP insertion, growth rates were compared between v1 parasites and the parental clone (1G5DC) with no significant differences observed (P\u2009>\u20090.05; see Fig.\xa0S2 in the supplemental material).'], 'mBio.01460-19-f0003': ['To seek insights into the development defect in cycle 2 rings derived from the rapamycin-treated RON3loxP parasites, we examined the fate of the PTEX exported proteins RESA, knob-associated histidine-rich protein (KAHRP), and Maurer’s cleft-associated histidine-rich protein 1 (MAHRP1) (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03</xref>); RESA was of particular interest, as it is known to be exported early during ring stages (); RESA was of particular interest, as it is known to be exported early during ring stages (15). In DMSO-treated RON3loxP parasites, RESA was found distributed throughout the infected RBC, concentrating in the space below the RBC membrane, as is normal (16, 17). In contrast, in newly formed rings of both v1 and v2 excised parasites, RESA was found surrounding the parasite, indicating failure of translocon function for protein export to the RBC (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03A</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0003">B</xref>). In addition, KAHRP, the electron-dense histidine-rich protein that normally localizes to below the knobs on the RBC surface, and MAHRP1, located at Maurer’s clefts, remained close to the parasite and were not translocated to the erythrocyte cytoplasm (). In addition, KAHRP, the electron-dense histidine-rich protein that normally localizes to below the knobs on the RBC surface, and MAHRP1, located at Maurer’s clefts, remained close to the parasite and were not translocated to the erythrocyte cytoplasm (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03A</xref>). Further analysis with antibodies to other exported proteins showed that the rapamycin-treated parasites were incapable of exporting other parasite proteins (skeleton binding protein 1 [SBP1], histidine-rich protein 2 [HRP2], and ). Further analysis with antibodies to other exported proteins showed that the rapamycin-treated parasites were incapable of exporting other parasite proteins (skeleton binding protein 1 [SBP1], histidine-rich protein 2 [HRP2], and P. falciparum erythrocyte membrane protein 1 [PfEMP1]; Fig.\xa0S3). Furthermore, in addition to RESA, the PTEX translocon components, EXP2 and PTEX150 (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03A</xref>), were detected in both DMSO- and rapamycin-treated parasites. EXP2, alongside the other PTEX components, are found in the dense granules (), were detected in both DMSO- and rapamycin-treated parasites. EXP2, alongside the other PTEX components, are found in the dense granules (18) and allow the export of proteins, such as RESA, in healthy (normal) parasites.'], 'mBio.01460-19-f0004': ['To examine whether loss of RON3 also interfered with import of molecules into parasitized cells (a function of EXP2 in a role independent of the PTEX complex [11]), an assay was devised to directly detect import of a labeled glucose analogue, 2-(N-(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Cells were further stained with Hoechst 33342 (DNA marker) and MitoTracker Deep Red (mitochondrial marker), which allowed parasites to be identified. Study of 2-NBDG uptake was not interfered with by the use of these two markers as determined using fluorescent controls (Fig.\xa0S4). RON3-deficient intracellular parasites were observed to take up less glucose than control DMSO-treated counterparts (<xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04A</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0004">C</xref>). After invasion, glucose uptake into the parasite was significantly lower than glucose uptake into the DMSO control, as quantified by comparing the mean fluorescent intensity (MFI) of 2-NBDG in DMSO- and rapamycin-treated parasites, wherein the MFI of background 2-NBDG signal in the infected RBC was subtracted from the 2-NBDG MFI in the parasite (change in MFI [ΔMFI]) (). After invasion, glucose uptake into the parasite was significantly lower than glucose uptake into the DMSO control, as quantified by comparing the mean fluorescent intensity (MFI) of 2-NBDG in DMSO- and rapamycin-treated parasites, wherein the MFI of background 2-NBDG signal in the infected RBC was subtracted from the 2-NBDG MFI in the parasite (change in MFI [ΔMFI]) (<xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04B</xref>). In two independent experiments (). In two independent experiments (<xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04C</xref> and and S5B), the ΔMFI of rapamycin-treated parasites was significantly less than that of the control, suggesting impairment of glucose uptake through the PVM. We further observed an outlier in the rapamycin-treated group; this is likely attributable to ∼2% of parasites not undergoing excision during rapamycin treatment and has been previously observed in other DiCre mutants (14). As 2-NBDG is reported to metabolize to a nonfluorescent form, we repeated the experiment with a nonmetabolizable analogue, 6-NBDG (19), and found that DMSO-treated parasites still significantly took up more glucose than the rapamycin-treated parasites did (Fig.\xa0S5C). We further compared the background MFI of 2-NBDG or 6-NBDG in the infected blood cell (Fig.\xa0S5D) and found a significant difference between DMSO- and rapamycin-treated conditions. As demonstrated in this paper, the loss of RON3 leads to no parasite protein export through the PTEX translocon. The absence of these parasite proteins in the RBC may lead to reduced uptake in glucose by the infected RBC. However, as there is only ∼20% reduction in uptake into the RBC between groups, we do not believe that this difference is enough to explain the ∼50% reduction in glucose uptake into the RON3-deficient parasite.', 'To exclude a role for RON3 in the function of the hexose transporter (HT) that is located on the parasite plasma membrane (PPM), released merozoites that were treated with DMSO or rapamycin were investigated. Free rapamycin-treated merozoites were observed to take up glucose similarly to DMSO-treated parasites (P\u2009>\u20090.05; <xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04D</xref>), indicating that the HT present on the PPM is functional and exhibits similar activity.), indicating that the HT present on the PPM is functional and exhibits similar activity.']}
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{'@content-type': 'genus-species', '#text': 'Plasmodium falciparum'} Deletion of Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake
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[
"PTEX",
"malaria",
"parasite proteins",
"parasitophorous vacuolar space"
] |
mBio
|
1562655600
|
The survival of spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane. The malarial parasite within the erythrocyte is surrounded by two membranes. translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We now describe a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We find that RON3 is required for the protein transport function of the PTEX and for glucose transport from the RBC cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2.
|
[
"Antigens, Neoplasm",
"Biological Transport",
"Erythrocytes",
"Gene Deletion",
"Glucose",
"Host-Parasite Interactions",
"Humans",
"Malaria, Falciparum",
"Plasmodium falciparum",
"Protein Transport",
"Protozoan Proteins",
"Translocation, Genetic"
] |
other
|
PMC6747712
| null |
33
|
[
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] |
mBio. 2019 Jul 9; 10(4):e01460-19
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NO-CC CODE
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RON3 is essential for the export of proteins into the infected RBC. Ring infected RBCs from RON3loxP v1 (A) and v2 (B) parasites were treated with DMSO or rapamycin (Rapa) for 4 h, washed, and cultured in cRPMI for ∼40 to 44 h. The parasites were fixed and stained with antibodies to RON3, KAHRP, RESA, MAHRP1, EXP2, or PTEX150 antibodies. RON3, KAHRP, and EXP2 staining is shown in green, and RESA, MAHRP1, and PTEX150 staining is shown in red. The nuclei staining (4′,6′-diamidino-2-phenylindole [DAPI]) is shown in blue. MAHRP1, KAHRP, or RESA was exported into the DMSO-treated infected RBCs while they appeared to be around the rapamycin-treated parasite. All images are representative of the majority of parasites over several fields and/or of three independent experiments. DIC, differential interference contrast. Bars, 2 μm.
|
mBio.01460-19-f0003
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2
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20867e4f79f8ad67196a6a84478fa59418e72b3435aa389a5e0bc286e9b86a9a
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mBio.01460-19-f0003.jpg
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multiple
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multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
750,
540
] |
[{'image_id': 'mBio.01460-19-f0002', 'image_file_name': 'mBio.01460-19-f0002.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0002.jpg', 'caption': 'RON3-deficient parasites invade normally but stall at the ring stage. (A and B) Version 1 (v1) clone 7C4 (A) or version 2 (v2) clone B11 (B) parasites were treated with DMSO or rapamycin (Rapa) for 4\u2009h during ring stage and allowed to mature and progress into the next cycle. Microscopic images of Giemsa-stained thin blood smears were taken at 46\u2009h, 52\u2009h, 72\u2009h, and 94\u2009h following the commencement of cycle 1. Images are representative of at least three independent experiments. (C) v1 clones 7C4 and 3E10 and the parental line, 1G5DC, were treated with DMSO or rapamycin to investigate invasive capacity after loss of RON3. Schizonts were purified after treatment and allowed to undergo egress and invasion into fresh RBCs for 4\u2009h before ring-stage parasitemia was determined by flow cytometry. Data were averaged from three biological replicate experiments, using blood samples from different donors. Values are means ± 1 standard deviation (error bars). Statistical significance was determined by a two-tailed t test where P\u2009>\u20090.05 is considered nonsignificant (ns). (D) Differential count of the DMSO- or rapamycin-treated v2 B11 parasite blood stages (rings, trophozoites, or schizonts) at 46\u2009h, 52\u2009h, and 94\u2009h. Percentages of each blood stage (rings, trophozoites, or schizonts) were normalized to total percentage parasitemia at each time point. Data represent two independent experiments, and error bars depict standard deviations (SD).', 'hash': '5d6422b939143cf9345d7890a84d936e993cd6f56d4e66700b39cafba7653e6d'}, {'image_id': 'mBio.01460-19-f0004', 'image_file_name': 'mBio.01460-19-f0004.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0004.jpg', 'caption': 'Glucose uptake is decreased in RON3loxP parasites. The glucose analogue 2-NBDG was used to track glucose uptake into v2 cycle 2 DMSO- or rapamycin (Rapa)-treated parasites. (A) Representative images of DMSO- and Rapa-treated parasites after 2 h of incubation with 2-NBDG. Parasites were additionally stained with Hoechst 33342 (DNA marker) and MitoTracker Deep Red (Mito DR), a mitochondrial marker. Bars, 2\u2009μm. (B) Example illustration of strategy for determining change in mean fluorescent intensity (ΔMFI) of 2-NBDG. MFI (green) was measured by first measuring fluorescent intensity attributed to the parasite (red circle as determined by localization with Hoechst/MitoTracker [blue/red] localization). Background MFI in the blood cell in which the parasite resides (blue circle) was subsequently subtracted to provide ΔMFI. (C) ΔMFI of 2-NBDG in DMSO-treated and rapamycin-treated ring-stage parasites (n\u2009=\u200935/group). (D) ΔMFI of 2-NBDG in DMSO-treated and rapamycin-treated merozoites was measured by subtracting background MFI from merozoite MFI. (E) Representative images of DMSO- and Rapa-treated merozoites stained with 2-NBDG (n\u2009=\u200935/group). Data represent the mean ± standard error of the mean (SEM). Statistical significance was determined by two-tailed unpaired t test where P\u2009<\u20090.05 is considered significant (****, P\u2009<\u20090.0001).', 'hash': 'aaf12af922b05d486babb2af6dc5791a359d453e340673567fee60bb55c8c41b'}, {'image_id': 'mBio.01460-19-f0003', 'image_file_name': 'mBio.01460-19-f0003.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0003.jpg', 'caption': 'RON3 is essential for the export of proteins into the infected RBC. Ring infected RBCs from RON3loxP v1 (A) and v2 (B) parasites were treated with DMSO or rapamycin (Rapa) for 4\u2009h, washed, and cultured in cRPMI for ∼40 to 44\u2009h. The parasites were fixed and stained with antibodies to RON3, KAHRP, RESA, MAHRP1, EXP2, or PTEX150 antibodies. RON3, KAHRP, and EXP2 staining is shown in green, and RESA, MAHRP1, and PTEX150 staining is shown in red. The nuclei staining (4′,6′-diamidino-2-phenylindole [DAPI]) is shown in blue. MAHRP1, KAHRP, or RESA was exported into the DMSO-treated infected RBCs while they appeared to be around the rapamycin-treated parasite. All images are representative of the majority of parasites over several fields and/or of three independent experiments. DIC, differential interference contrast. Bars, 2\u2009μm.', 'hash': '20867e4f79f8ad67196a6a84478fa59418e72b3435aa389a5e0bc286e9b86a9a'}, {'image_id': 'mBio.01460-19-f0001', 'image_file_name': 'mBio.01460-19-f0001.jpg', 'image_path': '../data/media_files/PMC6747712/mBio.01460-19-f0001.jpg', 'caption': 'Rapid and efficient conditional disruption of the P. falciparum\nRON3 gene. The native PfRON3 gene on chromosome 12 comprises 8 exons (indicated as gray boxes) and 7 introns. (A) Version 1 (v1). Schematic representation of pre- and postintegration events after CRISPR-Cas9 editing to produce the modified RON3loxP and the expected locus architecture before and after rapamycin treatment (pre- and postexcision). Recodonized exons 2 to 6 are shown as a green box. Red arrows indicate target areas of primers RON3_exon1_F (solid arrow)/RON3_exon7_R (dashed arrow). (B) PCR RON3loxP v1 clones 3E10 and 7C4 were treated with DMSO (D) or rapamycin (R), and schizonts were analyzed ∼44\u2009h after treatment to determine excision. DNA markers (M) ranged from 200 to 10,000 bp. Excision leads to a decrease in amplicon size from 1,615\u2009bp to 422\u2009bp. (C) Version 2 (v2). Schematic representation of pre- and postintegration events after CRISPR-Cas9 editing to produce the modified RON3loxP and the expected locus architecture before and after rapamycin treatment (pre- and postexcision). Recodonized exons 6 to 7 are shown as a yellow box. Blue arrows indicate target areas of primers RON3_exon4_F (solid arrow)/RON3_exon8_R (dashed arrow). (D) PCR RON3loxP v2 clone B11 was treated with DMSO (D) or rapamycin (R); excision leads to s decrease in amplicon size from 2,364\u2009bp to 757\u2009bp. DNA markers (M) ranged from 250 to 10,000 bp. Samples were run in duplicate on the same gel, with the white line indicating splicing of the image to show only one replicate.', 'hash': '95eecc550e8fa1a1ec3aa16a91218c917bd530043c98e7cf9bee07b338670fbc'}]
|
{'mBio.01460-19-f0001': ['To investigate the role of RON3 in the Plasmodium falciparum life cycle, we conditionally disrupted the gene in the 3D7 parasite clone 1G5DC using the dimerizable P1 bacteriophage cyclization recombination (DiCre) recombinase system (14). Two forms of RON3loxP were created by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 (Cas9)-mediated gene editing, whereby the locus of X-over P1 (loxP) sites were inserted into different segments of the RON3 gene. The RON3 gene contains eight exons with the signal sequence located in exon 1. For version 1 (v1), the RON3 gene was modified such that exons 2 to 6 were recodonized without introns between them. This was flanked by synthetic introns containing loxP sites (<xref ref-type="fig" rid="mBio.01460-19-f0001">Fig.\xa01A</xref>). In version 2 (v2), ). In version 2 (v2), RON3 was edited further toward the 3′ end of the gene by replacing exons 6 to 7 with a recodonized version containing no introns. The endogenous introns on either side of exons 6 to 7 were replaced with synthetic loxP-containing introns (<xref ref-type="fig" rid="mBio.01460-19-f0001">Fig.\xa01C</xref>).).', 'Disruption of the RON3 gene was induced in ring-stage parasites; excision of the gene is mediated by brief rapamycin treatment, allowing the activation of the Cre recombinase that recognizes loxP sequences by dimerization of the DiCre components (14). Rapamycin treatment was predicted to lead to the excision of the recodonized exons. In v1, this would eliminate the first two transmembrane domains, while in v2, this would lead to excision of the third transmembrane domain. Diagnostic PCR, using DNA extracted from dimethyl sulfoxide (DMSO)- or rapamycin-treated v1 (clones 3E10 and 7C4) and v2 (clone B11), showed the expected loss of the intervening recodonized sequence (<xref ref-type="fig" rid="mBio.01460-19-f0001">Fig.\xa01B</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0001">D</xref>); v2 parasites were further sequenced, confirming the excision after rapamycin treatment and demonstrating efficient excision of the floxed sequences (data not shown).); v2 parasites were further sequenced, confirming the excision after rapamycin treatment and demonstrating efficient excision of the floxed sequences (data not shown).'], 'mBio.01460-19-f0002': ['Rapamycin-treated v1 or v2 parasites were observed to progress normally through the cycle in which they were treated (cycle 1; <xref ref-type="fig" rid="mBio.01460-19-f0002">Fig.\xa02A</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0002">B</xref>). However, following invasion into cycle 2, rapamycin-treated parasites did not develop beyond the ring stage (). However, following invasion into cycle 2, rapamycin-treated parasites did not develop beyond the ring stage (<xref ref-type="fig" rid="mBio.01460-19-f0002">Fig. 2A</xref>, , <xref ref-type="fig" rid="mBio.01460-19-f0002">B</xref>, and , and <xref ref-type="fig" rid="mBio.01460-19-f0002">D</xref>). Comparison of ring-stage parasites at 94\u2009h was found to be significantly different between treatment conditions (). Comparison of ring-stage parasites at 94\u2009h was found to be significantly different between treatment conditions (P\u2009<\u20090.001). Examination of the invasion capacity of v1 parasites (clones 3E10 and 7C4) found that rapamycin-treated parasites invaded new RBCs at a similar rate to DMSO-treated parasites of the same clone (<xref ref-type="fig" rid="mBio.01460-19-f0002">Fig.\xa02C</xref>). The parental parasite clone, 1G5DC, was also included in these experiments to assess whether rapamycin treatment itself could affect invasion; this confirmed that there were no significant differences between DMSO and rapamycin treatment as previously shown (). The parental parasite clone, 1G5DC, was also included in these experiments to assess whether rapamycin treatment itself could affect invasion; this confirmed that there were no significant differences between DMSO and rapamycin treatment as previously shown (14). To rule out any effects of loxP insertion, growth rates were compared between v1 parasites and the parental clone (1G5DC) with no significant differences observed (P\u2009>\u20090.05; see Fig.\xa0S2 in the supplemental material).'], 'mBio.01460-19-f0003': ['To seek insights into the development defect in cycle 2 rings derived from the rapamycin-treated RON3loxP parasites, we examined the fate of the PTEX exported proteins RESA, knob-associated histidine-rich protein (KAHRP), and Maurer’s cleft-associated histidine-rich protein 1 (MAHRP1) (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03</xref>); RESA was of particular interest, as it is known to be exported early during ring stages (); RESA was of particular interest, as it is known to be exported early during ring stages (15). In DMSO-treated RON3loxP parasites, RESA was found distributed throughout the infected RBC, concentrating in the space below the RBC membrane, as is normal (16, 17). In contrast, in newly formed rings of both v1 and v2 excised parasites, RESA was found surrounding the parasite, indicating failure of translocon function for protein export to the RBC (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03A</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0003">B</xref>). In addition, KAHRP, the electron-dense histidine-rich protein that normally localizes to below the knobs on the RBC surface, and MAHRP1, located at Maurer’s clefts, remained close to the parasite and were not translocated to the erythrocyte cytoplasm (). In addition, KAHRP, the electron-dense histidine-rich protein that normally localizes to below the knobs on the RBC surface, and MAHRP1, located at Maurer’s clefts, remained close to the parasite and were not translocated to the erythrocyte cytoplasm (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03A</xref>). Further analysis with antibodies to other exported proteins showed that the rapamycin-treated parasites were incapable of exporting other parasite proteins (skeleton binding protein 1 [SBP1], histidine-rich protein 2 [HRP2], and ). Further analysis with antibodies to other exported proteins showed that the rapamycin-treated parasites were incapable of exporting other parasite proteins (skeleton binding protein 1 [SBP1], histidine-rich protein 2 [HRP2], and P. falciparum erythrocyte membrane protein 1 [PfEMP1]; Fig.\xa0S3). Furthermore, in addition to RESA, the PTEX translocon components, EXP2 and PTEX150 (<xref ref-type="fig" rid="mBio.01460-19-f0003">Fig.\xa03A</xref>), were detected in both DMSO- and rapamycin-treated parasites. EXP2, alongside the other PTEX components, are found in the dense granules (), were detected in both DMSO- and rapamycin-treated parasites. EXP2, alongside the other PTEX components, are found in the dense granules (18) and allow the export of proteins, such as RESA, in healthy (normal) parasites.'], 'mBio.01460-19-f0004': ['To examine whether loss of RON3 also interfered with import of molecules into parasitized cells (a function of EXP2 in a role independent of the PTEX complex [11]), an assay was devised to directly detect import of a labeled glucose analogue, 2-(N-(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Cells were further stained with Hoechst 33342 (DNA marker) and MitoTracker Deep Red (mitochondrial marker), which allowed parasites to be identified. Study of 2-NBDG uptake was not interfered with by the use of these two markers as determined using fluorescent controls (Fig.\xa0S4). RON3-deficient intracellular parasites were observed to take up less glucose than control DMSO-treated counterparts (<xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04A</xref> and and <xref ref-type="fig" rid="mBio.01460-19-f0004">C</xref>). After invasion, glucose uptake into the parasite was significantly lower than glucose uptake into the DMSO control, as quantified by comparing the mean fluorescent intensity (MFI) of 2-NBDG in DMSO- and rapamycin-treated parasites, wherein the MFI of background 2-NBDG signal in the infected RBC was subtracted from the 2-NBDG MFI in the parasite (change in MFI [ΔMFI]) (). After invasion, glucose uptake into the parasite was significantly lower than glucose uptake into the DMSO control, as quantified by comparing the mean fluorescent intensity (MFI) of 2-NBDG in DMSO- and rapamycin-treated parasites, wherein the MFI of background 2-NBDG signal in the infected RBC was subtracted from the 2-NBDG MFI in the parasite (change in MFI [ΔMFI]) (<xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04B</xref>). In two independent experiments (). In two independent experiments (<xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04C</xref> and and S5B), the ΔMFI of rapamycin-treated parasites was significantly less than that of the control, suggesting impairment of glucose uptake through the PVM. We further observed an outlier in the rapamycin-treated group; this is likely attributable to ∼2% of parasites not undergoing excision during rapamycin treatment and has been previously observed in other DiCre mutants (14). As 2-NBDG is reported to metabolize to a nonfluorescent form, we repeated the experiment with a nonmetabolizable analogue, 6-NBDG (19), and found that DMSO-treated parasites still significantly took up more glucose than the rapamycin-treated parasites did (Fig.\xa0S5C). We further compared the background MFI of 2-NBDG or 6-NBDG in the infected blood cell (Fig.\xa0S5D) and found a significant difference between DMSO- and rapamycin-treated conditions. As demonstrated in this paper, the loss of RON3 leads to no parasite protein export through the PTEX translocon. The absence of these parasite proteins in the RBC may lead to reduced uptake in glucose by the infected RBC. However, as there is only ∼20% reduction in uptake into the RBC between groups, we do not believe that this difference is enough to explain the ∼50% reduction in glucose uptake into the RON3-deficient parasite.', 'To exclude a role for RON3 in the function of the hexose transporter (HT) that is located on the parasite plasma membrane (PPM), released merozoites that were treated with DMSO or rapamycin were investigated. Free rapamycin-treated merozoites were observed to take up glucose similarly to DMSO-treated parasites (P\u2009>\u20090.05; <xref ref-type="fig" rid="mBio.01460-19-f0004">Fig.\xa04D</xref>), indicating that the HT present on the PPM is functional and exhibits similar activity.), indicating that the HT present on the PPM is functional and exhibits similar activity.']}
|
{'@content-type': 'genus-species', '#text': 'Plasmodium falciparum'} Deletion of Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake
|
[
"PTEX",
"malaria",
"parasite proteins",
"parasitophorous vacuolar space"
] |
mBio
|
1562655600
|
The survival of spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane. The malarial parasite within the erythrocyte is surrounded by two membranes. translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We now describe a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We find that RON3 is required for the protein transport function of the PTEX and for glucose transport from the RBC cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2.
|
[
"Antigens, Neoplasm",
"Biological Transport",
"Erythrocytes",
"Gene Deletion",
"Glucose",
"Host-Parasite Interactions",
"Humans",
"Malaria, Falciparum",
"Plasmodium falciparum",
"Protein Transport",
"Protozoan Proteins",
"Translocation, Genetic"
] |
other
|
PMC6747712
| null |
33
|
[
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"{'Citation': 'Moss DK, Remarque EJ, Faber BW, Cavanagh DR, Arnot DE, Thomas AW, Holder AA. 2012. Plasmodium falciparum 19-kilodalton merozoite surface protein 1 (MSP1)-specific antibodies that interfere with parasite growth in vitro can inhibit MSP1 processing, merozoite invasion, and intracellular parasite development. Infect Immun 80:1280–1287. doi:10.1128/IAI.05887-11.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1128/IAI.05887-11'}, {'@IdType': 'pmc', '#text': 'PMC3294643'}, {'@IdType': 'pubmed', '#text': '22202121'}]}}",
"{'Citation': 'Yoshioka K, Saito M, Oh KB, Nemoto Y, Matsuoka H, Natsume M, Abe H. 1996. Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells. Biosci Biotechnol Biochem 60:1899–1901. doi:10.1271/bbb.60.1899.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1271/bbb.60.1899'}, {'@IdType': 'pubmed', '#text': '8987871'}]}}"
] |
mBio. 2019 Jul 9; 10(4):e01460-19
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NO-CC CODE
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Dual-channel ratiometric
fluorescence imaging of living cells in
the presence of different concentrations of exogenous ONOO– (A); the fluorescence intensity in two channels in the presence
of different concentrations of SIN-1 (B); and the linear relationship
between the fluorescence intensity ratio (F670/F780) of the two channels and the concentrations
of SIN-1 (C).
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ao0c01320_0004
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2
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6cddedeccc7fa7af038b8c424bb0b68f83574db430ffac90cf8754a9249a854e
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ao0c01320_0004.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
664,
963
] |
[{'image_id': 'ao0c01320_0002', 'image_file_name': 'ao0c01320_0002.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0002.jpg', 'caption': '(A) Fluorescence excitation (1) and emission (2) spectra of NI-BQDs,\nand absorption spectra (3) of Cy7. (B) With the increase of the Cy7\nconcentration, the fluorescence intensity of NI-BQDs decreased, while\nthe fluorescence intensity of Cy7 increased. (C) Fluorescence spectra\nof NI-BQD-Cy7 in the presence of different concentrations of ONOO– (0–15 μM). (D) Linear relationship between\nthe logarithm value of the fluorescence intensity ratio (I670/I780) and the ONOO– concentration.', 'hash': '55074ff8697410e340c70b4f7fd35c42ede082d56365927b735cf2d86964514d'}, {'image_id': 'ao0c01320_0005', 'image_file_name': 'ao0c01320_0005.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0005.jpg', 'caption': '(A) Real-time tracing the generation of endogenous\nONOO– in a single living cell by ratiometric fluorescence\nimages. (B)\nChange of the single-cell fluorescence intensity with time in two\nfluorescence channels. (C) Linear relationship between the logarithmic\nvalue of the fluorescence intensity ratio (F670/F780) at the two channels and\nincubation time within 0–40 min.', 'hash': '072cbf703e30220b15897393b035b2dca11c72f65592c422cac26bd04edff4c4'}, {'image_id': 'ao0c01320_0004', 'image_file_name': 'ao0c01320_0004.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0004.jpg', 'caption': 'Dual-channel ratiometric\nfluorescence imaging of living cells in\nthe presence of different concentrations of exogenous ONOO– (A); the fluorescence intensity in two channels in the presence\nof different concentrations of SIN-1 (B); and the linear relationship\nbetween the fluorescence intensity ratio (F670/F780) of the two channels and the concentrations\nof SIN-1 (C).', 'hash': '6cddedeccc7fa7af038b8c424bb0b68f83574db430ffac90cf8754a9249a854e'}, {'image_id': 'ao0c01320_0003', 'image_file_name': 'ao0c01320_0003.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0003.jpg', 'caption': 'Colocalization imaging of suborganelle\nin RAW264.7 cells after\nco-incubation with NI-BQDs-Cy7 nanoprobe and localization reagents.', 'hash': 'f0a80d358eeaac32133707ed9e82e1afca589ab4709712004baea9448d4217fb'}, {'image_id': 'ao0c01320_0009', 'image_file_name': 'ao0c01320_0009.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0009.jpg', 'caption': 'No caption found', 'hash': '3d0dd62747189506872ff1de017a3562deb6cf8cdead3c4d5cca5367ba734b6d'}, {'image_id': 'ao0c01320_0007', 'image_file_name': 'ao0c01320_0007.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0007.jpg', 'caption': '(A) Real-time tracing the generation of endogenous ONOO– in vivo by ratiometric fluorescence images. The imaging parameters\nare the same as those in Figure 6. (B) Change of fluorescence intensity of endogenous\nONOO– in vivo with time in two channels. (C) Relationship\nbetween the fluorescence intensity ratio (F700/F790) of two channels and incubation\ntime within 0–70 min.', 'hash': '21b4763e629d49f4b2ed9e339aaa6d1e2a4a227c908336eb7f418869eac6b1f5'}, {'image_id': 'ao0c01320_0006', 'image_file_name': 'ao0c01320_0006.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0006.jpg', 'caption': 'Stability of the fluorescent NI-BQD-Cy7 nanoprobe in vivo. The\nimaging parameters are the excitation wavelength: 650 nm; receiving\nfilter wavelength: 700 and 790 nm; power: 400 W; and irradiation time:\n20 s.', 'hash': '70e2f1bfcc0387d4f398ff1f2cab5087291eb43a096e5a952952526ae20f3653'}, {'image_id': 'ao0c01320_0001', 'image_file_name': 'ao0c01320_0001.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0001.jpg', 'caption': '(A) TEM and\nHRTEM images of NI-BQDs. (B) XRD diffractogram of NI-BQDs\n(a) and NI-BQDs-Cy7 (b). (C) XPS spectrum of NI-BQDs. (D) FTIR spectra\nof Cy7 (a), NI-BQDs (b), and NI-BQD-Cy7 (c).', 'hash': '698ffe9e292f32728f6b92bdbbef7ccc6ab8db8712cff4730ef9bbe94aa4f1b4'}, {'image_id': 'ao0c01320_0008', 'image_file_name': 'ao0c01320_0008.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0008.jpg', 'caption': 'Schematic Representation for the Fabrication\nand Application of the\nNI-BQD-Cy7 NIR Dual-Emission Nanoprobe', 'hash': 'd8dc06d43ebc140d76349f74d907cfa9232e15fd5dc26e7a25d03cb7f963b92a'}]
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{'ao0c01320_0001': ['The morphology,\ndispersion, and particle size of NI-BQDs were first characterized\nby transmission electron microscopy (TEM). The TEM image shown in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>A reveals that NI-BQDs\nhave good dispersion with a diameter of about 2 nm. The lattice constant\nof the high-resolution transmission electron microscopy (HRTEM) diagram\nof NI-BQDs is 0.22 μm, which corresponds to the crystal plane\nof graphene (100), indicating that the graphitization and crystallinity\nof NI-BQDs are relatively high. The crystal forms of NI-BQDs and NI-BQD-Cy7\nwere investigated by X-ray diffraction (XRD) analysis. The diffractograms\ndisplayed in <xref rid="ao0c01320_0001" ref-type="fig">1</xref>A reveals that NI-BQDs\nhave good dispersion with a diameter of about 2 nm. The lattice constant\nof the high-resolution transmission electron microscopy (HRTEM) diagram\nof NI-BQDs is 0.22 μm, which corresponds to the crystal plane\nof graphene (100), indicating that the graphitization and crystallinity\nof NI-BQDs are relatively high. The crystal forms of NI-BQDs and NI-BQD-Cy7\nwere investigated by X-ray diffraction (XRD) analysis. The diffractograms\ndisplayed in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>B show that there is an obvious wide diffraction peak at the position\nof 2θ = 23.43°, and the corresponding carbon structure\nmirror is (002). There are no characteristic diffraction peaks of\nother graphites in the XRD patterns. A comparison of the two diffractograms\nreveals that the XRD patterns of NI-BQDs and NI-BQDs-Cy7 are almost\nidentical, indicating that the modification of Cy7 does not change\nthe structure and morphology of NI-BQDs. The surface element composition\nof NI-BQDs was characterized by X-ray photoelectron spectroscopy (XPS).\nThe XPS spectrum of NI-BQDs shown in <xref rid="ao0c01320_0001" ref-type="fig">1</xref>B show that there is an obvious wide diffraction peak at the position\nof 2θ = 23.43°, and the corresponding carbon structure\nmirror is (002). There are no characteristic diffraction peaks of\nother graphites in the XRD patterns. A comparison of the two diffractograms\nreveals that the XRD patterns of NI-BQDs and NI-BQDs-Cy7 are almost\nidentical, indicating that the modification of Cy7 does not change\nthe structure and morphology of NI-BQDs. The surface element composition\nof NI-BQDs was characterized by X-ray photoelectron spectroscopy (XPS).\nThe XPS spectrum of NI-BQDs shown in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>C reveals that it is mainly composed of C,\nN, and O, accounting for 57.31, 4.33, and 31.67%, respectively. A\nstrong O 1s characteristic peak appeared at 532.7 eV, a second strong\nC 1s characteristic peak is present at 285.1 eV, and a weak N 1s characteristic\npeak is present at 399.1 eV. The high-resolution XPS spectra of C\n1s, N 1s, and O 1s are displayed in <xref rid="ao0c01320_0001" ref-type="fig">1</xref>C reveals that it is mainly composed of C,\nN, and O, accounting for 57.31, 4.33, and 31.67%, respectively. A\nstrong O 1s characteristic peak appeared at 532.7 eV, a second strong\nC 1s characteristic peak is present at 285.1 eV, and a weak N 1s characteristic\npeak is present at 399.1 eV. The high-resolution XPS spectra of C\n1s, N 1s, and O 1s are displayed in Figure S1 (Supporting Information) that indicate the presence of C–H/C–C,\nC–N, C–O, and C=O bonds in the C 1s spectrum,\nthe existence of pyrrolic-N and pyridine-N bonds in the N 1s spectrum,\nand the presence of O=C, O–H, O–C, and O=C–O\nbonds in the C 1s spectrum. The composition of functional groups on\nthe surface of NI-BQDs was characterized by Fourier transform infrared\n(FTIR) spectroscopy. The FTIR spectra of Cy7, NI-BQDs, and NI-BQDs-Cy7\ndisplayed in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>D show a peak at 3410 cm<xref rid="ao0c01320_0001" ref-type="fig">1</xref>D show a peak at 3410 cm–1 that corresponds to\nthe stretching vibration peak of −O–H/N–H, and\nits intensity in NI-BQD-Cy7 (trace c) is clearly stronger than those\nin NI-BQDs (trace b) and Cy7 (trace a), indicating that Cy7 is covalently\ncoupled with NI-BQDs through an amide bond, which is consistent with\nthe experimental principle. The peak located at 2848 cm–1 corresponds to the telescopic vibration of C–H, and that\nat 1634 cm–1 corresponds to C=C and C=N\nbending vibration. Additionally, the peak at 1360–1020 cm–1 corresponds to C–O and C–N. The absorption\nof NI-BQD-Cy7 was significantly higher than that of NI-BQDs, and the\nwide peak around 1190 cm–1 is the characteristic\nabsorption peak of the sulfonic group, which indicates that Cy7 was\nsuccessfully conjugated to NI-BQDs. The surface charge of NI-BQDs\nand NI-BQD-Cy7 was further investigated, and the results showed that\nsurfaces of both were positively charged in a pH 7.4 neutral environment\n(Figure S2, Supporting Information).'], 'ao0c01320_0002': ['To determine\nthe optical properties of NI-BQDs and NI-BQD-Cy7, we first investigated\ntheir ultraviolet–visible (UV–vis) absorption spectra.\nThe UV–vis spectra shown in Figure S3 in the Supporting Information reveal an obvious absorption peak\nat 279 nm, which is the formation of π–π* electron\ntransition of C=O in NI-BQDs. The NI-BQDs-Cy7 has an obvious\nabsorption peak at 750 nm, which is due to the characteristic absorption\nof the Cy7 molecule, further indicating that Cy7 had been successfully\ncoupled on the surface of NI-BQDs. We studied the feasibility of FRET\nbetween NI-BQDs and Cy7, and the results revealed that the maximum\nexcitation and emission wavelengths of NI-BQDs are at 405 and 678\nnm, respectively. The maximum absorption wavelength of Cy7 is at 710\nnm, and the absorption spectrum of Cy7 overlaps with the fluorescence\nemission spectrum of NI-BQDs, which indicates that they can be a pair\nof donors and receptors of FRET (<xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>A). After the covalent coupling of NI-BQDs\nwith Cy7, the fluorescence emission spectrum of NI-BQD-Cy7 exhibited\nemission peaks at 670 and 780 nm, and the fluorescence intensity of\nthe peak at 670 nm gradually decreased with the increase of the Cy7\nconcentration, while the fluorescence intensity of the peak at 780\nnm was gradually increased (<xref rid="ao0c01320_0002" ref-type="fig">2</xref>A). After the covalent coupling of NI-BQDs\nwith Cy7, the fluorescence emission spectrum of NI-BQD-Cy7 exhibited\nemission peaks at 670 and 780 nm, and the fluorescence intensity of\nthe peak at 670 nm gradually decreased with the increase of the Cy7\nconcentration, while the fluorescence intensity of the peak at 780\nnm was gradually increased (<xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>B). These results showed that the coupling of NI-BQDs\nwith Cy7 was successful and a dual-emission NIR ratiometric fluorescent\nnanoprobe was formed by FRET interactions. The FRET efficiency can\nbe measured and calculated by a reported method.<xref rid="ao0c01320_0002" ref-type="fig">2</xref>B). These results showed that the coupling of NI-BQDs\nwith Cy7 was successful and a dual-emission NIR ratiometric fluorescent\nnanoprobe was formed by FRET interactions. The FRET efficiency can\nbe measured and calculated by a reported method.27', 'The response of the nanoprobe to ONOO– was investigated\nby mixing a certain amount of the NI-BQD-Cy7 solution with various\nsolutions containing different concentrations of ONOO–. The results shown in <xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>C reveal that as the ONOO<xref rid="ao0c01320_0002" ref-type="fig">2</xref>C reveal that as the ONOO– concentration\nincreases, the fluorescence intensity of the nanoprobe at 780 nm is\ngradually reduced, while the fluorescence intensity at 670 nm is gradually\nincreased. Additionally, the results also show that there is a good\nlinear relationship between the logarithm value of the fluorescence\nintensity ratio (I670/I780) and the ONOO– concentration in\nthe range of 0.02–15 μM (<xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>D) with a detection limit of 8.5 nM (S/N\n= 3), as indicated by the linear regression equation: log (<xref rid="ao0c01320_0002" ref-type="fig">2</xref>D) with a detection limit of 8.5 nM (S/N\n= 3), as indicated by the linear regression equation: log (I670/I780) = 0.05788CONOO– – 0.4474, R2 = 0.9959, which is comparable to the previously reported\nONOO– ratiometric fluorescent probe.14,15 In addition, the response time of the NI-BQD-Cy7 nanoprobe toward\nONOO– was also investigated via a kinetics method.\nAfter the addition of ONOO– to the NI-BQD-Cy7 nanoprobe\nsolution for 4 min, the fluorescence intensity of NI-BQD increased\nto its maximum and that of Cy7 decreased to its minimum (Figure S6, Supporting Information).'], 'ao0c01320_0003': ['Since endogenous ONOO– is produced in the mitochondria\nof living cells, it is necessary to confirm that the nanoprobe can\neffectively enter the cell mitochondria through a cell colocalization\nassay. Accordingly, RAW264.7 cells were incubated for 8 h with the\nNI-BQD-Cy7 nanoprobe, as well as lysosome, nucleus, and mitochondrial\nlocalization reagent, separately. Then, laser confocal imaging was\nperformed. The results, which are shown in <xref rid="ao0c01320_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>, indicate that the NI-BQD-Cy7 nanoprobe\nhas excellent mitochondria-targeting ability (colocalization coefficient\nis 0.89), while the colocalization coefficients for lysosome and nucleus\nare only 0.56 and 0.58, respectively.<xref rid="ao0c01320_0003" ref-type="fig">3</xref>, indicate that the NI-BQD-Cy7 nanoprobe\nhas excellent mitochondria-targeting ability (colocalization coefficient\nis 0.89), while the colocalization coefficients for lysosome and nucleus\nare only 0.56 and 0.58, respectively.'], 'ao0c01320_0004': ['To investigate the kinetic\nrange for ONOO– detection by the NI-BQD-Cy7 nanoprobe\nin living cells, RAW264.7 cells at the appropriate density were seeded\nin five 35 mm confocal imaging dishes and incubated for 20 h. Subsequently,\n50 mL of the PBS solution containing 0, 0.1, 0.3, 0.6, and 1 mM SIN-1\nwas added, respectively, into the above five cell culture dishes,\nand after incubation for 2 h, each group of cells was subjected to\ndual-channel fluorescence imaging. The results shown in <xref rid="ao0c01320_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>A reveal that with the increase\nof SIN-1 concentration, the fluorescence intensity at the 780 nm channel\ngradually decreases and the fluorescence intensity at the 670 nm channel\ngradually increases (<xref rid="ao0c01320_0004" ref-type="fig">4</xref>A reveal that with the increase\nof SIN-1 concentration, the fluorescence intensity at the 780 nm channel\ngradually decreases and the fluorescence intensity at the 670 nm channel\ngradually increases (<xref rid="ao0c01320_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>B). The results also show that there is a good linear relationship\nbetween the logarithmic value of the fluorescence intensity ratio\n(<xref rid="ao0c01320_0004" ref-type="fig">4</xref>B). The results also show that there is a good linear relationship\nbetween the logarithmic value of the fluorescence intensity ratio\n(F670/F780) of two channels and the concentration of SIN-1 in the range of\n0–1000 μM (<xref rid="ao0c01320_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>C), as indicated by the linear regression equation: log(<xref rid="ao0c01320_0004" ref-type="fig">4</xref>C), as indicated by the linear regression equation: log(F670/F780) = 4.453\n× 10–4CSIN-1 + 0.1765, R2 = 0.9964.'], 'ao0c01320_0005': ['The generation of endogenous ONOO– in single living\ncells was monitored by in situ ratiometric\nfluorescence imaging using the developed nanoprobe. RAW264.7 cells\nwere inoculated in 35 mm confocal imaging dishes. When the cells reached\na suitable density, the NI-BQD-Cy7 nanoprobe was added and the cells\nwere incubated for 6 h. Subsequently, the cells were incubated with\na mixed solution containing 50 ng/mL INF-γ and 1 μg/mL\nLPS for 4 h. After adding 25 nM PMA, a single cell was imaged on a\nconfocal microscopic imaging system at 0, 5, 10, 20, 30, and 40 min.\nThe results displayed in <xref rid="ao0c01320_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref> show that with the increase of incubation time, the\nfluorescence intensity of the single living cell at the 780 nm channel\ngradually decreases, while the fluorescence intensity at the 670 nm\nchannel gradually increases (<xref rid="ao0c01320_0005" ref-type="fig">5</xref> show that with the increase of incubation time, the\nfluorescence intensity of the single living cell at the 780 nm channel\ngradually decreases, while the fluorescence intensity at the 670 nm\nchannel gradually increases (<xref rid="ao0c01320_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>A,<xref rid="ao0c01320_0005" ref-type="fig">5</xref>A,<xref rid="ao0c01320_0005" ref-type="fig">5</xref>B). The results also show\nthat there was a good linear relationship between the logarithmic\nvalue of the fluorescence intensity ratio (B). The results also show\nthat there was a good linear relationship between the logarithmic\nvalue of the fluorescence intensity ratio (F670/F780) of the two channels and\nthe incubation time within the 0–40 min period (<xref rid="ao0c01320_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>C), as indicated by the linear\nregression equation: log(<xref rid="ao0c01320_0005" ref-type="fig">5</xref>C), as indicated by the linear\nregression equation: log(F670/F780) = 0.01383T (min) + 0.08668, R2 = 0.9995. These results showed that the NI-BQD-Cy7\nnanoprobe can real-time trace the generation of endogenous ONOO– in a single cell. Moreover, it was also found that\nthe generation of endogenous ONOO– increased linearly\nwithin 0–40 min.'], 'ao0c01320_0006': ['To\nachieve the ratiometric fluorescence imaging of ONOO– in vivo, the stability of the fluorescent nanoprobe in vivo was\ninvestigated. Two male nude mice (10 week old, weight of about 20\ng) were evaluated. One was injected with saline (150 μL), and\nthe other with the NI-BQD-Cy7 nanoprobe (3 mg/mL,150 μL) in\nthe abdominal cavity at ca. 2–4 mm depth. After anesthesia\nwith isoflurane and halothane, the mice were imaged using a small\nanimal imaging system. After injecting NI-BQD-Cy7, the fluorescence\nat the 700 ± 30 and 790 ± 30 nm channels was collected at\n0.5, 1.5, and 4 h under excitation at 650 nm.28 The results shown in <xref rid="ao0c01320_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref> reveal that the fluorescence intensity at two channels remains\nstable for 4 h, indicating that the developed nanoprobe is suitable\nfor the ratiometric fluorescence imaging of ONOO<xref rid="ao0c01320_0006" ref-type="fig">6</xref> reveal that the fluorescence intensity at two channels remains\nstable for 4 h, indicating that the developed nanoprobe is suitable\nfor the ratiometric fluorescence imaging of ONOO– in vivo.', '(A) Real-time tracing the generation of endogenous ONOO– in vivo by ratiometric fluorescence images. The imaging parameters\nare the same as those in <xref rid="ao0c01320_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>. (B) Change of fluorescence intensity of endogenous\nONOO<xref rid="ao0c01320_0006" ref-type="fig">6</xref>. (B) Change of fluorescence intensity of endogenous\nONOO– in vivo with time in two channels. (C) Relationship\nbetween the fluorescence intensity ratio (F700/F790) of two channels and incubation\ntime within 0–70 min.'], 'ao0c01320_0007': ['In situ ratiometric\nfluorescence imaging monitoring\nthe generation of endogenous ONOO– in vivo was investigated\nusing NI-BQD-Cy7 as the nanoprobe and acetaminophen (APAP) as the\nONOO–-inducing agent.26 The NI-BQD-Cy7 nanoprobe was injected into the diaphragm of male\nnude mice (3 mg/mL, 150 μL), and after 30 min, the APAP (500\nmg/kg) was also injected into the diaphragm of the mice. The mice\nwere anesthetized with isoflurane and halothane, and then the fluorescence\nat the 700 ± 30 and 790 ± 30 nm channels was collected at\n10, 30, 50, and 70 min after injection of APAP. The results presented\nin <xref rid="ao0c01320_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>A show that\nwith prolongation of time after treatment with APAP, the fluorescence\nintensity at the 700 ± 30 nm channel gradually increases, while\nthe fluorescence intensity at the 790 ± 30 nm channel gradually\ndecreases (<xref rid="ao0c01320_0007" ref-type="fig">7</xref>A show that\nwith prolongation of time after treatment with APAP, the fluorescence\nintensity at the 700 ± 30 nm channel gradually increases, while\nthe fluorescence intensity at the 790 ± 30 nm channel gradually\ndecreases (<xref rid="ao0c01320_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>B). The fluorescence intensity ratio of two channels (<xref rid="ao0c01320_0007" ref-type="fig">7</xref>B). The fluorescence intensity ratio of two channels (F700/F790) gradually increases\nwith the prolongation of time but slowed down after 50 min (<xref rid="ao0c01320_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>C). These results\nshow that the NI-BQD-Cy7 nanoprobe can be used for in situ ratiometric\nimaging to monitor the production of ONOO<xref rid="ao0c01320_0007" ref-type="fig">7</xref>C). These results\nshow that the NI-BQD-Cy7 nanoprobe can be used for in situ ratiometric\nimaging to monitor the production of ONOO– in vivo\nas well as in the diagnosis of ONOO– related diseases\nin vivo.']}
|
Near-Infrared Dual-Emission Ratiometric Fluorescence
Imaging Nanoprobe for Real-Time Tracing the Generation of Endogenous
Peroxynitrite in Single Living Cells and In Vivo
| null |
ACS Omega
|
1590649200
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Peroxynitrite (ONOO) is a highly reactive nitrogen species with potent oxidant and nitrating properties. Its excessive generation can cause DNA and protein damage, thereby contributing to cell injury, and it is closely related to the development of many diseases. Thus, there is an urgent need for a reliable method to determine changes in the steady-state levels of ONOO in vivo. Ratiometric imaging, due to its built-in self-calibration system, can reduce artifacts and enable reliable in vivo imaging. In this study, we designed and prepared near-infrared (NIR) biomass quantum dots (NI-BQDs) and covalently coupled them with the NIR dye Cyanine7 (Cy7) to construct an NIR dual-emission nanoprobe (NI-BQD-Cy7) for real-time tracing the generation of endogenous ONOO in single living cells and in vivo by ratiometric fluorescence imaging. NI-BQD-Cy7 exhibited high detection sensitivity and selectivity for ONOO in the mitochondria. Additionally, it can produce dual NIR fluorescence emission, thus allowing in situ ratiometric fluorescence imaging to real-time trace the generation and concentration changes of ONOO in vivo. The application of the proposed NIR dual-emission nanoprobe can provide accurate information for the study of the biological function of ONOO in single living cells and in vivo, and it is very useful to explain the mechanism of cell damage caused by ONOO.
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PMC7288700
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[
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] |
ACS Omega. 2020 May 28; 5(22):13278-13286
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NO-CC CODE
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Colocalization imaging of suborganelle
in RAW264.7 cells after
co-incubation with NI-BQDs-Cy7 nanoprobe and localization reagents.
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ao0c01320_0003
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2
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f0a80d358eeaac32133707ed9e82e1afca589ab4709712004baea9448d4217fb
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ao0c01320_0003.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
566,
394
] |
[{'image_id': 'ao0c01320_0002', 'image_file_name': 'ao0c01320_0002.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0002.jpg', 'caption': '(A) Fluorescence excitation (1) and emission (2) spectra of NI-BQDs,\nand absorption spectra (3) of Cy7. (B) With the increase of the Cy7\nconcentration, the fluorescence intensity of NI-BQDs decreased, while\nthe fluorescence intensity of Cy7 increased. (C) Fluorescence spectra\nof NI-BQD-Cy7 in the presence of different concentrations of ONOO– (0–15 μM). (D) Linear relationship between\nthe logarithm value of the fluorescence intensity ratio (I670/I780) and the ONOO– concentration.', 'hash': '55074ff8697410e340c70b4f7fd35c42ede082d56365927b735cf2d86964514d'}, {'image_id': 'ao0c01320_0005', 'image_file_name': 'ao0c01320_0005.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0005.jpg', 'caption': '(A) Real-time tracing the generation of endogenous\nONOO– in a single living cell by ratiometric fluorescence\nimages. (B)\nChange of the single-cell fluorescence intensity with time in two\nfluorescence channels. (C) Linear relationship between the logarithmic\nvalue of the fluorescence intensity ratio (F670/F780) at the two channels and\nincubation time within 0–40 min.', 'hash': '072cbf703e30220b15897393b035b2dca11c72f65592c422cac26bd04edff4c4'}, {'image_id': 'ao0c01320_0004', 'image_file_name': 'ao0c01320_0004.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0004.jpg', 'caption': 'Dual-channel ratiometric\nfluorescence imaging of living cells in\nthe presence of different concentrations of exogenous ONOO– (A); the fluorescence intensity in two channels in the presence\nof different concentrations of SIN-1 (B); and the linear relationship\nbetween the fluorescence intensity ratio (F670/F780) of the two channels and the concentrations\nof SIN-1 (C).', 'hash': '6cddedeccc7fa7af038b8c424bb0b68f83574db430ffac90cf8754a9249a854e'}, {'image_id': 'ao0c01320_0003', 'image_file_name': 'ao0c01320_0003.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0003.jpg', 'caption': 'Colocalization imaging of suborganelle\nin RAW264.7 cells after\nco-incubation with NI-BQDs-Cy7 nanoprobe and localization reagents.', 'hash': 'f0a80d358eeaac32133707ed9e82e1afca589ab4709712004baea9448d4217fb'}, {'image_id': 'ao0c01320_0009', 'image_file_name': 'ao0c01320_0009.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0009.jpg', 'caption': 'No caption found', 'hash': '3d0dd62747189506872ff1de017a3562deb6cf8cdead3c4d5cca5367ba734b6d'}, {'image_id': 'ao0c01320_0007', 'image_file_name': 'ao0c01320_0007.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0007.jpg', 'caption': '(A) Real-time tracing the generation of endogenous ONOO– in vivo by ratiometric fluorescence images. The imaging parameters\nare the same as those in Figure 6. (B) Change of fluorescence intensity of endogenous\nONOO– in vivo with time in two channels. (C) Relationship\nbetween the fluorescence intensity ratio (F700/F790) of two channels and incubation\ntime within 0–70 min.', 'hash': '21b4763e629d49f4b2ed9e339aaa6d1e2a4a227c908336eb7f418869eac6b1f5'}, {'image_id': 'ao0c01320_0006', 'image_file_name': 'ao0c01320_0006.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0006.jpg', 'caption': 'Stability of the fluorescent NI-BQD-Cy7 nanoprobe in vivo. The\nimaging parameters are the excitation wavelength: 650 nm; receiving\nfilter wavelength: 700 and 790 nm; power: 400 W; and irradiation time:\n20 s.', 'hash': '70e2f1bfcc0387d4f398ff1f2cab5087291eb43a096e5a952952526ae20f3653'}, {'image_id': 'ao0c01320_0001', 'image_file_name': 'ao0c01320_0001.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0001.jpg', 'caption': '(A) TEM and\nHRTEM images of NI-BQDs. (B) XRD diffractogram of NI-BQDs\n(a) and NI-BQDs-Cy7 (b). (C) XPS spectrum of NI-BQDs. (D) FTIR spectra\nof Cy7 (a), NI-BQDs (b), and NI-BQD-Cy7 (c).', 'hash': '698ffe9e292f32728f6b92bdbbef7ccc6ab8db8712cff4730ef9bbe94aa4f1b4'}, {'image_id': 'ao0c01320_0008', 'image_file_name': 'ao0c01320_0008.jpg', 'image_path': '../data/media_files/PMC7288700/ao0c01320_0008.jpg', 'caption': 'Schematic Representation for the Fabrication\nand Application of the\nNI-BQD-Cy7 NIR Dual-Emission Nanoprobe', 'hash': 'd8dc06d43ebc140d76349f74d907cfa9232e15fd5dc26e7a25d03cb7f963b92a'}]
|
{'ao0c01320_0001': ['The morphology,\ndispersion, and particle size of NI-BQDs were first characterized\nby transmission electron microscopy (TEM). The TEM image shown in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>A reveals that NI-BQDs\nhave good dispersion with a diameter of about 2 nm. The lattice constant\nof the high-resolution transmission electron microscopy (HRTEM) diagram\nof NI-BQDs is 0.22 μm, which corresponds to the crystal plane\nof graphene (100), indicating that the graphitization and crystallinity\nof NI-BQDs are relatively high. The crystal forms of NI-BQDs and NI-BQD-Cy7\nwere investigated by X-ray diffraction (XRD) analysis. The diffractograms\ndisplayed in <xref rid="ao0c01320_0001" ref-type="fig">1</xref>A reveals that NI-BQDs\nhave good dispersion with a diameter of about 2 nm. The lattice constant\nof the high-resolution transmission electron microscopy (HRTEM) diagram\nof NI-BQDs is 0.22 μm, which corresponds to the crystal plane\nof graphene (100), indicating that the graphitization and crystallinity\nof NI-BQDs are relatively high. The crystal forms of NI-BQDs and NI-BQD-Cy7\nwere investigated by X-ray diffraction (XRD) analysis. The diffractograms\ndisplayed in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>B show that there is an obvious wide diffraction peak at the position\nof 2θ = 23.43°, and the corresponding carbon structure\nmirror is (002). There are no characteristic diffraction peaks of\nother graphites in the XRD patterns. A comparison of the two diffractograms\nreveals that the XRD patterns of NI-BQDs and NI-BQDs-Cy7 are almost\nidentical, indicating that the modification of Cy7 does not change\nthe structure and morphology of NI-BQDs. The surface element composition\nof NI-BQDs was characterized by X-ray photoelectron spectroscopy (XPS).\nThe XPS spectrum of NI-BQDs shown in <xref rid="ao0c01320_0001" ref-type="fig">1</xref>B show that there is an obvious wide diffraction peak at the position\nof 2θ = 23.43°, and the corresponding carbon structure\nmirror is (002). There are no characteristic diffraction peaks of\nother graphites in the XRD patterns. A comparison of the two diffractograms\nreveals that the XRD patterns of NI-BQDs and NI-BQDs-Cy7 are almost\nidentical, indicating that the modification of Cy7 does not change\nthe structure and morphology of NI-BQDs. The surface element composition\nof NI-BQDs was characterized by X-ray photoelectron spectroscopy (XPS).\nThe XPS spectrum of NI-BQDs shown in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>C reveals that it is mainly composed of C,\nN, and O, accounting for 57.31, 4.33, and 31.67%, respectively. A\nstrong O 1s characteristic peak appeared at 532.7 eV, a second strong\nC 1s characteristic peak is present at 285.1 eV, and a weak N 1s characteristic\npeak is present at 399.1 eV. The high-resolution XPS spectra of C\n1s, N 1s, and O 1s are displayed in <xref rid="ao0c01320_0001" ref-type="fig">1</xref>C reveals that it is mainly composed of C,\nN, and O, accounting for 57.31, 4.33, and 31.67%, respectively. A\nstrong O 1s characteristic peak appeared at 532.7 eV, a second strong\nC 1s characteristic peak is present at 285.1 eV, and a weak N 1s characteristic\npeak is present at 399.1 eV. The high-resolution XPS spectra of C\n1s, N 1s, and O 1s are displayed in Figure S1 (Supporting Information) that indicate the presence of C–H/C–C,\nC–N, C–O, and C=O bonds in the C 1s spectrum,\nthe existence of pyrrolic-N and pyridine-N bonds in the N 1s spectrum,\nand the presence of O=C, O–H, O–C, and O=C–O\nbonds in the C 1s spectrum. The composition of functional groups on\nthe surface of NI-BQDs was characterized by Fourier transform infrared\n(FTIR) spectroscopy. The FTIR spectra of Cy7, NI-BQDs, and NI-BQDs-Cy7\ndisplayed in <xref rid="ao0c01320_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>D show a peak at 3410 cm<xref rid="ao0c01320_0001" ref-type="fig">1</xref>D show a peak at 3410 cm–1 that corresponds to\nthe stretching vibration peak of −O–H/N–H, and\nits intensity in NI-BQD-Cy7 (trace c) is clearly stronger than those\nin NI-BQDs (trace b) and Cy7 (trace a), indicating that Cy7 is covalently\ncoupled with NI-BQDs through an amide bond, which is consistent with\nthe experimental principle. The peak located at 2848 cm–1 corresponds to the telescopic vibration of C–H, and that\nat 1634 cm–1 corresponds to C=C and C=N\nbending vibration. Additionally, the peak at 1360–1020 cm–1 corresponds to C–O and C–N. The absorption\nof NI-BQD-Cy7 was significantly higher than that of NI-BQDs, and the\nwide peak around 1190 cm–1 is the characteristic\nabsorption peak of the sulfonic group, which indicates that Cy7 was\nsuccessfully conjugated to NI-BQDs. The surface charge of NI-BQDs\nand NI-BQD-Cy7 was further investigated, and the results showed that\nsurfaces of both were positively charged in a pH 7.4 neutral environment\n(Figure S2, Supporting Information).'], 'ao0c01320_0002': ['To determine\nthe optical properties of NI-BQDs and NI-BQD-Cy7, we first investigated\ntheir ultraviolet–visible (UV–vis) absorption spectra.\nThe UV–vis spectra shown in Figure S3 in the Supporting Information reveal an obvious absorption peak\nat 279 nm, which is the formation of π–π* electron\ntransition of C=O in NI-BQDs. The NI-BQDs-Cy7 has an obvious\nabsorption peak at 750 nm, which is due to the characteristic absorption\nof the Cy7 molecule, further indicating that Cy7 had been successfully\ncoupled on the surface of NI-BQDs. We studied the feasibility of FRET\nbetween NI-BQDs and Cy7, and the results revealed that the maximum\nexcitation and emission wavelengths of NI-BQDs are at 405 and 678\nnm, respectively. The maximum absorption wavelength of Cy7 is at 710\nnm, and the absorption spectrum of Cy7 overlaps with the fluorescence\nemission spectrum of NI-BQDs, which indicates that they can be a pair\nof donors and receptors of FRET (<xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>A). After the covalent coupling of NI-BQDs\nwith Cy7, the fluorescence emission spectrum of NI-BQD-Cy7 exhibited\nemission peaks at 670 and 780 nm, and the fluorescence intensity of\nthe peak at 670 nm gradually decreased with the increase of the Cy7\nconcentration, while the fluorescence intensity of the peak at 780\nnm was gradually increased (<xref rid="ao0c01320_0002" ref-type="fig">2</xref>A). After the covalent coupling of NI-BQDs\nwith Cy7, the fluorescence emission spectrum of NI-BQD-Cy7 exhibited\nemission peaks at 670 and 780 nm, and the fluorescence intensity of\nthe peak at 670 nm gradually decreased with the increase of the Cy7\nconcentration, while the fluorescence intensity of the peak at 780\nnm was gradually increased (<xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>B). These results showed that the coupling of NI-BQDs\nwith Cy7 was successful and a dual-emission NIR ratiometric fluorescent\nnanoprobe was formed by FRET interactions. The FRET efficiency can\nbe measured and calculated by a reported method.<xref rid="ao0c01320_0002" ref-type="fig">2</xref>B). These results showed that the coupling of NI-BQDs\nwith Cy7 was successful and a dual-emission NIR ratiometric fluorescent\nnanoprobe was formed by FRET interactions. The FRET efficiency can\nbe measured and calculated by a reported method.27', 'The response of the nanoprobe to ONOO– was investigated\nby mixing a certain amount of the NI-BQD-Cy7 solution with various\nsolutions containing different concentrations of ONOO–. The results shown in <xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>C reveal that as the ONOO<xref rid="ao0c01320_0002" ref-type="fig">2</xref>C reveal that as the ONOO– concentration\nincreases, the fluorescence intensity of the nanoprobe at 780 nm is\ngradually reduced, while the fluorescence intensity at 670 nm is gradually\nincreased. Additionally, the results also show that there is a good\nlinear relationship between the logarithm value of the fluorescence\nintensity ratio (I670/I780) and the ONOO– concentration in\nthe range of 0.02–15 μM (<xref rid="ao0c01320_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>D) with a detection limit of 8.5 nM (S/N\n= 3), as indicated by the linear regression equation: log (<xref rid="ao0c01320_0002" ref-type="fig">2</xref>D) with a detection limit of 8.5 nM (S/N\n= 3), as indicated by the linear regression equation: log (I670/I780) = 0.05788CONOO– – 0.4474, R2 = 0.9959, which is comparable to the previously reported\nONOO– ratiometric fluorescent probe.14,15 In addition, the response time of the NI-BQD-Cy7 nanoprobe toward\nONOO– was also investigated via a kinetics method.\nAfter the addition of ONOO– to the NI-BQD-Cy7 nanoprobe\nsolution for 4 min, the fluorescence intensity of NI-BQD increased\nto its maximum and that of Cy7 decreased to its minimum (Figure S6, Supporting Information).'], 'ao0c01320_0003': ['Since endogenous ONOO– is produced in the mitochondria\nof living cells, it is necessary to confirm that the nanoprobe can\neffectively enter the cell mitochondria through a cell colocalization\nassay. Accordingly, RAW264.7 cells were incubated for 8 h with the\nNI-BQD-Cy7 nanoprobe, as well as lysosome, nucleus, and mitochondrial\nlocalization reagent, separately. Then, laser confocal imaging was\nperformed. The results, which are shown in <xref rid="ao0c01320_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>, indicate that the NI-BQD-Cy7 nanoprobe\nhas excellent mitochondria-targeting ability (colocalization coefficient\nis 0.89), while the colocalization coefficients for lysosome and nucleus\nare only 0.56 and 0.58, respectively.<xref rid="ao0c01320_0003" ref-type="fig">3</xref>, indicate that the NI-BQD-Cy7 nanoprobe\nhas excellent mitochondria-targeting ability (colocalization coefficient\nis 0.89), while the colocalization coefficients for lysosome and nucleus\nare only 0.56 and 0.58, respectively.'], 'ao0c01320_0004': ['To investigate the kinetic\nrange for ONOO– detection by the NI-BQD-Cy7 nanoprobe\nin living cells, RAW264.7 cells at the appropriate density were seeded\nin five 35 mm confocal imaging dishes and incubated for 20 h. Subsequently,\n50 mL of the PBS solution containing 0, 0.1, 0.3, 0.6, and 1 mM SIN-1\nwas added, respectively, into the above five cell culture dishes,\nand after incubation for 2 h, each group of cells was subjected to\ndual-channel fluorescence imaging. The results shown in <xref rid="ao0c01320_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>A reveal that with the increase\nof SIN-1 concentration, the fluorescence intensity at the 780 nm channel\ngradually decreases and the fluorescence intensity at the 670 nm channel\ngradually increases (<xref rid="ao0c01320_0004" ref-type="fig">4</xref>A reveal that with the increase\nof SIN-1 concentration, the fluorescence intensity at the 780 nm channel\ngradually decreases and the fluorescence intensity at the 670 nm channel\ngradually increases (<xref rid="ao0c01320_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>B). The results also show that there is a good linear relationship\nbetween the logarithmic value of the fluorescence intensity ratio\n(<xref rid="ao0c01320_0004" ref-type="fig">4</xref>B). The results also show that there is a good linear relationship\nbetween the logarithmic value of the fluorescence intensity ratio\n(F670/F780) of two channels and the concentration of SIN-1 in the range of\n0–1000 μM (<xref rid="ao0c01320_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>C), as indicated by the linear regression equation: log(<xref rid="ao0c01320_0004" ref-type="fig">4</xref>C), as indicated by the linear regression equation: log(F670/F780) = 4.453\n× 10–4CSIN-1 + 0.1765, R2 = 0.9964.'], 'ao0c01320_0005': ['The generation of endogenous ONOO– in single living\ncells was monitored by in situ ratiometric\nfluorescence imaging using the developed nanoprobe. RAW264.7 cells\nwere inoculated in 35 mm confocal imaging dishes. When the cells reached\na suitable density, the NI-BQD-Cy7 nanoprobe was added and the cells\nwere incubated for 6 h. Subsequently, the cells were incubated with\na mixed solution containing 50 ng/mL INF-γ and 1 μg/mL\nLPS for 4 h. After adding 25 nM PMA, a single cell was imaged on a\nconfocal microscopic imaging system at 0, 5, 10, 20, 30, and 40 min.\nThe results displayed in <xref rid="ao0c01320_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref> show that with the increase of incubation time, the\nfluorescence intensity of the single living cell at the 780 nm channel\ngradually decreases, while the fluorescence intensity at the 670 nm\nchannel gradually increases (<xref rid="ao0c01320_0005" ref-type="fig">5</xref> show that with the increase of incubation time, the\nfluorescence intensity of the single living cell at the 780 nm channel\ngradually decreases, while the fluorescence intensity at the 670 nm\nchannel gradually increases (<xref rid="ao0c01320_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>A,<xref rid="ao0c01320_0005" ref-type="fig">5</xref>A,<xref rid="ao0c01320_0005" ref-type="fig">5</xref>B). The results also show\nthat there was a good linear relationship between the logarithmic\nvalue of the fluorescence intensity ratio (B). The results also show\nthat there was a good linear relationship between the logarithmic\nvalue of the fluorescence intensity ratio (F670/F780) of the two channels and\nthe incubation time within the 0–40 min period (<xref rid="ao0c01320_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>C), as indicated by the linear\nregression equation: log(<xref rid="ao0c01320_0005" ref-type="fig">5</xref>C), as indicated by the linear\nregression equation: log(F670/F780) = 0.01383T (min) + 0.08668, R2 = 0.9995. These results showed that the NI-BQD-Cy7\nnanoprobe can real-time trace the generation of endogenous ONOO– in a single cell. Moreover, it was also found that\nthe generation of endogenous ONOO– increased linearly\nwithin 0–40 min.'], 'ao0c01320_0006': ['To\nachieve the ratiometric fluorescence imaging of ONOO– in vivo, the stability of the fluorescent nanoprobe in vivo was\ninvestigated. Two male nude mice (10 week old, weight of about 20\ng) were evaluated. One was injected with saline (150 μL), and\nthe other with the NI-BQD-Cy7 nanoprobe (3 mg/mL,150 μL) in\nthe abdominal cavity at ca. 2–4 mm depth. After anesthesia\nwith isoflurane and halothane, the mice were imaged using a small\nanimal imaging system. After injecting NI-BQD-Cy7, the fluorescence\nat the 700 ± 30 and 790 ± 30 nm channels was collected at\n0.5, 1.5, and 4 h under excitation at 650 nm.28 The results shown in <xref rid="ao0c01320_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref> reveal that the fluorescence intensity at two channels remains\nstable for 4 h, indicating that the developed nanoprobe is suitable\nfor the ratiometric fluorescence imaging of ONOO<xref rid="ao0c01320_0006" ref-type="fig">6</xref> reveal that the fluorescence intensity at two channels remains\nstable for 4 h, indicating that the developed nanoprobe is suitable\nfor the ratiometric fluorescence imaging of ONOO– in vivo.', '(A) Real-time tracing the generation of endogenous ONOO– in vivo by ratiometric fluorescence images. The imaging parameters\nare the same as those in <xref rid="ao0c01320_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>. (B) Change of fluorescence intensity of endogenous\nONOO<xref rid="ao0c01320_0006" ref-type="fig">6</xref>. (B) Change of fluorescence intensity of endogenous\nONOO– in vivo with time in two channels. (C) Relationship\nbetween the fluorescence intensity ratio (F700/F790) of two channels and incubation\ntime within 0–70 min.'], 'ao0c01320_0007': ['In situ ratiometric\nfluorescence imaging monitoring\nthe generation of endogenous ONOO– in vivo was investigated\nusing NI-BQD-Cy7 as the nanoprobe and acetaminophen (APAP) as the\nONOO–-inducing agent.26 The NI-BQD-Cy7 nanoprobe was injected into the diaphragm of male\nnude mice (3 mg/mL, 150 μL), and after 30 min, the APAP (500\nmg/kg) was also injected into the diaphragm of the mice. The mice\nwere anesthetized with isoflurane and halothane, and then the fluorescence\nat the 700 ± 30 and 790 ± 30 nm channels was collected at\n10, 30, 50, and 70 min after injection of APAP. The results presented\nin <xref rid="ao0c01320_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>A show that\nwith prolongation of time after treatment with APAP, the fluorescence\nintensity at the 700 ± 30 nm channel gradually increases, while\nthe fluorescence intensity at the 790 ± 30 nm channel gradually\ndecreases (<xref rid="ao0c01320_0007" ref-type="fig">7</xref>A show that\nwith prolongation of time after treatment with APAP, the fluorescence\nintensity at the 700 ± 30 nm channel gradually increases, while\nthe fluorescence intensity at the 790 ± 30 nm channel gradually\ndecreases (<xref rid="ao0c01320_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>B). The fluorescence intensity ratio of two channels (<xref rid="ao0c01320_0007" ref-type="fig">7</xref>B). The fluorescence intensity ratio of two channels (F700/F790) gradually increases\nwith the prolongation of time but slowed down after 50 min (<xref rid="ao0c01320_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>C). These results\nshow that the NI-BQD-Cy7 nanoprobe can be used for in situ ratiometric\nimaging to monitor the production of ONOO<xref rid="ao0c01320_0007" ref-type="fig">7</xref>C). These results\nshow that the NI-BQD-Cy7 nanoprobe can be used for in situ ratiometric\nimaging to monitor the production of ONOO– in vivo\nas well as in the diagnosis of ONOO– related diseases\nin vivo.']}
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Near-Infrared Dual-Emission Ratiometric Fluorescence
Imaging Nanoprobe for Real-Time Tracing the Generation of Endogenous
Peroxynitrite in Single Living Cells and In Vivo
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ACS Omega
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1590649200
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Peroxynitrite (ONOO) is a highly reactive nitrogen species with potent oxidant and nitrating properties. Its excessive generation can cause DNA and protein damage, thereby contributing to cell injury, and it is closely related to the development of many diseases. Thus, there is an urgent need for a reliable method to determine changes in the steady-state levels of ONOO in vivo. Ratiometric imaging, due to its built-in self-calibration system, can reduce artifacts and enable reliable in vivo imaging. In this study, we designed and prepared near-infrared (NIR) biomass quantum dots (NI-BQDs) and covalently coupled them with the NIR dye Cyanine7 (Cy7) to construct an NIR dual-emission nanoprobe (NI-BQD-Cy7) for real-time tracing the generation of endogenous ONOO in single living cells and in vivo by ratiometric fluorescence imaging. NI-BQD-Cy7 exhibited high detection sensitivity and selectivity for ONOO in the mitochondria. Additionally, it can produce dual NIR fluorescence emission, thus allowing in situ ratiometric fluorescence imaging to real-time trace the generation and concentration changes of ONOO in vivo. The application of the proposed NIR dual-emission nanoprobe can provide accurate information for the study of the biological function of ONOO in single living cells and in vivo, and it is very useful to explain the mechanism of cell damage caused by ONOO.
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other
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PMC7288700
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28
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[
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"{'Citation': 'Wang S.; Fan Y.; Li D.; Sun C.; Lei Z.; Lu L.; Wang T.; Zhang F. Anti-quenching NIR-II molecular fluorophores for in vivo high-contrast imaging and pH sensing. Nat Commun. 2019, 10, 105810.1038/s41467-019-09043-x.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1038/s41467-019-09043-x'}, {'@IdType': 'pmc', '#text': 'PMC6401027'}, {'@IdType': 'pubmed', '#text': '30837470'}]}}"
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ACS Omega. 2020 May 28; 5(22):13278-13286
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NO-CC CODE
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Retrograde CTB labeling of motor pools connects transcriptional subpopulations with motor poolsa, Proportion of CTB-labeled cells from GLUT and IF that are labeled with Cdh8 and Sema3e. The GLUT has a significantly larger proportion of Cdh8+ and Sema3e+ cells than the IF. n=5 biologically independent animals. b, Lower power view of in situ hybridization against Prkcd/Sv2a, and Kcnq5/Chodl (insets=Fig. 4h) in longitudinal sections demonstrates the specificity of CTB injections into the Soleus (SOL) and Tibialis anterior (TA). n=5 (TA) and 4 (SOL) biologically independent animals. One-way ANOVA with post-hoc Sidak multiple comparison test between same-gene conditions. Adjusted p-values=0.0212 (Cdh8) and 0.0499 (Sema3e). c, Expression of FF and SF gene modules overlaid on UMAP of all α motor neurons. d, Average log-normalized canonical marker expression of Chodl (fast-firing) and Sv2a (slow-firing). Scale bars = 250 µm. e, Proportion of Prkcd+ cells positive for Sv2a (left) and proportion of Chodl+ cells that are positive for Kcnq5 (right). n=3 biologically independent animals. f, Representative in situ hybridization showing Kcnq5 is expressed in a subset of Chodl+ cells. n=4 biologically independent animals. Scale bar=50 µm inset and 200 µm overview. *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.
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nihms-1657468-f0010
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2
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5e766291ff9a5c2f159ebf3fe2be5b707dd8756d93f19b94dcd6fef9084272ca
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nihms-1657468-f0010.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
1445,
1800
] |
[{'image_id': 'nihms-1657468-f0009', 'image_file_name': 'nihms-1657468-f0009.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0009.jpg', 'caption': 'Discovery of a fundamental transcriptional bifurcation among γ motor neuronsa, UMAP with 3 subclustered γ motor neurons populations. b, Novel marker gene expression across γ motor neuron subpopulations. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level. c, Average log-normalized expression of genes enriched in γ* motor neurons over γ overlaid on UMAP. α, γ, and γ* populations are labeled. d, Average log-normalized expression of genes enriched in γ motor neurons over γ* overlaid on UMAP. α, γ, and γ* populations are labeled. e-h, Average expression of novel γ markers Stxbp6 (e) and Plch1 (f), as well as novel γ* markers Pard3b (g) and Creb5 (h) by cluster. i, Representative in situ hybridization against Htr1d/Creb5/Stxbp6 in transverse spinal cord shows mutual exclusion of novel markers Creb5 and Stxbp6 in Htr1d+ cells. n=4 biologically independent animals. Scale bar=20 µm (inset) and 200 µm (overview). j, Representative in situ hybridization against Htr1d/Pard3b/Stxbp6 in transverse spinal cord shows mutual exclusion of novel markers Pard3b and Stxbp6 in Htr1d+ cells. Arrowheads label canonical γ motor neurons and *labels γ*. n=5 biologically independent animals. Scale bar=20 µm (inset) and 100 µm (overview). Differentially expressed genes determined by Wilcoxon rank sum test implementation in Seurat and adjusted for multiple comparisons (Bonferroni method) (p_adj<0.01, log2-fold change >0.5).', 'hash': '9940edbb91face958f92f3c2cb74f2df57c5886ac44b5058f894c8598e41cb57'}, {'image_id': 'nihms-1657468-f0007', 'image_file_name': 'nihms-1657468-f0007.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0007.jpg', 'caption': 'Visceral motor neuron populations express selective repertoires of neuropeptides and are spatially distincta, Estimated relative density of visceral motor neurons along the rostral-caudal axis of the spinal cord based on Allen Spinal Cord Atlas10. Density functions are combined density estimates of marker genes for each cluster (see Methods). Clusters were grouped according to shape of density function, with clusters 3,7, and 10 clearly enriched in the sacral spinal cord. b, Validation of visceral spatial modeling from (a) via high-resolution in situ hybridization for Chat and visceral cluster markers (Piezo2, Cdh8, Creb5, Cpne4, Fbn2). Plots show number of motor neurons counted in the autonomic column, added across three counted slides in each region. Individual data points for total visceral motor neurons shown with filled circles, while marker gene-positive cell numbers shown with filled triangles. n=2 (Piezo2, Cdh8, Creb5, Cpne4) or 5 (Fbn2) biologically independent replicates. Error bars are SEM. c, Average log-normalized expression of Rxfp1 and Nts across all visceral motor neuron clusters (labeled), overlaid on UMAP. d, Representative in situ hybridization against Chat/Fbn2/Rxfp1 in transverse sacral spinal cord shows coexpression in the autonomic column but not in the ventral horn (VH). n=3 biologically independent animals. Scale bar=100 µm. e, Average log-normalized expression of Adra2a across all visceral clusters (labeled) shows that sporadic expression exists across populations, overlaid on UMAP. f, Representative in situ hybridization against Chat/Piezo2/Cdh8 in cholinergic cells around the central canal (CC). Scale bar=50 µm. n=2 biologically independent animals. g, Average log-normalized expression of Gldn across all cells in spinal cord shows clear enrichment in partition cell cluster (arrowhead), overlaid on UMAP. h, Average log-normalized expression of Nrxn3 across cholinergic interneurons shows that Nrxn3 expression is limited to half of partition cells (arrowhead), overlaid on UMAP.', 'hash': 'e2fb19106bd2313ce48be8d38ef748b185df068f3b8deb798c6f440d04bb6aee'}, {'image_id': 'nihms-1657468-f0011', 'image_file_name': 'nihms-1657468-f0011.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0011.jpg', 'caption': 'Retrograde CTB labeling of motor pools enables the identification of transcriptionally distinct classes of fast and slow-firing motor neurons in the adult spinal corda, Representative in situ hybridization against Chat/Mmp9/Kcnq5 in transverse spinal cord shows that Kcnq5 is expressed in a subset of Mmp9+ fast-firing motor neurons. n=4 biologically independent animals. Scale bar=20 µm inset and 200 µm overview. b, Representative in situ hybridization against Chat/Sv2a/Prkcd in transverse spinal cord shows that Prkcd is expressed in almost every Chat+/Sv2a+ slow-firing motor neuron. n=2 biologically independent animals. Scale bar=20 µm inset and 200 µm overview. c, Representative in situ hybridization against Chat/Mmp9/Prkcd in transverse spinal cord shows that Prkcd is excluded from almost every Chat+/Mmp9+ fast-firing motor neuron. n=4 biologically independent animals. Scale bar=30 µm inset and 200 µm overview. d-e, Proportion of cells expressing fast and slow-firing markers in the CTB-labeled TA (d) and SOL (e) motor pools. There is a significantly higher proportion of cells expressing both known and novel fast-firing markers in TA than SOL (d), and a higher proportion of cells expressing both known and novel slow-firing markers in SOL than TA. Adjusted p-value=0.0456 (Chodl+>Kcnq5+). f, Total number of CTB-positive cells labeled across biologically independent animals. One-way ANOVA with post-hoc Tukey multiple comparison test among all conditions. n=4–5 biologically independent animals (d-f). *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.', 'hash': '32dd8b7a5ac001d71a45ee4b4e762d1f1027848c095e4e8e03dc1d3e29f16371'}, {'image_id': 'nihms-1657468-f0006', 'image_file_name': 'nihms-1657468-f0006.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0006.jpg', 'caption': 'Novel markers of skeletal motor neurons confirmed by Allen Spinal Cord Atlas in situ hybridizationsa-d, Transverse schematic illustrating expected positions of skeletal motor neurons in ventral horn (VH, green) in lumbar spinal cord. Second row—corresponding in situ hybridization against Tns1 (a), Bcl6 (b), Syn1 (c), and Actb (d). Third row—expression mask shows relative enrichment of Tns1 and Bcl6 in small and large cell bodies in the VH.', 'hash': 'c7d19138117748b8c23b071c08bbc2d27c764bebb0241edec1f24ce8607e13f4'}, {'image_id': 'nihms-1657468-f0001', 'image_file_name': 'nihms-1657468-f0001.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0001.jpg', 'caption': 'Motor neuron enrichment and single-nucleus transcriptional analysis of the adult mouse spinal cord uncovers novel skeletal and visceral motor neuron markers.a, Workflow for cholinergic nucleus enrichment and single-nucleus RNA sequencing (snRNAseq)—GFP+ and TdTomato+ cells were mixed at a ratio between 1:3 and 2:3. Plot shows the distribution of canonical cell types with their proportional representation. FACS was used to select appropriate proportion of singlet, DAPI+/GFP+/tdTomato− nuclei. b, UMAP of clustered snRNAseq data from 43,890 transcriptomes. c, Average expression levels per cluster for marker genes of each canonical cell population. Cell type labels based on expression patterns of marker genes. d, Schematic depicting expected cholinergic cell types in the spinal cord. Visceral motor neurons (blue) innervate sympathetic ganglia, skeletal motor neurons (green) directly innervate muscle fibers, and cholinergic interneurons (red) innervate motor neurons and other cells. e, UMAP with graph-based clustering of all cholinergic neurons reveals 21 clusters. f, Ranked expression of known marker genes Pax2 (interneurons) and Nos1 (visceral motor neurons), as well as novel marker gene Anxa4 (skeletal motor neurons) by cluster. Cell labels were assigned hierarchically by expression levels of Pax2, Nos1, and Bcl6 and are reported below each plot. Cholinergic interneuron clusters that also express Nos1 are denoted with a (*). g, Enriched differentially expressed genes for cholinergic interneurons, skeletal motor neurons, and visceral motor neurons. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level. All expression values were log-normalized in Seurat50.', 'hash': '7aa65c7534bf7626e3571ade100b2243618535ea676e931588451b27cedbcfd0'}, {'image_id': 'nihms-1657468-f0008', 'image_file_name': 'nihms-1657468-f0008.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0008.jpg', 'caption': 'Vipr2 and Npas1 are novel, robust, and specific markers of α and γ motor neurons in the spinal corda, Average expression of Vipr2 and Npas1 across all spinal cord cell populations (labeled), overlaid on UMAP. Arrow points to α and γ motor neuron clusters, respectively. b, Representative in situ hybridization against Chat/Rbfox3/Vipr2 in transverse spinal cord shows coexpression in the ventral horn (VH). n=4 biologically independent animals. Scale bar=100 µm. c, Representative in situ hybridization against Chat/Htr1d/Vipr2 in transverse spinal cord shows mutual exclusion. Scale bar=50 µm (inset) and 200 µm (overview). n=4 biologically independent animals. d, Representative in situ hybridization against Chat/Npas1/Rbfox3 in transverse spinal cord shows mutual exclusion of Rbfox3 and Npas1 in Chat+ cells. n=5 biologically independent animals. Scale bar=20 µm (inset) and 200 µm (overview). e, Representative in situ hybridization against Chat/Npas1/Vipr2 in transverse spinal cord shows mutual exclusion of novel markers Vipr2 and Npas1 in Chat+ cells. n=4 biologically independent animals. Scale bar=20 µm (inset) and 200 µm (overview).', 'hash': '227268a33f53d708de3c86e49a856d66e29183fd3e958d61492c325cacc905f0'}, {'image_id': 'nihms-1657468-f0010', 'image_file_name': 'nihms-1657468-f0010.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0010.jpg', 'caption': 'Retrograde CTB labeling of motor pools connects transcriptional subpopulations with motor poolsa, Proportion of CTB-labeled cells from GLUT and IF that are labeled with Cdh8 and Sema3e. The GLUT has a significantly larger proportion of Cdh8+ and Sema3e+ cells than the IF. n=5 biologically independent animals. b, Lower power view of in situ hybridization against Prkcd/Sv2a, and Kcnq5/Chodl (insets=Fig. 4h) in longitudinal sections demonstrates the specificity of CTB injections into the Soleus (SOL) and Tibialis anterior (TA). n=5 (TA) and 4 (SOL) biologically independent animals. One-way ANOVA with post-hoc Sidak multiple comparison test between same-gene conditions. Adjusted p-values=0.0212 (Cdh8) and 0.0499 (Sema3e). c, Expression of FF and SF gene modules overlaid on UMAP of all α motor neurons. d, Average log-normalized canonical marker expression of Chodl (fast-firing) and Sv2a (slow-firing). Scale bars = 250 µm. e, Proportion of Prkcd+ cells positive for Sv2a (left) and proportion of Chodl+ cells that are positive for Kcnq5 (right). n=3 biologically independent animals. f, Representative in situ hybridization showing Kcnq5 is expressed in a subset of Chodl+ cells. n=4 biologically independent animals. Scale bar=50 µm inset and 200 µm overview. *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.', 'hash': '5e766291ff9a5c2f159ebf3fe2be5b707dd8756d93f19b94dcd6fef9084272ca'}, {'image_id': 'nihms-1657468-f0002', 'image_file_name': 'nihms-1657468-f0002.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0002.jpg', 'caption': 'Single-nucleus transcriptomics reveals immense diversity within the autonomic nervous system and partition cells.a, Schematic illustrating the position of sympathetic visceral motor neurons (blue) in the lateral autonomic column of the spinal cord. b, Diagram adapted from Espinosa-Medina 2016 showing innervation targets of the sympathetic nervous system12. c, UMAP with 16 visceral motor neuron subclusters. Inset shows all cells from Fig. 1e that were subclustered. d, Frequency of visceral motor neuron subpopulations (Rxfp1+ and Nts+) along the rostral-caudal axis of the spinal cord. Individual data points for total visceral motor neurons shown with filled circles, while marker gene-positive cell numbers shown with filled triangles. n=3 biologically independent animals. e, Novel marker genes for each cluster of visceral motor neurons. f, Representative in situ hybridization of Chat, Nts, and Fbn2 demonstrating Nts expression in visceral motor neurons. 200 µm scale bar in overview and 20 µm in inset. n=3 biologically independent animals. g, Schematic showing cholinergic interneuron innervation of skeletal motor neurons as demonstrated previously25. h, UMAP with graph-based clustering labels for cholinergic interneurons. Inset shows all cells from Fig. 1e that were subclustered. i, Novel marker genes for cholinergic interneuron clusters identified. j, Expression of Pitx2 in cholinergic interneuron populations, overlaid on UMAP projection from (h). Cluster 0 and 1 are Pitx2-positive. All expression values were log-normalized in Seurat50. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level in (e,i).', 'hash': 'eb234ce166cd7c78de109fc323a3a3b8d91ff13c1af57499f412ba3273a1bff4'}, {'image_id': 'nihms-1657468-f0005', 'image_file_name': 'nihms-1657468-f0005.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0005.jpg', 'caption': 'Single-nucleus transcriptional analysis of the adult mouse spinal cord reveals canonical cell types.a, Canonical cell class labels, visualized on UMAP. b, Average log-normalized marker gene expression across canonical cell classes. c–d, Representative in situ hybridization against Chat/Nos1 in transverse sacral (c) and thoracic (d) spinal cord hemi-sections. n=3 biologically independent animals. e-f, Average log-normalized expression of Zeb2 (e) and Fbn2 (f) across all cholinergic clusters (labeled), overlaid on UMAP. Dotted line surrounds clusters corresponding to visceral motor neurons. g, Representative in situ hybridization against Chat/Fbn2 in transverse thoracic spinal cord hemi-section. n=3 biologically independent animals. Scale bars=200 µm (c–d) and 100 µm (g). LAC=lateral autonomic column (c,d), VH=ventral horn (c,d).', 'hash': '07f71fd984b48abc0b5f1947ac5dae33aa222814777646a4676b2e6c019d7170'}, {'image_id': 'nihms-1657468-f0012', 'image_file_name': 'nihms-1657468-f0012.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0012.jpg', 'caption': 'Cross-replicate variability in single-nucleus transcriptomic experimentsa–e, Each spinal cord sequencing replicate, plotted side-by-side and visualized by UMAP. Note that we observe minimal batch-to-batch variability along sex, age, or replicate number axes in terms of cluster identification and overall shape of the dimensionality reduced data. This does not preclude sex or age-related transcriptional changes but demonstrates that they do not fundamentally alter the transcriptomic classes that we focus on in the manuscript.', 'hash': '848dda9f9bee6f6adef6a72deece4f2d510c296e4014d2d0e0df1c32b0b4accb'}, {'image_id': 'nihms-1657468-f0004', 'image_file_name': 'nihms-1657468-f0004.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0004.jpg', 'caption': 'Alpha (α) motor neuron pool, position, and electrophysiological subtype reflect transcriptional differences.a, Transverse schematic shows α motor neurons (blue) innervating extrafusal muscle fibers. b, UMAP with 12 subclustered α motor neuron populations. Inset shows all α motor neurons from Fig. 3b that were subclustered. c, Novel marker gene expression across α motor neuron subpopulations. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level. d, Representative in situ hybridization against novel (Hcrtr2) and known (Cpne4) intrinsic-foot (IF) motor pool markers in longitudinal sections, overlaid with CTb labeled cells that innervate the gluteus (GLUT) and IF. n=5 (IF) and 4 (GLUT) biologically independent animals. e, Proportion of CTB-labeled cells from GLUT and IF that are labeled with Hcrtr2 and Cpne4 shows novel and known markers selectively label the IF motor pool. n=5 (IF) and 4 (GLUT) biologically independent animals. f, Schematic illustrating slow-firing (SF, blue), fast fatigue-resistant (FR, purple) and fast fatigable (FF, red) α motor neuron populations innervating type I, IIa, and IIb fibers respectively. g, Heatmap showing all α motor neurons hierarchically clustered and colored by expression of differentially expressed genes between fast (Chodl+) and slow (Sv2a+) α motor neurons shows three cell populations corresponding to SF, FR, and FF motor neurons. Red and blue bars show gene modules enriched in fast- and slow-firing α motor neurons, respectively. h, Representative in situ hybridization in longitudinal sections against novel (Kcnq5, Prkcd) and known (Chodl, Sv2a) fast and slow-firing motor neuron markers, respectively. Images show CTb labeled cells that innervate a muscle with predominantly fast-twitch fibers (TA) and slow-twitch fibers (SOL). n=5 (TA) and 4 (SOL) biologically independent animals. i, Proportion of CTB-labeled cells from TA and SOL that are labeled with Chodl and Kcnq5. The TA has a significantly larger proportion of Chodl+ and Kcnq5+ cells than the SOL, though Kcnq5+ cells are significantly less frequently found in both populations. n=5 (TA) and 4 (SOL) biologically independent animals. Adjusted p-values=0.0197 (Chodl), 0.0262 (Kcnq5). j, Proportion of CTB-labeled cells from TA and SOL that are labeled with Sv2a and Prkcd. The SOL pool has a significantly larger proportion of Prkcd+ and Sv2a+ cells than the TA. n=5 (TA) and 4 (SOL) biologically independent animals. All differential expression calculated using Wilcoxon rank sum test and adjusted for multiple comparisons (Bonferroni method) (p_adj < 0.01, log2fc>0.5). All expression values were log-normalized in Seurat50. Scale bars=50µm (h), 75 µm (all others). One-way ANOVA with post-hoc Sidak multiple comparison test between same-gene conditions. *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.', 'hash': 'cc5533a2e1e7ddd4336d30ee321b35c620f91d750d9e4a8f000b74655a451eca'}, {'image_id': 'nihms-1657468-f0003', 'image_file_name': 'nihms-1657468-f0003.jpg', 'image_path': '../data/media_files/PMC8016743/nihms-1657468-f0003.jpg', 'caption': 'Transcriptional differences between alpha (α) and gamma (γ) motor neurons.a, Transverse schematic illustrating position of skeletal motor neurons (blue) in the ventral horn of the spinal cord. Gamma motor neurons are small and innervate intrafusal muscle fibers. α motor neurons are large and innervate extrafusal fibers. b, UMAP with 11 subclustered skeletal motor neurons populations. Inset shows all cells from Fig 1e that were subclustered. c, Average expression of known γ marker Htr1d and α markers Rbfox3 and Spp1 by cluster. d, Heatmap with average expression by cluster of differentially expressed genes in α and γ populations. Differentially expressed genes between γ and α populations. e, Representative in situ hybridization against Chat/Htr1d/Npas1 and Chat/Rbfox3/Vipr2 in transverse lumbar spinal cord sections. Arrowheads indicate γ motor neurons in both images. Scale bars are 200 µm (overview) and 50 µm (inset). n=4 biologically independent animals. f, Transverse schematic illustrating γ motor neurons (blue) innervating intrafusal muscle fibers. Inset shows all cells from Fig 3b that were subsequently subclustered. g, Heatmap showing fundamental subdivision between γ and γ* motor neurons, hierarchically clustered by expression of highly variable genes among all classes of γ motor neurons (see Methods). h, Differentially expressed membrane receptors between two main populations of γ motor neurons, as well as novel markers that delineate them. i, Representative in situ hybridization against Htr1d/Plch1/Creb5 in transverse lumbar spinal cord. Plch1 and Creb5 are expressed reciprocally in Htr1d+ cells and represent γ and γ* motor neurons. Arrow heads demarcate Creb5+ γ motor neurons and * indicates γ* motor neurons. n=5 biologically independent animals. All differential expression calculated using Wilcoxon rank sum test and adjusted for multiple comparisons (Bonferroni method) (p_adj < 0.01, log2fc>0.5). All expression values were log-normalized in Seurat50. Scale bars=50 µm.', 'hash': 'e77762f8806c029c6acf0e2e3f18f9af62513c32c9105169ae8c5ab1c3387bf7'}]
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{'nihms-1657468-f0001': ['Because spinal motor neurons are so scarce, we enriched for motor neuron nuclei using a transgenic fluorescent reporter mouse9. This technique enabled us to selectively isolate cholinergic nuclei (Chat+), a population that encompasses all motor neurons and several interneuron subtypes in the adult mouse spinal cord (see Methods). Given the important role of non-cell autonomous mechanisms in neurodegeneration, we also isolated non-motor neuron cells – including interneurons, astrocytes, microglia, and oligodendrocytes. In total, we transcriptionally profiled 43,890 nuclei from a collection of male, female, and mixed cohorts of wild-type adult mice, with 20–40% of nuclei coming from motor neurons and 60–80% from other cells in the spinal cord (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1a</xref>). We used graph-based methods to cluster nuclei and then annotate cell types across clusters based on averaged expression of common marker genes, including genes encoding neurotransmitter signaling machinery (). We used graph-based methods to cluster nuclei and then annotate cell types across clusters based on averaged expression of common marker genes, including genes encoding neurotransmitter signaling machinery (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1b</xref>,,<xref rid="nihms-1657468-f0001" ref-type="fig">c</xref>, , Methods). This approach enabled us to simultaneously characterize motor neurons, while also comparing their transcriptomes with other cells within the spinal cord, to find marker genes that are exclusively expressed in cell populations of interest. We assigned all profiled nuclei into seven broad categories: excitatory interneurons, inhibitory interneurons, cholinergic neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1c</xref>, , <xref rid="nihms-1657468-f0005" ref-type="fig">Extended Data Fig. 1a</xref>,,<xref rid="nihms-1657468-f0005" ref-type="fig">b</xref>, , Supplementary Table 1). Based on these categories, we estimate that ~30% (13,589 cells) of the profiled single-nucleus transcriptomes correspond to cholinergic neurons. This is a considerable improvement in representation over prior efforts7 and provides unparalleled access to the transcriptional heterogeneity of spinal motor neurons. We provide an interactive web portal to access and search all of the spinal cord transcriptome data: http://spinalcordatlas.org.', 'We next asked whether the observed transcriptional diversity of spinal motor neurons corresponds to functionally defined cell types, as in development (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1d</xref>). We computationally isolated and used graph-based clustering (see ). We computationally isolated and used graph-based clustering (see Methods) to segregate all cholinergic neurons into 20 clusters (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1e</xref>). We annotated these subpopulations as skeletal motor neurons, cholinergic interneurons, and visceral motor neurons (). We annotated these subpopulations as skeletal motor neurons, cholinergic interneurons, and visceral motor neurons (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1f</xref>) based on expression of known marker genes as well as expression patterns of uncharacterized genes in the publicly available Allen Mouse Spinal Cord Atlas) based on expression of known marker genes as well as expression patterns of uncharacterized genes in the publicly available Allen Mouse Spinal Cord Atlas10. Specifically, we identified cholinergic interneurons based on their expression of Pax211 and visceral motor neurons (which are part of the autonomic nervous system) by their expression of neuronal nitric oxide synthase (Nos1+)12. We performed double label in situ hybridization with Chat and Nos1 to confirm that Nos1 is expressed specifically in the lateral autonomic columns of the thoracic and sacral spinal cord—where visceral motor neurons are located (<xref rid="nihms-1657468-f0005" ref-type="fig">Extended Data Fig. 1c</xref>,,<xref rid="nihms-1657468-f0005" ref-type="fig">d</xref>).).', 'This work was supported by NIH grants R35NS097263 (A.D.G.) and R01NS083998 (J.A.K.), the Robert Packard Center for ALS Research at Johns Hopkins (A.D.G.), the Blavatnik Family Foundation (J.A.B.), and the Brain Rejuvenation Project of the Wu Tsai Neurosciences Institute. Sorting was performed on an instrument in the Shared FACS Facility obtained using NIH S10 Shared Instrument Grant S10RR025518–01. Portions of <xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1d</xref> and and <xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2b</xref> were generated using objects from BioRender.com, and figures were generated using Adobe Illustrator. were generated using objects from BioRender.com, and figures were generated using Adobe Illustrator.', 'a, Schematic illustrating the position of sympathetic visceral motor neurons (blue) in the lateral autonomic column of the spinal cord. b, Diagram adapted from Espinosa-Medina 2016 showing innervation targets of the sympathetic nervous system12. c, UMAP with 16 visceral motor neuron subclusters. Inset shows all cells from <xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1e</xref> that were subclustered. that were subclustered. d, Frequency of visceral motor neuron subpopulations (Rxfp1+ and Nts+) along the rostral-caudal axis of the spinal cord. Individual data points for total visceral motor neurons shown with filled circles, while marker gene-positive cell numbers shown with filled triangles. n=3 biologically independent animals. e, Novel marker genes for each cluster of visceral motor neurons. f, Representative in situ hybridization of Chat, Nts, and Fbn2 demonstrating Nts expression in visceral motor neurons. 200 µm scale bar in overview and 20 µm in inset. n=3 biologically independent animals. g, Schematic showing cholinergic interneuron innervation of skeletal motor neurons as demonstrated previously25. h, UMAP with graph-based clustering labels for cholinergic interneurons. Inset shows all cells from <xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1e</xref> that were subclustered. that were subclustered. i, Novel marker genes for cholinergic interneuron clusters identified. j, Expression of Pitx2 in cholinergic interneuron populations, overlaid on UMAP projection from (h). Cluster 0 and 1 are Pitx2-positive. All expression values were log-normalized in Seurat50. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level in (e,i).', 'a, Transverse schematic illustrating position of skeletal motor neurons (blue) in the ventral horn of the spinal cord. Gamma motor neurons are small and innervate intrafusal muscle fibers. α motor neurons are large and innervate extrafusal fibers. b, UMAP with 11 subclustered skeletal motor neurons populations. Inset shows all cells from <xref rid="nihms-1657468-f0001" ref-type="fig">Fig 1e</xref> that were subclustered. that were subclustered. c, Average expression of known γ marker Htr1d and α markers Rbfox3 and Spp1 by cluster. d, Heatmap with average expression by cluster of differentially expressed genes in α and γ populations. Differentially expressed genes between γ and α populations. e, Representative in situ hybridization against Chat/Htr1d/Npas1 and Chat/Rbfox3/Vipr2 in transverse lumbar spinal cord sections. Arrowheads indicate γ motor neurons in both images. Scale bars are 200 µm (overview) and 50 µm (inset). n=4 biologically independent animals. f, Transverse schematic illustrating γ motor neurons (blue) innervating intrafusal muscle fibers. Inset shows all cells from Fig 3b that were subsequently subclustered. g, Heatmap showing fundamental subdivision between γ and γ* motor neurons, hierarchically clustered by expression of highly variable genes among all classes of γ motor neurons (see Methods). h, Differentially expressed membrane receptors between two main populations of γ motor neurons, as well as novel markers that delineate them. i, Representative in situ hybridization against Htr1d/Plch1/Creb5 in transverse lumbar spinal cord. Plch1 and Creb5 are expressed reciprocally in Htr1d+ cells and represent γ and γ* motor neurons. Arrow heads demarcate Creb5+ γ motor neurons and * indicates γ* motor neurons. n=5 biologically independent animals. All differential expression calculated using Wilcoxon rank sum test and adjusted for multiple comparisons (Bonferroni method) (p_adj < 0.01, log2fc>0.5). All expression values were log-normalized in Seurat50. Scale bars=50 µm.'], 'nihms-1657468-f0006': ['We hypothesized that the remaining three clusters represented skeletal motor neurons—a broad cell population with no known genetic markers that distinguish them from visceral motor neurons. To test this hypothesis, we examined the Allen Mouse Spinal Cord Atlas10 for expression of two genes that are highly expressed in those clusters—Bcl6 and Tns1. Indeed, in contrast to ubiquitous (Actb) and pan-neuronal (Syn1) transcripts, which show broad expression in transverse sections, it is apparent that Bcl6 and Tns1 are strongly expressed in small and large diameter neurons in the ventral horn of the spinal cord (<xref rid="nihms-1657468-f0006" ref-type="fig">Extended Data Fig. 2a</xref>––<xref rid="nihms-1657468-f0006" ref-type="fig">d</xref>). Future studies will investigate the expression patterns of these putative markers in greater depth, but this pattern is consistent with skeletal motor neurons. Our classification of skeletal and visceral motor neurons differs from a previous study, which postulated that two other genes, ). Future studies will investigate the expression patterns of these putative markers in greater depth, but this pattern is consistent with skeletal motor neurons. Our classification of skeletal and visceral motor neurons differs from a previous study, which postulated that two other genes, Fbn2 and Zeb2 were novel markers specifically expressed in alpha (α) motor neurons13. Instead, our transcriptional data indicate that that Zeb2 and Fbn2 are absent from skeletal motor neuron clusters (<xref rid="nihms-1657468-f0005" ref-type="fig">Extended Data Fig. 1e</xref>,,<xref rid="nihms-1657468-f0005" ref-type="fig">f</xref>), the latter of which we confirm by multiplexed ), the latter of which we confirm by multiplexed Chat/Fbn2 in situ hybridization (<xref rid="nihms-1657468-f0005" ref-type="fig">Extended Data Fig. 1g</xref>). Thus, these new transcriptional profiles reveal dozens of possible new marker genes that reliably distinguish skeletal motor neurons from other cells in the spinal cord. (). Thus, these new transcriptional profiles reveal dozens of possible new marker genes that reliably distinguish skeletal motor neurons from other cells in the spinal cord. (<xref rid="nihms-1657468-f0001" ref-type="fig">Fig. 1g</xref>; ; Supplementary Table 2a).'], 'nihms-1657468-f0002': ['Unlike skeletal motor neurons, which control voluntary movement, visceral motor neurons in the spinal cord control the activity of involuntary smooth muscles responsible for regulating homeostatic processes throughout the body. These cells are part of the sympathetic nervous system, while comparable cells in the brain stem are part of the parasympathetic nervous system12. These sympathetic visceral motor neurons are very closely related, developmentally, to skeletal motor neurons14,15, but do not innervate muscle fibers directly. Instead, they project from the lateral autonomic column of the spinal cord and synapse onto the peripheral ganglia in the sympathetic chain that control smooth muscle contraction (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2a</xref>))12. Neurons of the autonomic nervous system innervate nearly every organ in the body and thus have different functional requirements to fit the needs of each peripheral target (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2b</xref>). Could these functional differences be encoded by transcriptional heterogeneity?). Could these functional differences be encoded by transcriptional heterogeneity?', 'The sympathetic nervous system is organized along the rostral-caudal axis of the spinal cord, such that visceral motor neurons that control the same organs are coarsely grouped near one another within the spinal cord (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2b</xref>). Intriguingly, past electrophysiological studies have uncovered heterogeneity among visceral motor neurons with respect to their membrane properties, responsiveness to neurotransmitters). Intriguingly, past electrophysiological studies have uncovered heterogeneity among visceral motor neurons with respect to their membrane properties, responsiveness to neurotransmitters16, and sensitivity to hormones such as dopamine17 and noradrenaline18. This evidence strongly suggests that the visceral motor system contains physiologically distinct subpopulations, but the overarching molecular logic underlying the sympathetic nervous system remains unresolved.', 'We hypothesized that transcriptomic clusters may reflect distinct populations of visceral motor neurons that either innervate specific peripheral targets, are selectively responsive to hormones17, and/or utilize distinct classes of neuropeptides in transmitting signals to peripheral ganglia19. To test these hypotheses, we subclustered all visceral motor neurons. By limiting the diversity of cell types that are concurrently analyzed and subclustering along principal axes of variation specific to visceral motor neurons, we resolved 16 transcriptionally distinct subpopulations (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2c</xref>). We identified marker genes that are, according to the Allen Spinal Cord Atlas). We identified marker genes that are, according to the Allen Spinal Cord Atlas10, expressed in the adult mouse lateral autonomic column of the spinal cord and are significantly enriched in at least one of the 16 visceral motor neuron populations (Supplementary Table 3). For each marker gene we estimated a normalized spatial density of expressing cells along the rostral-caudal spinal cord using data from the Allen Spinal Cord Atlas (see Methods). We then estimated the positional distribution of cells within each visceral motor neuron cluster as an average of these spatial cell distributions, weighted by the relative expression levels of the marker genes within each cluster (see Methods). While most clusters showed no strong spatial bias along this axis (<xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3a</xref>), clusters 3, 7, and 10 showed a clear enrichment in the sacral spinal cord.), clusters 3, 7, and 10 showed a clear enrichment in the sacral spinal cord.', 'To confirm these findings, we selected several genes that are enriched in distinct clusters (Rxfp1, Nts, Cdh8, Piezo2, Creb5, Fbn2) and performed in situ hybridizations on sections every 600 µm along the rostral-caudal axis of the adult spinal cord (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2d</xref>, , <xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3b</xref>). The results of this analysis were striking – we confirmed that ). The results of this analysis were striking – we confirmed that Rxfp1 (Clusters 3 and 7) is expressed exclusively in the sacral spinal cord (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2d</xref>, , <xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3c</xref>,,<xref rid="nihms-1657468-f0007" ref-type="fig">d</xref>). Among other roles, sacral visceral motor neurons modulate sexual function—an intriguing finding given that male and female sexual organs release relaxin family peptides). Among other roles, sacral visceral motor neurons modulate sexual function—an intriguing finding given that male and female sexual organs release relaxin family peptides20,21, which bind to Rxfp1. We provide Rxfp1 as an example for how these sequencing data can reveal functionally relevant receptor expression and there are other highly specific hormone receptor genes in distinct clusters (Table 1). We speculate that differentially expressed hormone receptor expression among visceral motor populations may be tuned to central presynaptic inputs and/or peripheral innervation targets.', 'We found preganglionic clusters to be highly transcriptionally divergent — with numerous individual markers capable of distinguishing specific clusters (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2e</xref>). Strikingly, the most common subtype of visceral motor neurons (cluster 0) expresses high levels of ). Strikingly, the most common subtype of visceral motor neurons (cluster 0) expresses high levels of Neurotensin (Nts) and are therefore neurotensinergic. These neurotensinergic motor neurons are distributed throughout the thoracic and sacral lateral autonomic columns (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2d</xref>). ). Nts expression was remarkably binary, with Ntson and Ntsoff visceral motor neurons frequently observed directly adjacent to one another in transverse sections (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2f</xref>). Neurotensin is a 13-amino acid peptide, which when injected into rats causes potent inhibition of sympathetic circuits that regulate blood pressure, heart rate, and inspiratory drive—among other effects on the sympathetic nervous system). Neurotensin is a 13-amino acid peptide, which when injected into rats causes potent inhibition of sympathetic circuits that regulate blood pressure, heart rate, and inspiratory drive—among other effects on the sympathetic nervous system22. Remarkably, although Nts is clearly involved in regulating sympathetic nervous function, its role in spinal preganglionic motor neurons has not been studied. We present strong evidence that Nts expression is a defining feature of preganglionic motor identity and provide the transcriptional roadmap for more detailed future characterization.', 'Because several studies have shown that hormones, neuropeptides, and monoaminergic signaling play a crucial role in the sympathetic nervous system17–23, we were interested in differentially expressed genes that would affect those pathways. We observed remarkable specificity of neuropeptides and receptors—as well as neurotransmitter receptors—across visceral motor neuron clusters (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2f</xref>, , Table 1). Adrenergic receptors, for example, show a remarkable degree of expression specificity in our single-nucleus sequencing data. Type I adrenergic receptor Adra1a is expressed in clusters 3 and 7, which we previously determined to correspond with sacral autonomic motor neurons (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2d</xref>). On the other hand, type II adrenergic receptor ). On the other hand, type II adrenergic receptor Adra2a is expressed at low levels among all visceral populations at (<xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3e</xref>). We also found highly specific expression of neuroactive peptide precursors, such as ). We also found highly specific expression of neuroactive peptide precursors, such as Penk and Sst in cluster 3 (Table 1). Together, these findings suggest that the repertoire of neuropeptide and hormone receptor expression is an organizing logic within the sympathetic preganglionic motor system.', 'Cholinergic interneurons are a rare cell population marked by Pax2 expression11. They play key roles in the circuits underlying locomotor behaviors24 (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2g</xref>). Subclustering revealed 7 distinct transcriptional populations of cholinergic interneurons (). Subclustering revealed 7 distinct transcriptional populations of cholinergic interneurons (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2h</xref>) —including clusters 2, 3, 5, 6, and 7 that express high levels of ) —including clusters 2, 3, 5, 6, and 7 that express high levels of Nos1, which is traditionally considered to be a marker of the autonomic nervous system in the spinal cord12 (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2i</xref>). It remains to be determined whether these ). It remains to be determined whether these Nos1+ cholinergic cells are interneurons or instead a preganglionic motor neuron population that projects into the periphery but also expresses the interneuron marker Pax2. A subset of this population of cells (clusters 2, 3, and 6) also expresses higher levels of Piezo2, a mechanosensitive ion channel involved in proprioception, than any other population in the adult spinal cord (Supplementary Table 2a). We performed in situ hybridization to demonstrate that cholinergic, Piezo2-high cells have large cell bodies and predominantly localize just lateral of the central canal (CC) (<xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3f</xref>). Notably, the localization and size of these cholinergic interneurons strongly resemble previously identified ). Notably, the localization and size of these cholinergic interneurons strongly resemble previously identified Pitx2- ‘C3’ cells25.', 'In contrast, clusters 0 and 1 do not express Nos1 but instead express Pitx2—an established marker of partition cells25,26 (<xref rid="nihms-1657468-f0002" ref-type="fig">Fig. 2j</xref>). Partition cells are a subset of cholinergic interneurons that make direct cholinergic synapses with motor neuron soma and proximal dendrites. These synapses, referred to as ‘C boutons,’ modulate motor neuron excitability during locomotor activity). Partition cells are a subset of cholinergic interneurons that make direct cholinergic synapses with motor neuron soma and proximal dendrites. These synapses, referred to as ‘C boutons,’ modulate motor neuron excitability during locomotor activity25. There is a robust transcriptional signature that separates partition cells from other cells in the spinal cord (Supplementary Table 2a,d). Of particular interest is Gldn, a gene that is selectively expressed in the cluster of cholinergic Pitx2+ cells that have previously been identified as partition cells (<xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3g</xref>). Mutations in ). Mutations in GLDN cause lethal congenital contracture syndrome, a crippling neurodegenerative disease in which joints become permanently fixed in a bent or straight position27. Future studies should seek to examine if GLDN mutations affect partition cells.'], 'nihms-1657468-f0007': ['Elegant viral tracing studies have delineated partition cells into ipsilaterally and contralaterally projecting populations that make exquisitely specific synaptic connections with motor neurons28. We find a parallel transcriptional bifurcation in partition cells, which segregate into two main clusters that are genetically delineated by numerous differentially expressed genes. One example is Nrxn3, which encodes a cell adhesion molecule responsible for establishing synaptic specificity (<xref rid="nihms-1657468-f0007" ref-type="fig">Extended Data Fig. 3h</xref>). Further ). Further in vivo experiments will be necessary to definitively show whether these transcriptionally distinct populations correspond to ipsilateral and contralateral projecting populations, and whether Nrxn3 plays a functional role in partition circuit assembly/maintenance. By identifying these populations in our data, we present a detailed molecular characterization of partition cells and reveal several novel marker genes of cholinergic interneurons cell classes.'], 'nihms-1657468-f0003': ['Traditionally, skeletal motor neurons have been defined based on their muscle innervation target4,29, developmental lineage30, morphology, and electrophysiological properties31,32. They are classified as α, beta (β), and gamma (γ) spinal motor neurons (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3a</xref>). α motor neurons directly innervate extrafusal muscle fiber neuromuscular junctions (NMJs). In contrast, γ MNs innervate intrafusal muscle spindles). α motor neurons directly innervate extrafusal muscle fiber neuromuscular junctions (NMJs). In contrast, γ MNs innervate intrafusal muscle spindles33. We identified skeletal motor neurons by Tns1/Bcl6 expression (as above, see Methods), and then subclustered them (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3b</xref>). We utilized the few robust genetic markers of α and γ motor neurons that have been confirmed in adult animals). We utilized the few robust genetic markers of α and γ motor neurons that have been confirmed in adult animals33–36. Thus, we identified γ motor neurons by their expression of Htr1d and α motor neurons by their high level of Rbfox3 and Spp1 expression.', 'To identify putative α and γ motor neuron populations, we examined marker gene expression within each cluster (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3c</xref>). Clusters 0, 2, and 6 express high levels of ). Clusters 0, 2, and 6 express high levels of Htr1d and low levels of Rbfox3 and Spp1 (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3c</xref>, , Supplementary Table 2e), suggesting that they represent γ motor neurons. The remaining clusters minimally express Htr1d, suggesting that they represent α motor neurons (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3c</xref>). We calculated differential gene expression between putative α and γ motor neuron clusters, yielding a collection of novel markers of each population (). We calculated differential gene expression between putative α and γ motor neuron clusters, yielding a collection of novel markers of each population (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3d</xref>). To validate a putative marker of γ motor neurons (). To validate a putative marker of γ motor neurons (Npas1), we performed multiplexed in situ hybridization with the canonical γ marker Htr1d and Chat in the adult spinal cord. The resulting images demonstrate robust coexpression in cholinergic cells in the ventral horn (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3e</xref>). Similarly, we performed ). Similarly, we performed in situ hybridization comparing expression of a novel α marker (Vipr2), Chat, and an established marker of α motor neurons (Rbfox3) to confirm that Vipr2 is expressed solely in α motor neurons (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3e</xref>, , <xref rid="nihms-1657468-f0008" ref-type="fig">Extended Data Fig. 4a</xref>,,<xref rid="nihms-1657468-f0008" ref-type="fig">b</xref>). Additionally, ). Additionally, Vipr2 and Htr1d have non-overlapping expression patterns in Chat+ cells (<xref rid="nihms-1657468-f0008" ref-type="fig">Extended Data Fig. 4c</xref>, as do , as do Npas1 and Rbfox3 (<xref rid="nihms-1657468-f0008" ref-type="fig">Extended Data Fig. 4d</xref>). As a final confirmation, we show that ). As a final confirmation, we show that Vipr2 and Npas1 are expressed in reciprocal populations of motor neurons in the ventral horn (<xref rid="nihms-1657468-f0008" ref-type="fig">Extended Data Fig. 4e</xref>). Notably, all existing α motor neuron markers are insufficient on their own to distinguish them from other cells in the spinal cord. In contrast, ). Notably, all existing α motor neuron markers are insufficient on their own to distinguish them from other cells in the spinal cord. In contrast, Vipr2 is expressed exclusively in α motor neurons. Together, these results provide a robust molecular basis for distinguishing α and γ motor neurons using newly described genetic markers.', 'Gamma motor neurons innervate intrafusal muscle fibers, which maintain the tension required for skeletal muscle to function properly (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3f</xref>). Hierarchical clustering of γ motor neuron marker genes (). Hierarchical clustering of γ motor neuron marker genes (Htr1d+) revealed two main clusters (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3g</xref>), which may be distinguished by the expression of numerous individual transcripts (), which may be distinguished by the expression of numerous individual transcripts (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3h</xref>). Clustering may also be taken to an even more granular level, segmenting γ motor neurons into 4 distinct populations (). Clustering may also be taken to an even more granular level, segmenting γ motor neurons into 4 distinct populations (<xref rid="nihms-1657468-f0009" ref-type="fig">Extended Data Fig. 5a</xref>,,<xref rid="nihms-1657468-f0009" ref-type="fig">b</xref>)—however, the majority of variation is captured by dividing them into two populations. We noticed that many of the genes enriched in the )—however, the majority of variation is captured by dividing them into two populations. We noticed that many of the genes enriched in the Stxbp6+ population are also expressed more broadly by α motor neurons (<xref rid="nihms-1657468-f0009" ref-type="fig">Extended Data Fig. 5c</xref>,,<xref rid="nihms-1657468-f0009" ref-type="fig">d</xref>). We named this new population γ* (pronounced gamma star), while all other ). We named this new population γ* (pronounced gamma star), while all other Htr1d+ cells are canonical γ motor neurons. We can reliably distinguish γ from γ* by reciprocal expression of either Stxbp6 or Plch1 (γ*) and Creb5 or Pard3b (γ), both in our single cell dataset (<xref rid="nihms-1657468-f0009" ref-type="fig">Extended Data Fig. 5e</xref>––<xref rid="nihms-1657468-f0009" ref-type="fig">h</xref>) and by ) and by in situ hybridization (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3i</xref>, , <xref rid="nihms-1657468-f0009" ref-type="fig">Extended Data Fig. 5i</xref>,,<xref rid="nihms-1657468-f0009" ref-type="fig">j</xref>). Owing to the extensive number of differentially expressed genes that separate γ from γ*, as well as the lack of an intermediate population between them that would suggest that they are two functional ‘states’ of γ motor neurons, we hypothesize that these cell types represent a fundamental subdivision of the fusimotor system. However, future work will be necessary to conclusively determine if this subdivision corresponds to transient ‘activity states’ of γ motor neurons or developmentally/functionally distinct populations of cells.). Owing to the extensive number of differentially expressed genes that separate γ from γ*, as well as the lack of an intermediate population between them that would suggest that they are two functional ‘states’ of γ motor neurons, we hypothesize that these cell types represent a fundamental subdivision of the fusimotor system. However, future work will be necessary to conclusively determine if this subdivision corresponds to transient ‘activity states’ of γ motor neurons or developmentally/functionally distinct populations of cells.', 'One kind of skeletal motor neuron that has been defined physiologically and anatomically, but not yet transcriptionally, is the β motor neuron. This cell type innervates both intrafusal and extrafusal fibers, and therefore has properties of both α and γ motor neurons37. Could γ* actually correspond to this historically elusive and long-sought skeletal motor neuron subtype? We present a list of novel markers that differentiate the γ and γ* populations (<xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3h</xref>), which will enable a more detailed exploration in the future.), which will enable a more detailed exploration in the future.', 'a, Transverse schematic shows α motor neurons (blue) innervating extrafusal muscle fibers. b, UMAP with 12 subclustered α motor neuron populations. Inset shows all α motor neurons from <xref rid="nihms-1657468-f0003" ref-type="fig">Fig. 3b</xref> that were subclustered. that were subclustered. c, Novel marker gene expression across α motor neuron subpopulations. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level. d, Representative in situ hybridization against novel (Hcrtr2) and known (Cpne4) intrinsic-foot (IF) motor pool markers in longitudinal sections, overlaid with CTb labeled cells that innervate the gluteus (GLUT) and IF. n=5 (IF) and 4 (GLUT) biologically independent animals. e, Proportion of CTB-labeled cells from GLUT and IF that are labeled with Hcrtr2 and Cpne4 shows novel and known markers selectively label the IF motor pool. n=5 (IF) and 4 (GLUT) biologically independent animals. f, Schematic illustrating slow-firing (SF, blue), fast fatigue-resistant (FR, purple) and fast fatigable (FF, red) α motor neuron populations innervating type I, IIa, and IIb fibers respectively. g, Heatmap showing all α motor neurons hierarchically clustered and colored by expression of differentially expressed genes between fast (Chodl+) and slow (Sv2a+) α motor neurons shows three cell populations corresponding to SF, FR, and FF motor neurons. Red and blue bars show gene modules enriched in fast- and slow-firing α motor neurons, respectively. h, Representative in situ hybridization in longitudinal sections against novel (Kcnq5, Prkcd) and known (Chodl, Sv2a) fast and slow-firing motor neuron markers, respectively. Images show CTb labeled cells that innervate a muscle with predominantly fast-twitch fibers (TA) and slow-twitch fibers (SOL). n=5 (TA) and 4 (SOL) biologically independent animals. i, Proportion of CTB-labeled cells from TA and SOL that are labeled with Chodl and Kcnq5. The TA has a significantly larger proportion of Chodl+ and Kcnq5+ cells than the SOL, though Kcnq5+ cells are significantly less frequently found in both populations. n=5 (TA) and 4 (SOL) biologically independent animals. Adjusted p-values=0.0197 (Chodl), 0.0262 (Kcnq5). j, Proportion of CTB-labeled cells from TA and SOL that are labeled with Sv2a and Prkcd. The SOL pool has a significantly larger proportion of Prkcd+ and Sv2a+ cells than the TA. n=5 (TA) and 4 (SOL) biologically independent animals. All differential expression calculated using Wilcoxon rank sum test and adjusted for multiple comparisons (Bonferroni method) (p_adj < 0.01, log2fc>0.5). All expression values were log-normalized in Seurat50. Scale bars=50µm (h), 75 µm (all others). One-way ANOVA with post-hoc Sidak multiple comparison test between same-gene conditions. *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.'], 'nihms-1657468-f0004': ['During development, motor neurons require cell-intrinsic and extrinsic cues that coordinate expression of transcription factors and cell adhesion molecules3,4. This molecular cascade enables α motor neurons to form into groups, known as motor pools, that cluster alongside one another in the spinal cord and collectively innervate the extrafusal fibers of distinct muscles34 (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4a</xref>). Owing to a vast heterogeneity in muscle location throughout the body and the types of muscle contractions required for coordinated movement, mature α motor neurons display substantial functional differences). Owing to a vast heterogeneity in muscle location throughout the body and the types of muscle contractions required for coordinated movement, mature α motor neurons display substantial functional differences38–40. However, a priori, it was unknown whether or not electrophysiological subtypes of α motor neurons with specific innervation targets express different transcriptional programs.', 'Foundational studies strongly suggest that motor neuron target specificity arises from transcriptional heterogeneity3 during development, but whether these differences persist into adulthood is unknown. We subclustered the α motor neuron transcriptomes (as above—see Methods; <xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4b</xref>). This analysis revealed 12 clusters. Most α motor neurons fall in one large population that consists of clusters 0, 1, and 4. Other clusters diverge transcriptionally from this main population, and express specific distinguishing markers (). This analysis revealed 12 clusters. Most α motor neurons fall in one large population that consists of clusters 0, 1, and 4. Other clusters diverge transcriptionally from this main population, and express specific distinguishing markers (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4c</xref>). Cluster 3 expresses ). Cluster 3 expresses Cpne4 and Fign specifically (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4c</xref>, , Supplementary Table 2f), genes which were recently shown to be highly expressed in intrinsic-foot (IF) motor neurons during development38. Furthermore, clusters 7 and 8 express high levels of Sema3e (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4c</xref>)—which within the developing lumbar spinal cord is a specific genetic marker for )—which within the developing lumbar spinal cord is a specific genetic marker for Gluteus maximus (Glut)-innervating motor neurons41, as well as shoulder-innervating motor neurons in the cervical spinal cord42. Sema3e encodes a protein that, together with its receptor PlexinD1, contributes to synaptic specificity between sensory and motor neurons in that pool41,42. These findings raise the hypothesis that single-nucleus transcriptomics is sufficient to distinguish subpopulations of α motor neurons that in the adult mouse specifically innervate unique muscle groups.', 'To test whether the α motor neuron clusters that we identified by transcriptomics correspond to functionally defined motor pools, which collectively innervate a specific muscle, we performed intramuscular injections of fluorescently conjugated cholera toxin beta subunit (CTB)—a recombinant protein that is taken up by motor neuron axon terminals and transported retrogradely to the cell body43. By performing simultaneous CTB labeling and in situ hybridization against candidate marker genes for a given population, we are able to perform whole-transcriptome characterization of functionally defined cell populations (akin to performing laser-capture microdissection on an anatomically distinct population of cells). In other words, the individual marker genes that we derive are not the end goal of these experiments, but rather a means to define the full transcriptome of functionally defined motor neuron populations. To accomplish this, we first demonstrated that IF adult motor neurons express the developmentally defined IF marker Cpne4 (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4d</xref>,,<xref rid="nihms-1657468-f0004" ref-type="fig">e</xref>), while other populations of motor neurons (GLUT) do not. This experiment enabled the tentative conclusion that cluster 3 from our transcriptional data corresponds with IF motor neurons labeled by CTB, because ), while other populations of motor neurons (GLUT) do not. This experiment enabled the tentative conclusion that cluster 3 from our transcriptional data corresponds with IF motor neurons labeled by CTB, because Cpne4 was exclusively expressed in that population using orthogonal methods. However, to conclusively demonstrate this finding, we tested a novel, functionally intriguing marker that we found specifically expressed in cluster 3 of our single-nucleus experiment (Hcrtr2). Similarly, this gene was a robust marker of IF motor neurons (87% of IF MNs are Hcrtr2+) and not substantially expressed in GLUT motor neurons (21% of GLUT MNs are Hcrtr2+) (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4d</xref>,,<xref rid="nihms-1657468-f0004" ref-type="fig">e</xref>). It is intriguing that a specific marker of IF motor neurons, ). It is intriguing that a specific marker of IF motor neurons, Hcrtr2, encodes a membrane-bound hypocretin (orexin) receptor, a gene whose disruption causes muscle weakness and cataplexy in animal models44.', 'Skeletal muscles innervated by motor neurons are composed of slow (type I), intermediate (type IIa), and fast (type IIx/IIb) twitch fibers, each of which requires different patterns of synaptic input (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4f</xref>))32. Likewise, α motor neurons innervating these fibers are divided into slow-firing (SF), fast-fatigue resistant (FR), and fast-fatigable (FF) cell types—each with specific electrophysiological and metabolic properties34. Importantly, these groups of α motor neurons have drastically different susceptibilities to degeneration in neuromuscular disorders such as ALS5,46. Thus, determining the transcriptional programs that define these classes of motor neurons may provide insight into divergent function and susceptibility to disease5,32. Past work has begun to address this question, leading to the discovery of several markers of FF/FR (Chodl, Mmp9)31 and SF (Sv2a) motor neurons. However, the broader transcriptional differences beyond these few validated markers has thus far remained elusive.', 'To identify the gene expression modules that underly differences between SF, FR, and FF motor neurons, we segmented all α motor neurons by their mutually exclusive expression of the known markers Chodl (fast-firing) and Sv2a (slow-firing)47. We identified differentially expressed genes between Chodl+ and Sv2a+ α motor neurons, and then hierarchically clustered cells based on expression of this gene set (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4g</xref>). This analysis was sufficient to segment α motor neurons into three main populations (). This analysis was sufficient to segment α motor neurons into three main populations (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4g</xref>). It is clear that fast and slow gene modules are expressed in reciprocal populations of α motor neurons across virtually all motor pools, but in different proportions (). It is clear that fast and slow gene modules are expressed in reciprocal populations of α motor neurons across virtually all motor pools, but in different proportions (<xref rid="nihms-1657468-f0010" ref-type="fig">Extended Data Fig. 6c</xref>,,<xref rid="nihms-1657468-f0010" ref-type="fig">d</xref>). Importantly, hierarchical clustering of cells revealed three main populations of cells, two of which are ). Importantly, hierarchical clustering of cells revealed three main populations of cells, two of which are Chodl+ and one that is Sv2a+. As Chodl is expressed in both FF and FR and Sv2a is expressed in SF motor neurons, this raises the possibility that the three populations may in fact correspond to FF, FR, and SF α motor neurons. We identified novel transcripts to distinguish each population, including a putative marker of FF α motor neurons (Kcnq5) and a specific marker of SF motor neurons (Prkcd). To confirm these findings, we demonstrated that Kcnq5 is expressed in a subpopulation of Chodl+ (FF/FR) and Mmp9+ (FF/FR) α motor neurons (<xref rid="nihms-1657468-f0010" ref-type="fig">Extended Data Fig. 6e</xref>,,<xref rid="nihms-1657468-f0010" ref-type="fig">f</xref>, , <xref rid="nihms-1657468-f0011" ref-type="fig">7a</xref>). We also validated that ). We also validated that Prkcd is expressed in Sv2a+ α motor neurons and is excluded from Mmp9+ motor neurons (<xref rid="nihms-1657468-f0011" ref-type="fig">Extended Data Fig. 7b</xref>,,<xref rid="nihms-1657468-f0011" ref-type="fig">c</xref>).).', 'To validate that the expression of the fast and slow-firing gene modules that we identified define electrophysiological subtypes of motor neurons, we leveraged unique properties of the soleus (Sol) and tibialis anterior (TA) muscles, which consist of predominantly slow and fast-twitch fibers, respectively31,48. We performed intramuscular CTB injections that label TA and Sol-innervating motor neurons and measured expression of novel FF and SF marker genes by in situ hybridization (<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4h</xref>––<xref rid="nihms-1657468-f0004" ref-type="fig">j</xref>, , <xref rid="nihms-1657468-f0010" ref-type="fig">Extended Data Figs. 6b</xref>, , <xref rid="nihms-1657468-f0011" ref-type="fig">7d</xref>,,<xref rid="nihms-1657468-f0011" ref-type="fig">e</xref>). In Sol-innervating motor neurons, ). In Sol-innervating motor neurons, Prkcd was expressed in 69% of cells, while Kcnq5 was expressed in just 29%. This trend was opposite in the TA, which contained 54% Kcnq5+ cells and only 28% Prkcd+ cells (<xref rid="nihms-1657468-f0011" ref-type="fig">Extended Data Fig. 7d</xref>,,<xref rid="nihms-1657468-f0011" ref-type="fig">e</xref>). We observed almost identical patterns of expression for ). We observed almost identical patterns of expression for Prkcd and Sv2a, however it was clear that within these motor pools Kcnq5 was only expressed in a subpopulation of fast-firing motor neurons. Intriguingly, the proportion of Kcnq5+ cells in the TA (54%) is quite close to the proportion of type IIb fibers in that muscle48. These data support the conclusion that Kcnq5 is expressed in FF motor neurons, while Chodl is more broadly expressed in FF and FR motor neurons—although future electrophysiological characterization will be required to demonstrate this conclusively. In addition, Prkcd is a robust and specific marker of SF motor neurons.', 'a, Proportion of CTB-labeled cells from GLUT and IF that are labeled with Cdh8 and Sema3e. The GLUT has a significantly larger proportion of Cdh8+ and Sema3e+ cells than the IF. n=5 biologically independent animals. b, Lower power view of in situ hybridization against Prkcd/Sv2a, and Kcnq5/Chodl (insets=<xref rid="nihms-1657468-f0004" ref-type="fig">Fig. 4h</xref>) in longitudinal sections demonstrates the specificity of CTB injections into the ) in longitudinal sections demonstrates the specificity of CTB injections into the Soleus (SOL) and Tibialis anterior (TA). n=5 (TA) and 4 (SOL) biologically independent animals. One-way ANOVA with post-hoc Sidak multiple comparison test between same-gene conditions. Adjusted p-values=0.0212 (Cdh8) and 0.0499 (Sema3e). c, Expression of FF and SF gene modules overlaid on UMAP of all α motor neurons. d, Average log-normalized canonical marker expression of Chodl (fast-firing) and Sv2a (slow-firing). Scale bars = 250 µm. e, Proportion of Prkcd+ cells positive for Sv2a (left) and proportion of Chodl+ cells that are positive for Kcnq5 (right). n=3 biologically independent animals. f, Representative in situ hybridization showing Kcnq5 is expressed in a subset of Chodl+ cells. n=4 biologically independent animals. Scale bar=50 µm inset and 200 µm overview. *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.'], 'nihms-1657468-f0010': ['We next examined whether Sema3e+ clusters present in our dataset correspond to the GLUT motor pool. Indeed, we found an increase in proportion of both Sema3e+ and Cdh8+ cells in GLUT-innervating motor neurons when compared to the IF motor pool (<xref rid="nihms-1657468-f0010" ref-type="fig">Extended Data Fig. 6a</xref>). Overall, our results demonstrate that motor pools in the adult mouse have distinct transcriptional properties, which can be resolved by single-nucleus sequencing of α motor neurons. Still, there are far more motor pools (~fifty)). Overall, our results demonstrate that motor pools in the adult mouse have distinct transcriptional properties, which can be resolved by single-nucleus sequencing of α motor neurons. Still, there are far more motor pools (~fifty)4 than clusters (twelve). To explain this discrepancy, we propose that transcriptional differences among adult α motor neurons are more subtle than those that emerge during embryonic development, as is the case in the olfactory system45, and single-nucleus profiling is only sufficient to delineate more dramatic transcriptional differences. We show that the embryonic marker for GLUT innervating motor neurons (Sema3e) is less specific in the adult mouse, suggesting that future work should seek to utilize more robust, novel markers (like the ones identified here) to map transcriptional subpopulations onto their peripheral targets. The abundance of specifically expressed genes in each novel population will empower further functional study of the adult motor system.'], 'nihms-1657468-f0012': ['To determine batch-to-batch variability and reproducibility of effects, all levels of clustering were plotted as UMAPs for each replicate, along with the age, sex, and number of mice that were pooled per replicate (<xref rid="nihms-1657468-f0012" ref-type="fig">Extended Data Fig. 8</xref>). This analysis demonstrated that cluster identity was largely invariant to sex, age, and number of mice pooled.). This analysis demonstrated that cluster identity was largely invariant to sex, age, and number of mice pooled.']}
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Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons
| null |
Nat Neurosci
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1618470000
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[{'@Label': 'OBJECTIVES', '@NlmCategory': 'OBJECTIVE', '#text': 'Acute type A aortic dissection complicated with brain ischemia is associated with significantly higher mortality risks. Even if rescued with central aortic repair, some patients develop permanent postoperative neurological deficiency postoperatively. We recently introduced direct common carotid artery perfusion for acute type A aortic dissection involving the common carotid artery. This study introduced this technique to prevent postoperative neurological deficiency by comparing brain protection strategies.'}, {'@Label': 'METHODS', '@NlmCategory': 'METHODS', '#text': 'Among 91 acute type A aortic dissection patients treated at our hospital during August 2015-October 2020, the common carotid artery was involved in 19 (21%), which had\u2009>\u200990% stenosis in either of the carotid arteries on preoperative contrast-enhanced computed tomography. Twelve patients underwent conventional selective cerebral perfusion during August 2015-December 2018 and seven patients underwent direct carotid artery perfusion during January 2019-October 2020. We assessed patient characteristics, surgical courses, clinical outcomes, and neurological outcomes.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'The mean age was 69 (range 39-84) years; 17 patients were male (89%). Eight patients (42%) had neurological symptoms. Concomitant procedures, postoperative neurological symptoms, and late mortality were significantly associated with conventional selective cerebral perfusion. Five selective cerebral perfusion patients experienced worsened neurological symptoms and two died of broad cerebral ischemia. No direct carotid artery perfusion patient died during hospitalization or experienced worsened neurological outcomes.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'Direct carotid artery perfusion may be useful in aortic dissection with brain ischemia because it does not aggravate neurological symptoms and is not associated with intraoperative cerebral infarction. However, it may be ineffective when cerebral infarction has already developed.'}]
|
[
"Adult",
"Aged",
"Aged, 80 and over",
"Aortic Dissection",
"Carotid Arteries",
"Carotid Artery, Common",
"Humans",
"Male",
"Middle Aged",
"Perfusion"
] |
other
|
PMC8016743
| null |
29
|
[
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] |
Nat Neurosci. 2021 Apr 15; 24(4):572-583
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NO-CC CODE
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Rec114–Mei4 and Mer2 form mixed condensates.
a. Rec114–Mei4 colocalizes with Mer2 in mixed condensates irrespective of DNA concentration. Reactions containing 16 nM Rec114–Mei4 and 100 nM Mer2 in the presence of 1, 10, or 100 ng/μl plasmid DNA were assembled for 20 minutes at 30 °C. DAPI (5 μg/ml) was added to the reaction before applying to glass slides. DNA enrichment within the condensates is visible at lower DNA concentrations (top and middle rows), but is not as clear at high DNA concentrations (bottom row). The ratios of Rec114–Mei4 (heterotrimers) and Mer2 (tetramers) to each 2.6-kb plasmid DNA molecule are indicated on the right. Colocalization of Rec114–Mei4 and Mer2 complexes is evident even with a molar excess of DNA molecules, demonstrating that formation of joint foci is not simply because both protein complexes are independently associating with a limiting number of DNA substrates. b. Correlated intensity of Rec114–Mei4 and Mer2 proteins within the condensates. Each point shows the fluorescence intensity in an individual focus (n = 950, 925 and 1000 foci from 2-3 fields of view for samples with 1, 10 and 100 ng/μl DNA, respectively), normalized to the average foci intensity per field of view. The strong correlation indicates that the composition of the condensates is highly uniform between foci. In the presence of high DNA concentration, the fraction of smaller foci increased and correlated intensities decreased. c. Recruitment of soluble Rec114–Mei4 (left) or Mer2 (right) into preassembled condensates of Mer2 (left) or Rec114–Mei4 (right). White arrowheads point to examples of the preassembled condensates. d. Pulldown of purified Mer2 on amylose resin with or without immobilized Rec114–Mei4 complexes. e. XL-MS of Rec114–Mei4–Mer2 condensates (620 crosslinked peptides, 229 distinct crosslinked pairs of lysines).
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EMS116953-f011
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2
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1345bbde57463eb8ceffefc4e0924f5690e5e5561cf6e1e3db55adfbc3266a8d
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EMS116953-f011.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
1139
] |
[{'image_id': 'EMS116953-f012', 'image_file_name': 'EMS116953-f012.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f012.jpg', 'caption': 'Recruitment of the Spo11 core complex to Rec114–Mei4–Mer2 condensates.\na. Quantification of core complex signal within Rec114–Mei4 foci in the presence (100 nM) or absence of Mer2. The average intensity within 20 foci is plotted for each reaction. Shaded areas represent 95% confidence intervals. b. Quantification of core complex signal within Mer2 foci in the presence (16 nM) or absence of Rec114–Mei4. Reactions contained 25 nM Mer2. The average intensity within 20 foci is plotted for each reaction. Shaded areas represent 95% confidence intervals. c. Effect of including 100 nM MBPRec102–Rec104HisFlag competitor on the recruitment of the core complex to RMM condensates (16 nM Rec114–Mei4, 100 nM Mer2). The fraction of Rec114–Mei4–Mer2 foci that contain detectable core complex signal is plotted (mean and SD from 10 fields of view). d. Intensity of core complex signal within Rec114–Mei4–Mer2 condensates in the absence or presence of Rec102–Rec104 competitor. The average core complex intensity within 20 foci is plotted for each reaction. Shaded areas represent 95% confidence intervals. e. Mapping regions of Rec114 required for interaction with Rec102 or Rec104 by Y2H analysis. ±-galactosidase units are measured for the interaction between truncated variants of Gal4AD-Rec114 and LexA-Rec102 or LexA-Rec104 (mean and SD from four replicates). The position of the HLS mutation within the Rec114 PH-fold is indicated. f. Impact of the HLS mutation on the formation of comingled RMM condensates. Error bars show mean ± SD from 10 fields of view. g. Spore viability of Rec114-WT and HLS mutant strains. h. Immunoblot analysis of meiotic protein extracts from myc-tagged Rec114-WT and HLS mutant strains. Samples from two biological replicates are shown. For gel source data, see Supplementary Figure 1.', 'hash': '3ba3a57979ece1aa504443476afec52534da9c6021a8b9101c04720cb37b83e0'}, {'image_id': 'EMS116953-f004', 'image_file_name': 'EMS116953-f004.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f004.jpg', 'caption': 'Tripartite Rec114–Mei4–Mer2 nucleoprotein condensates recruit the Spo11 core complex.\na. Fluorescently labeled Rec114–Mei4 (17 nM) and Mer2 (100 nM) were mixed prior to DNA-driven condensation (for 30 minutes, 5.6 nM pUC19) and imaging by epifluorescence microscopy. b. Rec114–Mei4 and Mer2 nucleoprotein condensates were assembled separately for 10 minutes then mixed. After mixing, reactions contained 5.6 nM pUC19, 8.5 nM Alexa594Rec114–Mei4 and 50 nM Alexa488Mer2. Samples were dropped on a microscope slide 10 seconds (top) or 20 minutes (bottom) after mixing. White arrowheads indicate Mer2 condensates. c. Time course of Rec114–Mei4 and Mer2 colocalization. The time to achieve 50% of Mer2 foci overlapping with Rec114–Mei4 is indicated (t½). Lines are one-phase association models fit to the data. Error bars show mean ± SD from 9–10 fields of view. d. Incorporation of Alexa488-labeled core complexes10 into Alexa594-labeled Rec114–Mei4–Mer2 condensates. e. Fraction of Rec114–Mei4 foci that contained detectable core complex signal as a function of Mer2 concentration. Error bars show mean ± SD from 10 fields of view. f. Fraction of Mer2 foci that contained detectable core complex signal as a function of Rec114–Mei4 concentration. Error bars show mean ± SD from 9–10 fields of view. g. Y2H interaction between Gal4AD-Rec114 wild type or H39A/L40A/S41A (HLS) mutant and LexA-Mei4, LexA-Rec114, LexA-Rec102, or LexA-Rec104 (mean and SD from four replicates). h. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot. i. Immunofluorescence microscopy of meiotic chromosome spreads with myc-tagged Rec114-WT and HLS mutant strains. Green, anti-myc (Rec114); red, anti-Zip1; blue, DAPI. Quantification of the number of Rec114 foci per leptotene or early zygotene cell is plotted (n = 24). Line and error bars represent mean ± SD. For gel source data, see Supplementary Figure 1. See Source Data for exact n values for panels c and f.', 'hash': '40773818a5415a02648da4c9042ae5004e23ad38e0ac89ea82743e9c7d827d0e'}, {'image_id': 'EMS116953-f003', 'image_file_name': 'EMS116953-f003.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f003.jpg', 'caption': 'DNA binding by Mer2 is important for macromolecular condensation in vitro and in vivo and for Spo11-induced break formation.\na. Gel shift assay of wild-type (WT) and mutant Mer2 complexes binding to an 80-bp DNA substrate. The Mer2-KRRR mutant has residues K265, R266, R267, and R268 mutated to alanine. Lines on the graph are a sigmoidal curve (WT) and a smooth spline (KRRR) fit to the data. b. Effect of the Mer2-KRRR mutation on condensation in vitro. Reactions included 5% PEG. Each point is the average of the intensities of foci in a field of view, normalized to the overall mean for wild type. Error bars show mean ± SD (n = 20 fields of view). c. Immunofluorescence on meiotic chromosome spreads for myc-tagged Mer2. The number of foci per leptotene or early zygotene cell is plotted. Error bars show mean ± SD (n = 48 and 95 cells for WT and KRRR, respectively). d. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot in wild type and mer2 mutant strains. For gel source data, see Supplementary Figure 1.', 'hash': 'e727d676e5c0d4b726e0b9eac75871c94a00661f59d66df82d6902255d96e6c2'}, {'image_id': 'EMS116953-f013', 'image_file_name': 'EMS116953-f013.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f013.jpg', 'caption': 'A condensate model for assembly of the meiotic DSB machinery and implications for the control of DSB formation and repair.\na. Assembly of the DSB machinery. (Left) Rec114–Mei4 and Mer2 complexes bind DNA in a highly cooperative manner to form large mixed nucleoprotein condensates. (Right) These condensates provide a platform to recruit the core complex through interactions that involve the N-terminal domain of Rec114 and the Rec102–Rec104 components of the core complex. Multiple Spo11 complexes are recruited and may engage an incoming DNA loop simultaneously. The molecular arrangement of the core complex proteins is based on ref 10. See Supplementary Discussion 4 for more detail. b. Hotspot competition and DSB interference. Competition arises prior to DSB formation as a consequence of the partitioning of RMM proteins into condensates. DSB interference is implemented through local inhibition of further DSB formation by DSB-activated Tel1. Inhibition could work on the same cluster that generated the activation DSB as well as on nearby clusters in cis. See Supplementary Discussion 5 for more detail. c. The coherence provided by the condensates may serve functions during repair, including the maintenance of a physical connection between the DNA ends that involves end-capping by condensate-embedded core complexes. See Supplementary Discussion 6 for more detail.', 'hash': 'd913eb21f906265af7568d0020f09d86f949800fed3e15c764f788ea65d8bb8d'}, {'image_id': 'EMS116953-f002', 'image_file_name': 'EMS116953-f002.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f002.jpg', 'caption': 'Rec114–Mei4 and Mer2 form condensates on DNA.\na. Gel shift analysis of Rec114–Mei4 and Mer2 binding to 80 bp DNA substrates (see also Extended Data Fig. 2a, b). b. Quantification of gel-shift analyses with 20-, 40- or 80 bp substrates. Error bars are ranges from two independent experiments. Lines are sigmoidal curves fit to the data, except for the 20 bp substrate (smooth spline fits). Apparent affinities of Rec114–Mei4 are: 6 ± 1.4 nM (80 bp, mean and range); 35 ± 1.3 nM (40 bp); ≈ 80 nM (20 bp). Apparent affinities of Mer2 are: 19 ± 1.5 nM (80 bp); 64 ± 15 nM (40 bp); > 400 nM (20 bp). Here and elsewhere, concentrations for Rec114–Mei4 refer to the trimeric complex, but for Mer2 they refer to the monomer. Therefore, the complexes have comparable affinities for DNA if the quaternary units (trimers and tetramers, respectively) are considered. c. AFM imaging of 50 nM Mer2 in the absence (left) or presence (right) of 1 nM plasmid DNA (pUC19). d. Time course of the assembly of Mer2 foci in the presence of plasmid DNA. The x axis indicates the time in solution before plating, upon which DNA is immobilized to the glass slide while soluble protein is still free to diffuse. Quantification is provided of focus numbers and average focus intensity (normalized to the mean at 30 min). Error bars show mean ± SD from 8–10 fields of view (see Source Data for exact n values). For gel source data, see Supplementary Figure 1.', 'hash': '7618140deaee39ecd206361aba8bf189c5b7b76d91cde36a5f6245005cb341ce'}, {'image_id': 'EMS116953-f005', 'image_file_name': 'EMS116953-f005.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f005.jpg', 'caption': 'Characterization of the Rec114–Mei4 complex.\na. Strategy for purification of a hypothetical Rec114–Mei4–Mer2 (RMM) complex. Combinations of MBP-tagged and HisFlag-tagged RMM subunits were co-expressed in insect cells. After cell lysis, complexes were purified by sequential affinity chromatography and analyzed by SDS-PAGE. Expression and solubility of the recombinant proteins are verified by western blotting (WB) of cell extracts. b. Analysis of purified complexes. Rec114–Mei4 complexes were apparent (lanes 1 and 3), but no Mer2 was co-purified. Lanes 2 and 4 show some enrichment of MBP-Mer2, but no co-purification of Rec114–Mei4. The presence of MBP-Mer2 in lanes 2 and 4 of the silver-stained gel may be due to background binding of MBP-Mer2 to the NiNTA resin (potentially via adsorption of DNA to the resin), or to low-affinity interactions to immobilized His-tagged Rec114–Mei4 complexes. Either way, none of the combinations tested yielded stoichiometric complexes of all three RMM subunits. Western blot controls of cell extracts showed that the tagged RMM proteins were expressed and soluble. c. Mass spectrometry analysis of Rec114–Mei4 complexes. Purified Rec114-Mei4 complexes were treated with trypsin and analyzed by LC-MS/MS. The ratio of spectral counts between Rec114 and Mei4 provides additional evidence supporting the 2:1 stoichiometry of the complex. d, e. Alignments and predicted secondary structures of the C-terminus of Rec114 (d) and the N-terminus of Mei4 (e). The positions of the conserved SSMs are indicated. f. Cartoon of the Rec114–Mei4 truncations analyzed. g. Purification of Rec114–Mei4 truncations. Proteins were expressed in E. coli and purified on NiNTA resin using a HisSUMO tag fused to the N-terminus of the Rec114 fragment. After removal of the tag by treatment with the SUMO protease Ulp1, complexes were further purified by gel filtration. A Coomassie-stained SDS-PAGE analysis of purified complexes is shown. 5 μg was loaded for each sample. Polypeptides containing Rec114(375-428) and Mei4(1-43) retained the ability to interact (combination #4). h. SEC-MALS analysis of Rec114–Mei4 truncations. The data are consistent with expectation for truncations that contain two Rec114 subunits and one Mei4 subunit. The C-terminus of Rec114 alone forms a dimer. i. Wild type and F411A-containing variants of HisSUMORec114(325-428) were co-expressed with Mei4(1-90) and purified by chromatography on NiNTA resin. The absence of the Mei4 fragment with Rec114-F411A shows that the mutation abolishes the interaction with Mei4. j. SEC-MALS analysis of untagged wild-type (WT, reproduced from panel H to aid comparison) and F411A Rec114(325-428) show that the mutation affects Rec114 dimerization. k. Y2H analysis of the interaction of Gal4BD-Rec114 (WT and F411A) with LexA-Mei4, LexA-Rec102, or LexA-Rec104 (mean and SD from four replicates). β-Gal units are quantified based on hydrolysis of ONPG. The F411A mutation abolishes the interaction of Rec114 with Mei4, but not with Rec102 and Rec104. l. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot, showing that rec114-F411A is defective in meiotic DSB formation. m. Spore viability of rec114-F411A mutant (n = 40). n. Western-blot analyses of meiotic protein extracts from myc-tagged REC114-WT and F411A strains. The F411A mutation does not compromise the expression of Rec114. o. Immunofluorescence microscopy analysis of meiotic chromosome spreads with wild-type and F411A myc-tagged Rec114. Green, anti-myc; red, synaptonemal complex component Zip1; blue, DNA. Quantification of the number of Rec114 foci per leptotene or early zygotene cell is plotted; error bars show mean ± SD (n = 20 and 38 cells for WT and F411A, respectively). The F411A mutation abolishes the formation of chromatin-associated Rec114 foci.', 'hash': 'ff99cac890103b5004400d235ee6a07acbcbe55d412a67e1b0e0cd9d11ff1db6'}, {'image_id': 'EMS116953-f006', 'image_file_name': 'EMS116953-f006.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f006.jpg', 'caption': 'DNA-binding properties of Rec114–Mei4 and Mer2 complexes.\na, b. Gel shift analysis of Rec114–Mei4 (a) or Mer2 (b) binding to 20- or 40- bp DNA substrates. Quantification is in Fig. 2b. c, d. Competition assay of Rec114–Mei4 (c) or Mer2 (d) binding to 80 bp radiolabeled DNA (1 nM) in the presence of 20- or 80 bp cold competitor. Fold excess is in nucleotides. Lines are one-phase decay fits. e, f. Binding to plasmid DNA analyzed by native agarose gel electrophoresis. Rec114–Mei4 (e) and Mer2 (f) were titrated with 2 nM plasmid DNA (pUC19) in the presence or absence of 5 mM MgCl2. Rec114–Mei4 complexes bound with roughly similar affinity independently of the presence of Mg2+ (apparent KD ≈ 50–80 nM). Note that the apparent affinity is significantly lower than suggested by the gel shift analyses with radiolabeled substrates presented in panel a and Fig. 2a, b (see apparent affinities in Fig. 2 legend). We interpret that this difference is because the proteins coalesce on a small fraction of the plasmid molecules, as illustrated in the cartoon below. Indeed, bound plasmids remained trapped in the wells, which is consistent with cooperative assembly of large nucleoprotein structures. Because each plasmid substrate provides many more binding sites than the short oligonucleotide substrates in panel a and Fig. 2a, a higher concentration of protein is required to reach complete binding of all of the plasmid molecules. In contrast to Rec114–Mei4, Mer2 showed efficient binding in the absence of Mg2+ in this assay (KD = 30 ± 2 nM) but binding appeared to be considerably inhibited in the presence of Mg2+ (KD ≈ 150 nM), as indicated by the persistence of unbound substrate at high protein concentrations. However, while the electrophoretic mobility of Mer2-bound plasmids decreased steadily as the concentration of Mer2 increased in the absence of Mg2+, no such steady progression was observed when Mg2+ was included. Instead, a minority of bound substrates shifted to a low-mobility species (labeled * in panel f, bottom), indicating that they were occupied by multiple Mer2 complexes. We interpret that, rather than inhibiting DNA binding, Mg2+ promotes cooperativity, in agreement with the fluorescence microscopy analysis (Extended Data Fig. 3b). The difference in migration distance of the plasmid between the +/- Mg2+ gels is due to the presence of Mg2+ in the electrophoresis buffer. g. AFM imaging of 12 nM Rec114–Mei4 in the absence (left) or in the presence (right) of 1 nM plasmid DNA (pUC19).', 'hash': 'cf4130e20e12ccde49af9716988da7bcbc588c3723cfffe55e136147badc3965'}, {'image_id': 'EMS116953-f001', 'image_file_name': 'EMS116953-f001.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f001.jpg', 'caption': 'Purification and subunit arrangement of the S. cerevisiae RMM proteins.\na. Prediction of protein disorder (IUPRED server42). The ANCHOR score predicts the transition from unstructured to structured depending on a binding partner. Previously identified SSMs are highlighted23,26. b. SDS-PAGE of purified tagged and untagged Rec114–Mei4 complexes. 4 μg was loaded. c. SEC-MALS analysis of tagged and untagged Rec114–Mei4. The traces show UV absorbance (left axis), circles are molar mass measurements across the peak (right axis). Elution positions of protein standards are marked. d. XL-MS analysis of Rec114–Mei4 (4812 crosslinked peptides, 258 distinct crosslinked pairs of lysines). Black loops are intermolecular self-links. Black vertical lines indicate lysines. e. Cartoon of the Rec114–Mei4 complex. f. Protein disorder prediction for Mer2. The predicted coiled coil and previously identified SSMs are highlighted23,26. g. SDS-PAGE of purified Mer2. 4 μg was loaded. h. SEC-MALS analysis of Mer2. i. XL-MS analysis of Mer2 (487 crosslinked peptides, 89 distinct crosslinked pairs of lysines). j. SEC-MALS analysis of the coiled coil domain of Mer2 and a single-chain dimer variant of the coiled coil domain. A tetramer of monomers and a dimer of single-chain dimers both have an expected MW of 70 kDa. The difference between the profiles of the monomer and single chain dimer can be explained by reduced degrees of freedom (tension) in the single-chain dimer and heterogeneity. k. Interpretive cartoon of the molecular arrangement of the coiled coil domain of Mer2. For gel source data, see Supplementary Figure 1.', 'hash': 'e068bb5592b3a4b6564a3ac7abbec71d6863c6f2f98cd4a58423444c62e5c576'}, {'image_id': 'EMS116953-f008', 'image_file_name': 'EMS116953-f008.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f008.jpg', 'caption': 'Properties of Rec114–Mei4 and Mer2 DNA-dependent condensates.\na, b. Effect of challenging Rec114–Mei4 (a) or Mer2 (b) nucleoprotein condensates with DNase I or 0.5 M NaCl. Condensates were assembled for 5 minutes prior to challenge. Quantification is provided of focus numbers per 1000 μm2 and of the total fluorescence intensity within foci within fields of view (normalized to mean of the no-treatment controls). Error bars show mean ± SD from 5–10 fields of view. c, e. Titrations of Rec114–Mei4 (c) and Mer2 (e) in the presence of DNA and PEG and various concentrations of NaCl. Heat maps represent the fraction of fluorescence signal found within foci. Condensed fractions are maximal at high protein and low salt concentrations. At all protein concentrations, condensation is essentially abolished beyond 250 mM NaCl. This suggests that electrostatic interactions, likely between the negatively charged DNA backbone and positively charged protein residues, are important for condensation. d, f. Time dependence for irreversibility of Rec114–Mei4 (d) and Mer2 (f) condensates. Some phase-separated liquid droplets have been shown to mature over time and progressively adopt gel-like or solid states35,37–39. Such sol-gel transitions may occur spontaneously through different mechanisms, including fibrillization and entanglement, and are thought to be counteracted in vivo to prevent the progressive accumulation of amyloid-like structures associated with pathological states35. To address whether our condensates are prone to progressive hardening, we queried the effect of assembly time on reversibility. We performed a time-course experiment where the condensates were challenged by treatment with 0.5 M NaCl after an indicated period of assembly in the presence or absence of PEG. The graph shows the total intensity summed for foci within fields of view, expressed as a percentage of the intensity without a salt challenge. Points and error bars are means ± SD for 6–10 fields of view. With Rec114–Mei4, 10% and 50% of fluorescent signal became refractory to the salt wash within 5 minutes of incubation time in the absence and presence of PEG, respectively (see panel a for example images and quantification). With Mer2, there was no evidence for the formation of irreversible structures in the absence of PEG during the course of the experiment. However, up to 25% of the focus intensity resisted the salt wash treatment after 8 minutes of incubation time in the presence of PEG. Therefore, both Rec114–Mei4 and Mer2 have a propensity to form more stable, perhaps gel-like, structures over time. Under our experimental conditions, this was more evident for Rec114–Mei4 than for Mer2, and was accentuated by molecular crowding. g, h. Assembly of Rec114–Mei4 (g) and Mer2 (h) with fluorescently labeled 9.6 kb and 100 bp linear DNA substrates. The overlap between the protein foci and puncta of DNA shows that the DNA is also enriched in the condensates. However, in contrast to the protein signal, the fluorescent signal of the DNA covers the slide because DNA is in excess and does not condense by itself. i, j. Competition between long and short DNA substrates for incorporation into condensates. Rec114–Mei4 (i) or Mer2 (j) condensates were assembled in the presence of a fluorescently labeled DNA substrate with or without 20-fold nucleotide excess of unlabeled competitor. The amount of fluorescent DNA signal averaged between ten foci is plotted. In each case, the 9.6-kb substrate was a more effective competitor than the 100-bp substrate. In addition, the 100-bp substrate was more successful at competing with the 100-bp fluorescent substrate than with the 9.6-kb fluorescent substrate. This preference for large DNA substrates is consistent with the hypothesis that the condensates form through multivalent interactions between the positively charged residues of Rec114-Mei4 or Mer2 and the sugar-phosphate backbone of the DNA. See Source Data for exact n values for panels a, b, d, and f.', 'hash': 'ecfa54c24d2bd2abf3fc3ea264efaa15a78d86ce8c902c1551e7118f8c8d5ece'}, {'image_id': 'EMS116953-f010', 'image_file_name': 'EMS116953-f010.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f010.jpg', 'caption': 'Identification of DNA-binding residues and effect of DNA binding on condensation in vitro and in vivo and on Spo11-induced break formation.\na. Mapping the DNA-binding domain of Rec114–Mei4 complexes. Gel-shift analysis was performed with pUC19 plasmid DNA and the Rec114–Mei4 protein constructs shown in Extended Data Fig. 1f. Constructs #2, #3 and #4, which include the C terminus of Rec114 and the N terminus of Mei4, were competent for DNA binding. The difference in mobility of shifted species between these constructs is in line with the difference in sizes of the protein complexes. Mei4 is dispensable for DNA binding by Rec114 (Construct #5 lacks Mei4). The N terminus of Rec114 alone, encompassing the PH domain, did not bind DNA (Construct #6). None of the constructs showed evidence for cooperative DNA binding (unlike the full-length protein, see Extended Data Fig. 2e), suggesting that they do not undergo DNA-driven condensation. b. Gel shift analysis wild-type (WT) and mutant Rec114–Mei4 complexes binding to an 80-bp DNA substrate. The Rec114-4KR mutant has residues R395, K396, K399, and R400 mutated to alanine. Lines on graphs are sigmoidal curve fits. c. Mapping the DNA-binding domain of Mer2. Gel-shift analysis was performed with pUC19 plasmid DNA and HisSUMO-tagged Mer2 protein that was either full-length (FL), had the N terminus removed (fragment 77-314), or had both the N and C termini removed (fragment 77-227). Deleting the N terminus alone had no significant effect on DNA binding, but further deleting the C terminus strongly reduced DNA binding. d. Effect of the Rec114-4KR mutation on condensation in vitro. Reactions included 5% PEG. Each point is the average of the intensities of foci in a field of view (n = 20 fields), normalized to the overall mean for wild type. Error bars show mean ± SD. e. Incorporation of Mer2-KRRR into preformed condensates. Condensates were assembled with 100 nM unlabeled Mer2. Reactions were then supplemented with the indicated amount eGFP-Mer2 (WT or KRRR) and plated immediately. Incorporation of eGFP-tagged complexes within condensates was quantified. Points and error bars are mean ± SD from 20 fields of view. f. Immunofluorescence on meiotic chromosome spreads for myc-tagged Rec114. The number of foci per leptotene or early zygotene cell is plotted. Error bars show mean ± SD (n = 44 and 40 cells for WT and 4KR, respectively). g. Immunoblotting of meiotic protein extracts for wild type and mutant Rec114 (left) or Mer2 (right). h. Partial proteolysis of wild-type and mutant Mer2 and Rec114–Mei4 complexes. i. Immunoblot analysis of Mer2-WT and Mer2-KRRR. Protein extracts of meiotic time courses were analyzed by SDS-PAGE followed by immunoblotting against Mer2-myc. Anti-Kar2 was used as a loading control. Quantification of immunoblot signal is plotted. Mer2myc-KRRR reached higher steady-state protein levels and persisted longer than wild-type Mer2myc. A previous study showed that mutating an essential CDK phosphorylation site of Mer2 (Ser30) or inhibiting CDK activity led to reduced turnover of Mer2, similar to the effect of the KRRR mutant15. This is consistent with the hypothesis that Mer2 turnover is tied to phosphorylation, which requires DNA binding. j. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot in strains expressing wild-type or mutant Rec114 protein. k. Labeling of Spo11-oligo complexes in wild type and mutant Rec114 (top) and Mer2 (bottom) strains. Error bars represent the range from two biological replicates. l. Spore viability of wild type and mutant Rec114 (left) and Mer2 (right) strains (n = 40). For gel source data, see Supplementary Figure 1.', 'hash': 'ad23579f6107dea392c5a1c422dd301f61f63af8d32478e9be0dff1f16900f26'}, {'image_id': 'EMS116953-f009', 'image_file_name': 'EMS116953-f009.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f009.jpg', 'caption': 'Growth of DNA-driven condensates by fusion.\na. Three scenarios for the assembly of DNA-driven condensates. (i) Nucleation could be limiting, with focus growth resulting principally from incorporation of protein from soluble pools. (ii) Frequent nucleation events could occur initially leading to large numbers of small foci, whereupon some foci dissolve and others grow. (iii) Frequent nucleation could yield numerous small foci that then collide and fuse to yield fewer, larger foci. See Supplementary Discussion 2 for more detail. b. Time course of the assembly of Rec114–Mei4 foci in the presence of plasmid DNA. The x axis indicates the time in solution before plating, upon which DNA is immobilized to the glass slide while soluble protein is still free to diffuse. Quantification is provided of focus numbers and average focus intensity (normalized to the mean at 30 min). Error bars show mean ± SD from 10 fields of view. c. FRAP experiments with Mer2 and Rec114–Mei4 condensates. Points and error bars are mean ± SD for six photobleached condensates.', 'hash': '8b6e961fc20964ea31462624318e1d96cf806d080138e88d40c712f696a71ae0'}, {'image_id': 'EMS116953-f007', 'image_file_name': 'EMS116953-f007.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f007.jpg', 'caption': 'Properties of Rec114–Mei4 and Mer2 DNA-dependent condensates.\na, b. Visualization of nucleoprotein condensates by epifluorescence microscopy using tagged Rec114–Mei4 (a) or Mer2 (b) in the presence or absence of 5 mM MgCl2. Foci were defined using a fixed intensity threshold between samples. Each point represents the measurement from a field of view. Error bars show mean ± SD from (a) 10 fields of view of 1.7 × 104 μm2 or (b) 27 and 26 sections of 400 μm2 with and without Mg2+, respectively. c-f. Effect of fluorophore labeling or tagging on the DNA-binding and DNA-driven condensation activities of Rec114–Mei4 and Mer2 complexes. Labeling with Alexa594 or Alexa488 was achieved using amine-reactive fluorophores. Tagging was achieved by fusion of Rec114 with the monomeric fluorescent protein mScarlet or fusion of Mer2 with the weakly dimerizing fluorescent protein eGFP. The results described here indicate that the covalent Alexa labeling has little if any effect on DNA binding properties of these complexes, whereas fluorescent protein tagging caused subtle alterations in DNA binding and/or condensation. In most subsequent experiments, we used the dye-labeled complexes to minimize steric effects or oligomerization of fluorescent protein tags. c. Gel-shift analysis of binding of unlabeled, Alexa594-labeled, or mScarlet-tagged Rec114–Mei4 complexes to an 80-bp radiolabeled DNA substrate. The three versions of the Rec114–Mei4 complex have the same intrinsic DNA-binding activity. d. Gel-shift analysis of binding of unlabeled, Alexa488-labeled, or eGFP-tagged Mer2 complexes to an 80-bp radiolabeled DNA substrate. The DNA-binding activity of the Alexa-labeled Mer2 complex is nearly identical to the untagged protein, but the eGFP-tagged complex has 3.5-fold reduced DNA-binding activity. e. A comparison between Alexa-labeled and mScarlet-tagged Rec114–Mei4 complexes for DNA-driven condensation. Focus numbers (left graphs) and total fluorescence intensity within foci normalized to the no-PEG samples (right graphs) are shown for the complexes in the presence or absence of 5% PEG. With and without PEG, mScarlet-tagged Rec114–Mei4 produced more foci than the Alexa-labeled version. Because intrinsic DNA binding was indistinguishable between the complexes (panel c), we infer that the mScarlet-tagged complexes had a reduced efficiency in the cooperative formation of large condensates compared to the Alexa-labeled version, producing more numerous foci. Asterisk indicates p < 0.0001 (two-tailed t test). Lines and error bars are mean ± SD from 8–10 fields of view. f. A comparison between Alexa-labeled and eGFP-tagged Mer2 complexes for DNA-driven condensation. Quantification is presented as in panel e. The two labeled complexes show different numbers and intensities of foci in the presence of PEG. It is likely that the DNA-binding defect of the eGFP construct (panel d) leads to the formation of fewer, brighter condensates. It is possible that the weak dimerization activity of eGFP also contributes. Asterisk indicates p < 0.0001 (two-tailed t test). Lines and error bars are mean ± SD from 9–10 fields of view. g, h. Effect of a crowding agent (PEG) on formation of nucleoprotein condensates visualized using covalently fluorophore-labeled Rec114–Mei4 (g) or Mer2 (h). Graphs show the effect of protein concentration on DNA-driven condensation in the presence or absence of 5% PEG. Left graphs show focus numbers and right graphs show the total fluorescence intensity within foci (normalized to the mean of the highest intensity sample). Points and error bars are means ± SD from 4–6 fields of view (g) or 7–10 fields of view (h). The titrations reveal complex behaviors: (g) In the presence of PEG, titration of Rec114–Mei4 from 4 to 32 nM led to a steady decrease in the number of foci, which was accompanied by a concomitant increase in focus intensity. In the absence of PEG, however, the number of Rec114–Mei4 foci first peaked at 8 nM before decreasing as the intensity of the foci started to increase. Nevertheless, focus intensity plateaued at a much lower intensity than in the presence of PEG. (h) In the case of Mer2, titration from 25 to 300 nM in the presence of PEG yielded a peak in the number of foci at ~100 nM, which then sharply declined and stabilized beyond 150 nM. Consistently, Mer2 foci remained at a constant, low intensity between 25 and 100 nM, then became abruptly brighter above 100 nM. In the absence of PEG, the number of Mer2 foci increased between 25 and 200 nM, then started to decrease beyond that threshold. These behaviors likely reflect complex combined effects of nucleation, growth, and collapse of the condensates, which are each affected differently by protein concentrations and by the crowding effect provided by PEG. See Source Data for exact n values for panels e, f, g, and h.', 'hash': 'cc3da794db8c40e30f12216b12d697606c7804d38c0e22878bfc7b95227ea6ab'}, {'image_id': 'EMS116953-f011', 'image_file_name': 'EMS116953-f011.jpg', 'image_path': '../data/media_files/PMC8016751/EMS116953-f011.jpg', 'caption': 'Rec114–Mei4 and Mer2 form mixed condensates.\na. Rec114–Mei4 colocalizes with Mer2 in mixed condensates irrespective of DNA concentration. Reactions containing 16 nM Rec114–Mei4 and 100 nM Mer2 in the presence of 1, 10, or 100 ng/μl plasmid DNA were assembled for 20 minutes at 30 °C. DAPI (5 μg/ml) was added to the reaction before applying to glass slides. DNA enrichment within the condensates is visible at lower DNA concentrations (top and middle rows), but is not as clear at high DNA concentrations (bottom row). The ratios of Rec114–Mei4 (heterotrimers) and Mer2 (tetramers) to each 2.6-kb plasmid DNA molecule are indicated on the right. Colocalization of Rec114–Mei4 and Mer2 complexes is evident even with a molar excess of DNA molecules, demonstrating that formation of joint foci is not simply because both protein complexes are independently associating with a limiting number of DNA substrates. b. Correlated intensity of Rec114–Mei4 and Mer2 proteins within the condensates. Each point shows the fluorescence intensity in an individual focus (n = 950, 925 and 1000 foci from 2-3 fields of view for samples with 1, 10 and 100 ng/μl DNA, respectively), normalized to the average foci intensity per field of view. The strong correlation indicates that the composition of the condensates is highly uniform between foci. In the presence of high DNA concentration, the fraction of smaller foci increased and correlated intensities decreased. c. Recruitment of soluble Rec114–Mei4 (left) or Mer2 (right) into preassembled condensates of Mer2 (left) or Rec114–Mei4 (right). White arrowheads point to examples of the preassembled condensates. d. Pulldown of purified Mer2 on amylose resin with or without immobilized Rec114–Mei4 complexes. e. XL-MS of Rec114–Mei4–Mer2 condensates (620 crosslinked peptides, 229 distinct crosslinked pairs of lysines).', 'hash': '1345bbde57463eb8ceffefc4e0924f5690e5e5561cf6e1e3db55adfbc3266a8d'}]
|
{'EMS116953-f005': ['Because of long-known functional relationships between Rec114, Mei4 and Mer211,12,17, confirmed in other species14,21,23,25, we sought to purify a tripartite complex. However, while Mer2 alone and a Rec114–Mei4 complex were readily purified, we could not obtain a stable RMM complex (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1a, b</xref>).).', 'Truncations retaining SSM7 of Rec114 and SSMs 1 and 2 of Mei4 (Rec114(375-428) and Mei4(1-43)) formed a 2:1 complex (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1d-h</xref>). Dimerization of Rec114 C-terminal fragments did not require Mei4 (). Dimerization of Rec114 C-terminal fragments did not require Mei4 (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1h</xref>). Mutating a conserved Rec114 residue (F411A) abolished dimerization, which disrupted the interaction with Mei4 similarly to an equivalent mutation in the ). Mutating a conserved Rec114 residue (F411A) abolished dimerization, which disrupted the interaction with Mei4 similarly to an equivalent mutation in the Schizosaccharomyces pombe Rec114 ortholog, Rec7 (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1i-k</xref>))13. Rec114-F411A was expressed at normal levels in vivo, but it eliminated Rec114 foci and DSBs, leading to spore death (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1l-o</xref>).).', 'Interaction of Mer2 and Rec114–Mei4 complexes within nucleoprotein condensates may account for their interactions in immunoprecipitation and Y2H experiments11–14,17 despite not forming a stable tripartite complex (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1a, b</xref>). We observed weak interactions between recombinant proteins by affinity pulldown (). We observed weak interactions between recombinant proteins by affinity pulldown (<xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7d</xref>). Moreover, XL-MS applied to mixtures of both complexes in the presence of DNA yielded numerous crosslinks between the Rec114 C-terminal domain and the coiled coil region of Mer2, and between Mei4 and Mer2 at multiple positions along their lengths (). Moreover, XL-MS applied to mixtures of both complexes in the presence of DNA yielded numerous crosslinks between the Rec114 C-terminal domain and the coiled coil region of Mer2, and between Mei4 and Mer2 at multiple positions along their lengths (<xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7e</xref>).).', '\na. Mapping the DNA-binding domain of Rec114–Mei4 complexes. Gel-shift analysis was performed with pUC19 plasmid DNA and the Rec114–Mei4 protein constructs shown in <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1f</xref>. Constructs #2, #3 and #4, which include the C terminus of Rec114 and the N terminus of Mei4, were competent for DNA binding. The difference in mobility of shifted species between these constructs is in line with the difference in sizes of the protein complexes. Mei4 is dispensable for DNA binding by Rec114 (Construct #5 lacks Mei4). The N terminus of Rec114 alone, encompassing the PH domain, did not bind DNA (Construct #6). None of the constructs showed evidence for cooperative DNA binding (unlike the full-length protein, see . Constructs #2, #3 and #4, which include the C terminus of Rec114 and the N terminus of Mei4, were competent for DNA binding. The difference in mobility of shifted species between these constructs is in line with the difference in sizes of the protein complexes. Mei4 is dispensable for DNA binding by Rec114 (Construct #5 lacks Mei4). The N terminus of Rec114 alone, encompassing the PH domain, did not bind DNA (Construct #6). None of the constructs showed evidence for cooperative DNA binding (unlike the full-length protein, see <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2e</xref>), suggesting that they do not undergo DNA-driven condensation. ), suggesting that they do not undergo DNA-driven condensation. b. Gel shift analysis wild-type (WT) and mutant Rec114–Mei4 complexes binding to an 80-bp DNA substrate. The Rec114-4KR mutant has residues R395, K396, K399, and R400 mutated to alanine. Lines on graphs are sigmoidal curve fits. c. Mapping the DNA-binding domain of Mer2. Gel-shift analysis was performed with pUC19 plasmid DNA and HisSUMO-tagged Mer2 protein that was either full-length (FL), had the N terminus removed (fragment 77-314), or had both the N and C termini removed (fragment 77-227). Deleting the N terminus alone had no significant effect on DNA binding, but further deleting the C terminus strongly reduced DNA binding. d. Effect of the Rec114-4KR mutation on condensation in vitro. Reactions included 5% PEG. Each point is the average of the intensities of foci in a field of view (n = 20 fields), normalized to the overall mean for wild type. Error bars show mean ± SD. e. Incorporation of Mer2-KRRR into preformed condensates. Condensates were assembled with 100 nM unlabeled Mer2. Reactions were then supplemented with the indicated amount eGFP-Mer2 (WT or KRRR) and plated immediately. Incorporation of eGFP-tagged complexes within condensates was quantified. Points and error bars are mean ± SD from 20 fields of view. f. Immunofluorescence on meiotic chromosome spreads for myc-tagged Rec114. The number of foci per leptotene or early zygotene cell is plotted. Error bars show mean ± SD (n = 44 and 40 cells for WT and 4KR, respectively). g. Immunoblotting of meiotic protein extracts for wild type and mutant Rec114 (left) or Mer2 (right). h. Partial proteolysis of wild-type and mutant Mer2 and Rec114–Mei4 complexes. i. Immunoblot analysis of Mer2-WT and Mer2-KRRR. Protein extracts of meiotic time courses were analyzed by SDS-PAGE followed by immunoblotting against Mer2-myc. Anti-Kar2 was used as a loading control. Quantification of immunoblot signal is plotted. Mer2myc-KRRR reached higher steady-state protein levels and persisted longer than wild-type Mer2myc. A previous study showed that mutating an essential CDK phosphorylation site of Mer2 (Ser30) or inhibiting CDK activity led to reduced turnover of Mer2, similar to the effect of the KRRR mutant15. This is consistent with the hypothesis that Mer2 turnover is tied to phosphorylation, which requires DNA binding. j. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot in strains expressing wild-type or mutant Rec114 protein. k. Labeling of Spo11-oligo complexes in wild type and mutant Rec114 (top) and Mer2 (bottom) strains. Error bars represent the range from two biological replicates. l. Spore viability of wild type and mutant Rec114 (left) and Mer2 (right) strains (n = 40). For gel source data, see Supplementary Figure 1.'], 'EMS116953-f001': ['Much of Rec114 is predicted as disordered (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1a</xref>, top). The N-terminal region contains six signature sequence motifs (SSMs), with a seventh located near the C terminus, top). The N-terminal region contains six signature sequence motifs (SSMs), with a seventh located near the C terminus12,23,26. The N-terminal SSMs of mouse REC114 form a pleckstrin homology (PH)-like fold25,27. Mei4 is mostly ordered (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1a</xref>, bottom), with six SSMs, bottom), with six SSMs26.', 'Purified Rec114–Mei4 had molar masses (MW) of 180 and 114 kDa for tagged and untagged complexes, respectively, on size-exclusion chromatography with multi-angle light scattering (SEC-MALS, <xref ref-type="fig" rid="EMS116953-f001">Fig. 1b, c</xref>). These results, plus intensities of Coomassie-stained bands and an observed 2:1 ratio of mass spectrometry spectral counts (). These results, plus intensities of Coomassie-stained bands and an observed 2:1 ratio of mass spectrometry spectral counts (<xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1c</xref>), suggested a stoichiometry of two Rec114 subunits and one Mei4 (expected 200 and 146 kDa for tagged and untagged, respectively).), suggested a stoichiometry of two Rec114 subunits and one Mei4 (expected 200 and 146 kDa for tagged and untagged, respectively).', 'We delineated the molecular arrangement within the complexes by crosslinking plus mass spectrometry (XL-MS), observing 258 distinct pairs of crosslinked lysines (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1d</xref> & & Supplementary Table 1). The Rec114 C-terminus crosslinked extensively to the Mei4 N-terminus (pink lines), implying that these are the primary interaction regions. Four inter-molecular self-links (crosslinking of two identical lysines) occurred near the C-terminal end of Rec114 (black loops in <xref ref-type="fig" rid="EMS116953-f001">Fig. 1d</xref>), supporting the 2:1 stoichiometry and suggesting that this domain homo-dimerizes (), supporting the 2:1 stoichiometry and suggesting that this domain homo-dimerizes (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1e</xref>).).', 'Mer2 has a predicted coiled coil and two SSMs23,28, with a disordered region between the coiled coil and SSM2 (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1f</xref>). Untagged Mer2 was 156 kDa by SEC-MALS, consistent with a tetramer (143 kDa) (). Untagged Mer2 was 156 kDa by SEC-MALS, consistent with a tetramer (143 kDa) (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1g, h</xref>), but the elution volume matched that of a considerably larger complex, suggesting an elongated shape (see marker positions in ), but the elution volume matched that of a considerably larger complex, suggesting an elongated shape (see marker positions in <xref ref-type="fig" rid="EMS116953-f001">Fig. 1h</xref>).).', 'XL-MS revealed nine intermolecular self-links (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1i</xref>). Self-links occurred along the coiled coil, consistent with parallel α-helices, but this domain also incurred long-range crosslinks. If the coiled coil forms uninterrupted helices, crosslinks further than ~ 18 amino acids cannot be explained by intra-molecular events or by intermolecular events within a parallel coiled coil. Therefore, it is likely that there are both parallel and antiparallel helices.). Self-links occurred along the coiled coil, consistent with parallel α-helices, but this domain also incurred long-range crosslinks. If the coiled coil forms uninterrupted helices, crosslinks further than ~ 18 amino acids cannot be explained by intra-molecular events or by intermolecular events within a parallel coiled coil. Therefore, it is likely that there are both parallel and antiparallel helices.', 'To address this, we first observed that the coiled-coil domain alone (residues 77-227) was still tetrameric (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1j</xref>). Next, we engineered a single-chain dimer with two copies of the coiled-coil domain separated by a 19 amino-acid linker, too short for a parallel intramolecular coiled coil. This assembled a similarly sized complex as the monomeric construct (99 vs. 84 kDa), consistent with two single-chain dimers, each folded in antiparallel (). Next, we engineered a single-chain dimer with two copies of the coiled-coil domain separated by a 19 amino-acid linker, too short for a parallel intramolecular coiled coil. This assembled a similarly sized complex as the monomeric construct (99 vs. 84 kDa), consistent with two single-chain dimers, each folded in antiparallel (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1j, k</xref>). Alternative scenarios predict an artificially elongated single-chain dimer leading to faster elution on size exclusion, which was not observed. A plausible configuration is thus a homotetrameric alpha-helical bundle with two pairs of parallel helices arranged in antiparallel fashion (). Alternative scenarios predict an artificially elongated single-chain dimer leading to faster elution on size exclusion, which was not observed. A plausible configuration is thus a homotetrameric alpha-helical bundle with two pairs of parallel helices arranged in antiparallel fashion (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1k</xref>).).', 'Light scattering data in <xref ref-type="fig" rid="EMS116953-f001">Fig. 1c, h</xref> were collected using a Superdex 200, 10/300, HR Size Exclusion Chromatography (SEC) column (GE Healthcare, Piscataway, NJ), connected to High Performance Liquid Chromatography System (HPLC), Agilent 1200, (Agilent Technologies, Wilmington, DE) equipped with an autosampler. The elution from SEC was monitored by a photodiode array (PDA) UV/VIS detector (Agilent Technologies, Wilmington, DE), differential refractometer (OPTI-Lab rEx Wyatt Corp., Santa Barbara, CA), static and dynamic, multiangle laser light scattering (LS) detector (HELEOS II with QELS capability, Wyatt Corp., Santa Barbara, CA). The SEC-UV/LS/RI system was equilibrated in buffer 25 mM Hepes pH 7.5, 500 mM NaCl, 10 % glycerol, 2 mM EDTA at the flow rate of 0.5 ml/min or 1.0 ml/min. Two software packages were used for data collection and analysis: the Chemstation software (version B.04.03-SP1, Agilent Technologies, Wilmington, DE) controlled the HPLC operation and data collection from the multi-wavelength UV/VIS detector, while the ASTRA V software (Wyatt Corp., Santa Barbara, CA) collected data from the refractive index detector, the light scattering detectors, and recorded the UV trace at 280 nm sent from the PDA detector. The weight average molecular masses were determined across the entire elution profile in intervals of 1 sec from static LS measurement using ASTRA software. were collected using a Superdex 200, 10/300, HR Size Exclusion Chromatography (SEC) column (GE Healthcare, Piscataway, NJ), connected to High Performance Liquid Chromatography System (HPLC), Agilent 1200, (Agilent Technologies, Wilmington, DE) equipped with an autosampler. The elution from SEC was monitored by a photodiode array (PDA) UV/VIS detector (Agilent Technologies, Wilmington, DE), differential refractometer (OPTI-Lab rEx Wyatt Corp., Santa Barbara, CA), static and dynamic, multiangle laser light scattering (LS) detector (HELEOS II with QELS capability, Wyatt Corp., Santa Barbara, CA). The SEC-UV/LS/RI system was equilibrated in buffer 25 mM Hepes pH 7.5, 500 mM NaCl, 10 % glycerol, 2 mM EDTA at the flow rate of 0.5 ml/min or 1.0 ml/min. Two software packages were used for data collection and analysis: the Chemstation software (version B.04.03-SP1, Agilent Technologies, Wilmington, DE) controlled the HPLC operation and data collection from the multi-wavelength UV/VIS detector, while the ASTRA V software (Wyatt Corp., Santa Barbara, CA) collected data from the refractive index detector, the light scattering detectors, and recorded the UV trace at 280 nm sent from the PDA detector. The weight average molecular masses were determined across the entire elution profile in intervals of 1 sec from static LS measurement using ASTRA software.', 'Micrographs shown in the article are representative images to illustrate the observations. Sample numbers in quantifications are indicated in the figure legends. <xref ref-type="fig" rid="EMS116953-f001">Fig. 1b, g</xref>: Purified proteins were analyzed by gel electrophoresis more than three times. : Purified proteins were analyzed by gel electrophoresis more than three times. <xref ref-type="fig" rid="EMS116953-f002">Fig. 2c</xref>: Condensates were imaged by AFM at least three times, typically with dozens of condensates observed for each experiment. Protein complexes without DNA were imaged at least twice in different buffers with similar results. : Condensates were imaged by AFM at least three times, typically with dozens of condensates observed for each experiment. Protein complexes without DNA were imaged at least twice in different buffers with similar results. <xref ref-type="fig" rid="EMS116953-f002">Fig. 2d</xref>: Quantification is shown for a time course performed once, but the pattern was confirmed at least once independently. : Quantification is shown for a time course performed once, but the pattern was confirmed at least once independently. <xref ref-type="fig" rid="EMS116953-f003">Fig. 3a</xref>: Quantification is shown for one experiment, but the DNA-binding defect of the mutant was confirmed at least twice independently using different substrates. : Quantification is shown for one experiment, but the DNA-binding defect of the mutant was confirmed at least twice independently using different substrates. <xref ref-type="fig" rid="EMS116953-f003">Fig. 3b</xref>: Quantification is shown for one experiment, but the condensation defect of the mutant was confirmed at least twice independently in different conditions. : Quantification is shown for one experiment, but the condensation defect of the mutant was confirmed at least twice independently in different conditions. <xref ref-type="fig" rid="EMS116953-f003">Fig. 3c</xref>: Quantification is shown with data pooled from two cultures. The observation was reproduced at least twice independently. : Quantification is shown with data pooled from two cultures. The observation was reproduced at least twice independently. <xref ref-type="fig" rid="EMS116953-f003">Fig. 3d</xref>: Southern blot analysis was performed with two independent cultures with identical results. : Southern blot analysis was performed with two independent cultures with identical results. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4a</xref>: Co-localization was observed more than three times in different conditions. : Co-localization was observed more than three times in different conditions. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4b</xref>: The pattern was observed at least twice independently. : The pattern was observed at least twice independently. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4c</xref>: Quantification is shown for a time course performed once. : Quantification is shown for a time course performed once. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4d</xref>: The observation was reproduced more than three times. : The observation was reproduced more than three times. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4e</xref>: Quantification is shown for a titration performed once. : Quantification is shown for a titration performed once. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4g</xref>: Quantification is shown for an experiment with four replicates. The experiment was repeated once with similar results. : Quantification is shown for an experiment with four replicates. The experiment was repeated once with similar results. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4h</xref>: Southern blot is shown for a time course performed once. : Southern blot is shown for a time course performed once. <xref ref-type="fig" rid="EMS116953-f004">Fig. 4i</xref>: Quantification is shown with data pooled from two independent cultures. : Quantification is shown with data pooled from two independent cultures. <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1b, g, i</xref>: Observations were reproduced at least once independently. : Observations were reproduced at least once independently. <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1l</xref>: Southern blot is shown for a time course performed once. : Southern blot is shown for a time course performed once. <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1g</xref>: Quantification is shown for an experiment with four replicates. The experiment was repeated once with similar results. : Quantification is shown for an experiment with four replicates. The experiment was repeated once with similar results. <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1n</xref>: The experiment was performed with two independent cultures with identical results. : The experiment was performed with two independent cultures with identical results. <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2a, b</xref>: Titrations were repeated at least once with identical results. : Titrations were repeated at least once with identical results. <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2c, d</xref>: Competition was performed once. : Competition was performed once. <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2e, f</xref>: The observations were reproduced at least twice independently. : The observations were reproduced at least twice independently. <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2g</xref>: Condensates were imaged by AFM at least three times, typically with dozens of condensates observed for each experiment. Protein complexes without DNA were imaged at least twice in different buffers with similar results. : Condensates were imaged by AFM at least three times, typically with dozens of condensates observed for each experiment. Protein complexes without DNA were imaged at least twice in different buffers with similar results. <xref ref-type="fig" rid="EMS116953-f008">Extended Data Fig. 4g, h</xref>: Observation reproduced at least once independently. : Observation reproduced at least once independently. <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6a, c</xref>: Truncation analyses were performed at least twice. : Truncation analyses were performed at least twice. <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6b, d</xref>: Quantifications are shown for one experiment, but the DNA-binding and condensation defects of the mutant was confirmed at least twice independently in different conditions. : Quantifications are shown for one experiment, but the DNA-binding and condensation defects of the mutant was confirmed at least twice independently in different conditions. <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6g</xref>: Experiment were performed with two independent cultures with identical results. : Experiment were performed with two independent cultures with identical results. <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6h</xref>: Patterns were confirmed at least once. : Patterns were confirmed at least once. <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6i</xref>: Time course was performed once. : Time course was performed once. <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6j</xref>: Southern blot analysis was performed with two independent cultures with identical results. : Southern blot analysis was performed with two independent cultures with identical results. <xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7a</xref>: Experiment was performed once. : Experiment was performed once. <xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7c</xref>: Experiment was performed at least twice. : Experiment was performed at least twice. <xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7d</xref>: Pulldown was repeated at least twice independently.: Pulldown was repeated at least twice independently.'], 'EMS116953-f002': ['In electrophoretic mobility shift assays, Rec114–Mei4 and Mer2 bound to 20-, 40-, and 80-bp substrates, with affinity increasing with DNA length (<xref ref-type="fig" rid="EMS116953-f002">Fig. 2a, b</xref>, , <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2a, b</xref>). Preference for longer substrates was confirmed in competition assays (). Preference for longer substrates was confirmed in competition assays (<xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2c, d</xref>). Protein titrations yielded well-shifts with no discrete bands and switch-like transitions from no binding to complete binding within narrow (2 to 4-fold) ranges, suggesting cooperative assembly of higher-order structures (). Protein titrations yielded well-shifts with no discrete bands and switch-like transitions from no binding to complete binding within narrow (2 to 4-fold) ranges, suggesting cooperative assembly of higher-order structures (<xref ref-type="fig" rid="EMS116953-f002">Fig. 2a, b</xref>, , <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2a, b</xref>).).', 'To directly visualize DNA-bound particles, we used atomic force microscopy (AFM). Rec114–Mei4 and Mer2 formed small, relatively homogeneous particles on the mica surface in the absence of DNA, but plasmid DNA caused Rec114–Mei4 and Mer2 to assemble large protein clusters with emanating DNA loops (<xref ref-type="fig" rid="EMS116953-f002">Fig. 2c</xref>, , <xref ref-type="fig" rid="EMS116953-f006">Extended Data Figure 2g</xref>). Most plasmid molecules remained unbound and the surface was devoid of free protein particles, showing that clustering is extremely cooperative. From the sizes (~0.2 μm diameter for Rec114–Mei4 and ~0.4 μm for Mer2), the clusters must contain many hundreds of proteins.). Most plasmid molecules remained unbound and the surface was devoid of free protein particles, showing that clustering is extremely cooperative. From the sizes (~0.2 μm diameter for Rec114–Mei4 and ~0.4 μm for Mer2), the clusters must contain many hundreds of proteins.', '\na, b. Gel shift analysis of Rec114–Mei4 (a) or Mer2 (b) binding to 20- or 40- bp DNA substrates. Quantification is in <xref ref-type="fig" rid="EMS116953-f002">Fig. 2b</xref>. . c, d. Competition assay of Rec114–Mei4 (c) or Mer2 (d) binding to 80 bp radiolabeled DNA (1 nM) in the presence of 20- or 80 bp cold competitor. Fold excess is in nucleotides. Lines are one-phase decay fits. e, f. Binding to plasmid DNA analyzed by native agarose gel electrophoresis. Rec114–Mei4 (e) and Mer2 (f) were titrated with 2 nM plasmid DNA (pUC19) in the presence or absence of 5 mM MgCl2. Rec114–Mei4 complexes bound with roughly similar affinity independently of the presence of Mg2+ (apparent KD ≈ 50–80 nM). Note that the apparent affinity is significantly lower than suggested by the gel shift analyses with radiolabeled substrates presented in panel a and <xref ref-type="fig" rid="EMS116953-f002">Fig. 2a, b</xref> (see apparent affinities in (see apparent affinities in <xref ref-type="fig" rid="EMS116953-f002">Fig. 2</xref> legend). We interpret that this difference is because the proteins coalesce on a small fraction of the plasmid molecules, as illustrated in the cartoon below. Indeed, bound plasmids remained trapped in the wells, which is consistent with cooperative assembly of large nucleoprotein structures. Because each plasmid substrate provides many more binding sites than the short oligonucleotide substrates in panel legend). We interpret that this difference is because the proteins coalesce on a small fraction of the plasmid molecules, as illustrated in the cartoon below. Indeed, bound plasmids remained trapped in the wells, which is consistent with cooperative assembly of large nucleoprotein structures. Because each plasmid substrate provides many more binding sites than the short oligonucleotide substrates in panel a and <xref ref-type="fig" rid="EMS116953-f002">Fig. 2a</xref>, a higher concentration of protein is required to reach complete binding of all of the plasmid molecules. In contrast to Rec114–Mei4, Mer2 showed efficient binding in the absence of Mg, a higher concentration of protein is required to reach complete binding of all of the plasmid molecules. In contrast to Rec114–Mei4, Mer2 showed efficient binding in the absence of Mg2+ in this assay (KD = 30 ± 2 nM) but binding appeared to be considerably inhibited in the presence of Mg2+ (KD ≈ 150 nM), as indicated by the persistence of unbound substrate at high protein concentrations. However, while the electrophoretic mobility of Mer2-bound plasmids decreased steadily as the concentration of Mer2 increased in the absence of Mg2+, no such steady progression was observed when Mg2+ was included. Instead, a minority of bound substrates shifted to a low-mobility species (labeled * in panel f, bottom), indicating that they were occupied by multiple Mer2 complexes. We interpret that, rather than inhibiting DNA binding, Mg2+ promotes cooperativity, in agreement with the fluorescence microscopy analysis (<xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3b</xref>). The difference in migration distance of the plasmid between the +/- Mg). The difference in migration distance of the plasmid between the +/- Mg2+ gels is due to the presence of Mg2+ in the electrophoresis buffer. g. AFM imaging of 12 nM Rec114–Mei4 in the absence (left) or in the presence (right) of 1 nM plasmid DNA (pUC19).'], 'EMS116953-f006': ['\na. Gel shift analysis of Rec114–Mei4 and Mer2 binding to 80 bp DNA substrates (see also <xref ref-type="fig" rid="EMS116953-f006">Extended Data Fig. 2a, b</xref>). ). b. Quantification of gel-shift analyses with 20-, 40- or 80 bp substrates. Error bars are ranges from two independent experiments. Lines are sigmoidal curves fit to the data, except for the 20 bp substrate (smooth spline fits). Apparent affinities of Rec114–Mei4 are: 6 ± 1.4 nM (80 bp, mean and range); 35 ± 1.3 nM (40 bp); ≈ 80 nM (20 bp). Apparent affinities of Mer2 are: 19 ± 1.5 nM (80 bp); 64 ± 15 nM (40 bp); > 400 nM (20 bp). Here and elsewhere, concentrations for Rec114–Mei4 refer to the trimeric complex, but for Mer2 they refer to the monomer. Therefore, the complexes have comparable affinities for DNA if the quaternary units (trimers and tetramers, respectively) are considered. c. AFM imaging of 50 nM Mer2 in the absence (left) or presence (right) of 1 nM plasmid DNA (pUC19). d. Time course of the assembly of Mer2 foci in the presence of plasmid DNA. The x axis indicates the time in solution before plating, upon which DNA is immobilized to the glass slide while soluble protein is still free to diffuse. Quantification is provided of focus numbers and average focus intensity (normalized to the mean at 30 min). Error bars show mean ± SD from 8–10 fields of view (see Source Data for exact n values). For gel source data, see Supplementary Figure 1.'], 'EMS116953-f007': ['Rec114–Mei4 complexes with mScarlet fused to the Rec114 N-terminus yielded bright epifluorescent foci in the presence of DNA, independent of Mg2+ (<xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3a</xref>). eGFP-tagged Mer2 complexes also produced DNA-dependent foci in the presence of Mg). eGFP-tagged Mer2 complexes also produced DNA-dependent foci in the presence of Mg2+, but gave only diffuse fluorescence signal without Mg2+ (<xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3b</xref> and and <xref ref-type="fig" rid="EMS116953-f006">2e, f</xref>).).', 'We asked whether Rec114–Mei4 and Mer2 condensates display behaviors typical of phase-separated systems, using fluorophore-conjugated complexes (<xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3c-f</xref>). The molecular crowding agent polyethylene glycol (5% PEG-8000) dramatically increased condensate intensity for both Rec114–Mei4 and Mer2 (). The molecular crowding agent polyethylene glycol (5% PEG-8000) dramatically increased condensate intensity for both Rec114–Mei4 and Mer2 (<xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3g, h</xref>). Protein titrations revealed complex, sometimes counter-intuitive behaviors, including a decrease in focus numbers with increasing protein concentrations. These behaviors likely reflect balances between nucleation, growth, and collapse of the condensates (see legend to ). Protein titrations revealed complex, sometimes counter-intuitive behaviors, including a decrease in focus numbers with increasing protein concentrations. These behaviors likely reflect balances between nucleation, growth, and collapse of the condensates (see legend to <xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3g, h</xref>).).'], 'EMS116953-f008': ['Condensation was inhibited by high salt, suggesting dependency on electrostatic interactions (<xref ref-type="fig" rid="EMS116953-f008">Extended Data Fig. 4c, e</xref>). Competition experiments revealed preferential incorporation of larger DNA molecules, consistent with multivalency of the substrate driving condensation (). Competition experiments revealed preferential incorporation of larger DNA molecules, consistent with multivalency of the substrate driving condensation (<xref ref-type="fig" rid="EMS116953-f008">Extended Data Fig. 4g-h</xref>).).', 'Pre-assembled condensates were almost completely dissolved when challenged with DNase I or 500 mM NaCl in the absence of PEG, showing that they are reversible (<xref ref-type="fig" rid="EMS116953-f008">Extended Data Fig. 4a, b</xref>). However, in the presence of PEG, about half of the condensate-associated Rec114–Mei4 fluorescence signal resisted challenge. Reversibility of Rec114–Mei4 condensates decreased over time, accentuated by molecular crowding (). However, in the presence of PEG, about half of the condensate-associated Rec114–Mei4 fluorescence signal resisted challenge. Reversibility of Rec114–Mei4 condensates decreased over time, accentuated by molecular crowding (<xref ref-type="fig" rid="EMS116953-f008">Extended Data Fig. 4d</xref>). After a short assembly time, Mer2 condensates were unable to resist dissolution whether PEG was present or not, but longer incubation times with PEG allowed Mer2 as well to form resistant foci (). After a short assembly time, Mer2 condensates were unable to resist dissolution whether PEG was present or not, but longer incubation times with PEG allowed Mer2 as well to form resistant foci (<xref ref-type="fig" rid="EMS116953-f008">Extended Data Fig. 4b, 4f</xref>). These results suggest that condensates of Rec114–Mei4 and, to a lesser extent, Mer2 may spontaneously mature into irreversible, perhaps gel-like, structures, as has been observed for other systems). These results suggest that condensates of Rec114–Mei4 and, to a lesser extent, Mer2 may spontaneously mature into irreversible, perhaps gel-like, structures, as has been observed for other systems35,37–39.'], 'EMS116953-f009': ['Several scenarios might account for condensate assembly, differing as to whether growth results principally from fusion of existing condensates or from incorporation of soluble protein that diffuses in and out of condensates (<xref ref-type="fig" rid="EMS116953-f009">Extended Data Fig. 5a</xref>). To distinguish between these possibilities, we immobilized DNA at varied time points by spreading assembly reactions on glass slides. Plating should prevent focus fusion, but not exchange of condensates with soluble protein pools. Images were captured at a late time point (>1 hour), so the time variable is the period that the DNA is free in solution before constraint. If foci grow by addition from soluble protein pools, DNA immobilization should have no effect and all reactions should be identical. In contrast, if fusion drives growth, focus numbers should decrease over time while their intensities increase. The latter outcome was observed for both Rec114–Mei4 and Mer2 (). To distinguish between these possibilities, we immobilized DNA at varied time points by spreading assembly reactions on glass slides. Plating should prevent focus fusion, but not exchange of condensates with soluble protein pools. Images were captured at a late time point (>1 hour), so the time variable is the period that the DNA is free in solution before constraint. If foci grow by addition from soluble protein pools, DNA immobilization should have no effect and all reactions should be identical. In contrast, if fusion drives growth, focus numbers should decrease over time while their intensities increase. The latter outcome was observed for both Rec114–Mei4 and Mer2 (<xref ref-type="fig" rid="EMS116953-f002">Fig. 2d</xref>, , <xref ref-type="fig" rid="EMS116953-f009">Extended Data Fig. 5b</xref>). Moreover, no fluorescence recovery was seen after photobleaching of immobilized foci (). Moreover, no fluorescence recovery was seen after photobleaching of immobilized foci (<xref ref-type="fig" rid="EMS116953-f009">Extended Data Fig. 5c</xref>). These findings thus establish that fusion can occur. However, they do not exclude exchange with soluble pools being important under other conditions, including ). These findings thus establish that fusion can occur. However, they do not exclude exchange with soluble pools being important under other conditions, including in vivo (Supplementary Discussion 2).'], 'EMS116953-f010': ['The Rec114 C-terminal domain is necessary and sufficient for DNA binding (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6a</xref>). Alanine substitution of four basic residues in this domain yielded a Rec114–Mei4 complex (“4KR”) with reduced DNA binding (). Alanine substitution of four basic residues in this domain yielded a Rec114–Mei4 complex (“4KR”) with reduced DNA binding (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6b</xref>). Similarly, alanine substitutions in a conserved basic patch towards the C-terminus of Mer2 (“KRRR”) yielded a DNA-binding defective mutant (). Similarly, alanine substitutions in a conserved basic patch towards the C-terminus of Mer2 (“KRRR”) yielded a DNA-binding defective mutant (<xref ref-type="fig" rid="EMS116953-f003">Fig. 3a</xref>, , <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6c</xref>). As expected if multivalent protein-DNA interactions contribute to condensation, both the Rec114-4KR and the Mer2-KRRR mutant proteins showed strongly reduced focus formation ). As expected if multivalent protein-DNA interactions contribute to condensation, both the Rec114-4KR and the Mer2-KRRR mutant proteins showed strongly reduced focus formation in vitro (<xref ref-type="fig" rid="EMS116953-f003">Fig. 3b</xref>, , <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6d</xref>). However, fluorescently tagged Mer2-KRRR protein was incorporated into pre-assembled Mer2 condensates as efficiently as wild-type Mer2 (). However, fluorescently tagged Mer2-KRRR protein was incorporated into pre-assembled Mer2 condensates as efficiently as wild-type Mer2 (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6e</xref>), indicating that the protein-protein interactions important for condensation are retained in the mutant.), indicating that the protein-protein interactions important for condensation are retained in the mutant.'], 'EMS116953-f003': ['\nIn vivo, the mutant proteins formed much fewer foci than wild type upon immunofluorescent staining of chromosome spreads (<xref ref-type="fig" rid="EMS116953-f003">Fig. 3c</xref>, , <xref ref-type="fig" rid="EMS116953-f010">Extended Data Figure 6f</xref>))11,12,15. This could not be attributed to protein destabilization because immunoblotting signal was not reduced compared to wild type (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6g</xref>) and purified recombinant proteins did not show increased sensitivity to digestion with trypsin () and purified recombinant proteins did not show increased sensitivity to digestion with trypsin (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6h</xref>). In fact, Mer2-KRRR mutant protein accumulated and persisted longer during meiosis (). In fact, Mer2-KRRR mutant protein accumulated and persisted longer during meiosis (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6i</xref>). The Mer2-KRRR protein also had faster electrophoretic mobility than wild type, probably because it failed to become phosphorylated. It therefore appears that DNA-binding is a prerequisite for Mer2 phosphorylation, which is known to promote turnover of the protein). The Mer2-KRRR protein also had faster electrophoretic mobility than wild type, probably because it failed to become phosphorylated. It therefore appears that DNA-binding is a prerequisite for Mer2 phosphorylation, which is known to promote turnover of the protein15.', 'Both mutations also conferred defects in meiotic DSB formation when assayed locally by Southern blotting at a DSB hotspot (<xref ref-type="fig" rid="EMS116953-f003">Fig. 3d</xref>, , <xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6j</xref>) or globally by quantification of Spo11-oligo complexes () or globally by quantification of Spo11-oligo complexes (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6k</xref>). These DSB defects caused low spore viability (). These DSB defects caused low spore viability (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6l</xref>). In conclusion, the DNA-binding activities of Rec114–Mei4 and Mer2 are essential for DNA-driven condensation ). In conclusion, the DNA-binding activities of Rec114–Mei4 and Mer2 are essential for DNA-driven condensation in vitro and in vivo and for their DSB-promoting activity, suggesting in turn that condensation itself is important for these proteins’ biological functions.'], 'EMS116953-f004': ['\nIn vivo, Rec114, Mei4 and Mer2 form partially overlapping foci11,12 and yield coincident ChIP signals16. We therefore tested whether they function together as joint condensates by mixing fluorescent Rec114–Mei4 and Mer2 either before or after DNA-driven condensation (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4a, b</xref>). Premixing the proteins led to joint foci with essentially perfect overlap (). Premixing the proteins led to joint foci with essentially perfect overlap (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4a</xref>). Colocalization was evident even with a large excess of DNA, thus joint foci were not from fortuitous overlap of independent assemblies on limiting numbers of substrate molecules (). Colocalization was evident even with a large excess of DNA, thus joint foci were not from fortuitous overlap of independent assemblies on limiting numbers of substrate molecules (<xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7a, b</xref>).).', 'Next, we asked if preassembled Rec114–Mei4 and Mer2 condensates can mingle. No overlap was seen between Rec114–Mei4 and Mer2 foci when preformed nucleoprotein condensates were mixed and then immediately plated (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4b</xref>, top). In contrast, when the mixtures were incubated for 20 minutes prior to plating, all of the Mer2 condensates overlapped with a Rec114–Mei4 focus (, top). In contrast, when the mixtures were incubated for 20 minutes prior to plating, all of the Mer2 condensates overlapped with a Rec114–Mei4 focus (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4b</xref>, bottom). The lack of overlap in samples that were plated immediately rules out the joint foci arising via a pool of soluble protein under these conditions, so we infer that existing condensates can fuse., bottom). The lack of overlap in samples that were plated immediately rules out the joint foci arising via a pool of soluble protein under these conditions, so we infer that existing condensates can fuse.', 'To further test this, we performed a time course experiment with different concentrations of Rec114–Mei4 (17 and 35 nM) (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4c</xref>). As shown above, the lower concentration yields more foci (). As shown above, the lower concentration yields more foci (<xref ref-type="fig" rid="EMS116953-f007">Extended Data Fig. 3g</xref>). If the likelihood of cluster fusion reflects contact probability, the rate of forming joint foci would be expected to be higher with the lower concentration of Rec114–Mei4. This was indeed the case: the halftime for detecting joint foci was 2.0 ± 0.3 min for 17 nM vs. 3.3 ± 0.6 min for 35 nM (). If the likelihood of cluster fusion reflects contact probability, the rate of forming joint foci would be expected to be higher with the lower concentration of Rec114–Mei4. This was indeed the case: the halftime for detecting joint foci was 2.0 ± 0.3 min for 17 nM vs. 3.3 ± 0.6 min for 35 nM (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4c</xref>, right panel)., right panel).', 'When fluorescently labeled Spo11 core complexes bound to DNA were mixed with preassembled RMM-DNA condensates, core complex signal overlapped with RMM foci (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4d</xref>). Recruitment of the core complex depended on Mer2 (). Recruitment of the core complex depended on Mer2 (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4e</xref>, , <xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig 8a</xref>). Rec114-Mei4 was also required when Mer2 was present at low concentration (25 nM), but was dispensable at high Mer2 concentration (100 nM) (). Rec114-Mei4 was also required when Mer2 was present at low concentration (25 nM), but was dispensable at high Mer2 concentration (100 nM) (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4f</xref>, , <xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig. 8b</xref>).).', '\nAuthor contributions: C.C.B. and S.K. designed the study and supervised the research; C.C.B. carried out all experiments except as noted; S.P. performed yeast-two-hybrid experiments (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4g</xref>, , <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig 1k</xref>, , <xref ref-type="fig" rid="EMS116953-f012">8e</xref>) and assisted C.C.B with the generation of expression constructs, virus preparations and protein purifications; J.W. performed SEC-MALS analyses of mutant protein constructs () and assisted C.C.B with the generation of expression constructs, virus preparations and protein purifications; J.W. performed SEC-MALS analyses of mutant protein constructs (<xref ref-type="fig" rid="EMS116953-f001">Fig. 1j</xref>, , <xref ref-type="fig" rid="EMS116953-f005">Extended Data Fig. 1h, j</xref>) and W.X. performed FRAP experiments () and W.X. performed FRAP experiments (<xref ref-type="fig" rid="EMS116953-f009">Extended Data Fig. 5c</xref>), under supervision of D.J.P.; C.O. performed MBP pulldown (), under supervision of D.J.P.; C.O. performed MBP pulldown (<xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7d</xref>) and trypsin proteolysis experiments () and trypsin proteolysis experiments (<xref ref-type="fig" rid="EMS116953-f010">Extended Data Fig. 6h</xref>) and D.D. performed the condensate mixing experiments in ) and D.D. performed the condensate mixing experiments in <xref ref-type="fig" rid="EMS116953-f010">Extended Data Figs. 6e</xref> and and <xref ref-type="fig" rid="EMS116953-f012">8f</xref>, under supervision of C.C.B.; C.C.B. and S.K. wrote the paper with input from the other authors; C.C.B., D.J.P. and S.K. secured funding., under supervision of C.C.B.; C.C.B. and S.K. wrote the paper with input from the other authors; C.C.B., D.J.P. and S.K. secured funding.'], 'EMS116953-f011': ['We also asked whether soluble protein can be recruited into condensates. Here, Rec114–Mei4 or Mer2 condensates were assembled, then the other protein was added in solution and the mixtures were immediately plated to prevent subsequent fusion. Preassembled Rec114–Mei4 foci incorporated Mer2 and vice versa (<xref ref-type="fig" rid="EMS116953-f011">Extended Data Fig. 7c</xref>), showing that condensates provide nucleation sites for the partner complexes.), showing that condensates provide nucleation sites for the partner complexes.'], 'EMS116953-f012': ['Rec114 interacts with Rec102 and Rec104 in Y2H assays12,17. Consistent with these interactions mediating recruitment of core complexes to condensates, an excess of Rec102–Rec104 subcomplexes was able to outcompete the full core complex (<xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig. 8c, d</xref>).).', 'We mapped the core complex interacting domain of Rec114 by Y2H truncation analysis (<xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig. 8e</xref>). Deleting ~50 amino acids from either the N or C termini of Rec114 abolished interaction with both Rec102 and Rec104, but deleting the disordered region did not (residues 152-377, see ). Deleting ~50 amino acids from either the N or C termini of Rec114 abolished interaction with both Rec102 and Rec104, but deleting the disordered region did not (residues 152-377, see <xref ref-type="fig" rid="EMS116953-f001">Fig. 1a</xref>). Altering conserved residues in the N-terminal PH domain identified a mutation (HLS, H39A/L40A/S41A) that specifically reduced interactions with Rec102 and Rec104 but did not affect the interaction with Mei4 or wild-type Rec114 (). Altering conserved residues in the N-terminal PH domain identified a mutation (HLS, H39A/L40A/S41A) that specifically reduced interactions with Rec102 and Rec104 but did not affect the interaction with Mei4 or wild-type Rec114 (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4g</xref>), or the ability to make comingled RMM condensates ), or the ability to make comingled RMM condensates in vitro (<xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig. 8f</xref>). The ). The rec114-HLS mutant was defective for DSB formation (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4h</xref>) and gave inviable spores () and gave inviable spores (<xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig. 8g</xref>) despite the mutant protein being expressed at normal levels () despite the mutant protein being expressed at normal levels (<xref ref-type="fig" rid="EMS116953-f012">Extended Data Fig. 8h</xref>) and forming normal-looking chromatin-associated foci () and forming normal-looking chromatin-associated foci (<xref ref-type="fig" rid="EMS116953-f004">Fig. 4i</xref>).).'], 'EMS116953-f013': ['Meiotic chromosomes form chromatin loops extending from a linear protein axis and it is thought that the DSB machinery assembled on axes captures and breaks loop DNA5,16. We propose that recruitment of Spo11 and regulatory components to RMM clusters forms the basis of this tethered loop-axis configuration (<xref ref-type="fig" rid="EMS116953-f013">Extended Data Fig. 9a</xref> and and Supplementary Discussion 4).', 'This model has important implications. First, each cluster likely recruits multiple core complexes, so it may explain how core complexes can be induced to dimerize10 and how Spo11 can sometimes cut the same chromatid more than once40,41. RMM condensates may also provide platforms that display co-oriented arrays of Spo11 complexes, which could account for observed 10-bp periodicity in the spacing between Spo11 cuts41 (<xref ref-type="fig" rid="EMS116953-f013">Extended Data Fig. 9a</xref> and and Supplementary Discussion 4).', 'Second, RMM condensates may explain two previously unclear aspects of DSB patterning: hotspot competition, where strong hotspots reduce activity of neighboring hotspots, and DSB interference, in which the DSB-responsive kinase Tel1 inhibits additional DSBs near an existing DSB4 (<xref ref-type="fig" rid="EMS116953-f013">Extended Data Fig. 9b</xref> and and Supplementary Discussion 5). Hotspot competition could arise if nucleation of a condensate plus highly cooperative assembly locally depletes Rec114–Mei4 and Mer2 proteins, reducing the probability of another nucleation event. After a DSB is made, Tel1 may suppress additional DSBs nearby by acting both within and between adjacent condensates.', 'Third, the condensates may regulate DSB repair, for example by tethering and controlling the broken DNA ends and/or by nucleating formation of the recombination nodules where repair takes place (<xref ref-type="fig" rid="EMS116953-f013">Extended Data Fig. 9c</xref> and and Supplementary Discussion 6).']}
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DNA-driven condensation assembles the meiotic DNA break machinery
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Nature
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1617260400
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With the drying process, the water activity and moisture content of the foods are reduced, so the growth of microorganisms in the foods is largely prevented/postponed. But low-aw foods should not be considered sterile they can be contaminated by fungi and other contaminants during the drying process under unhygienic conditions. If drying is not done to a sufficient degree of moisture during food processing and storage, where dried foods are processed, sometimes the minimum value is reached for the growth of microorganisms. In dry foods, some pathogens, yeast and molds can continue to grow during storage, transport and transportation until the sale and they causing spoilage. They can even cause health problems if enough pathogen or spore cells remain viable. Considering this situation today, it is attempted to obtain high-quality dried foods with good microbiologically and chemically properties. For this purpose, various drying methods have been developed. Most studies suggest that when foods are pre-treated with the ascorbic acid or sodium metabisulfite or applied with various combined methods such as UV irradiation, supercritical carbon dioxide (SCO), low-pressure superheated steam drying (LPSSD), and infrared (IR) drying, they can be effective on inactivation of microorganisms. We have reviewed in this study how these methods made dried products efficient of microbial inactivation and microbiologically safe.
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[] |
other
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PMC8016751
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99
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[
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Nature. 2021 Apr 1; 592(7852):144-149
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DARPP-32 interacts with IGF1R.A) Two-way co-immunoprecipitation and Western blot analysis demonstrates the interaction of DARPP-32 and IGF1R. B) in situ proximity ligation assay (PLA) showing co-localization of DARPP-32 with IGF1R (red dots, left panel, bottom row), no ligation signals were seen in controls. C) Using immunoprecipitation of p-SRC and IGF1R, the IGF1R-p-SRC interaction was examined following DARPP-32 siRNA knockdown in MKN45 cells.
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multiple panels: images & plots
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"Immuno Assays"
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[{'image_id': 'nihms-1522657-f0006', 'image_file_name': 'nihms-1522657-f0006.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0006.jpg', 'caption': 'DARPP-32 regulates IGFR-SRC pathway.A) Immunofluorescence analysis of p-STAT3 (green) and DARPP-32 (red) in gastric organoids established from wild-type, TFF1 KO, DP KO and TFF1 KO/DP KO mice. B) H&E staining of representative histological features of gastric mucosa from 2–4 month old mice; wild-type (n=9), TFF1 KO (n=14), DP KO (n=10), and TFF1 KO/DP KO (n=9). Lack of dysplastic gastric glands in wild-type, DP KO, and TFF1 KO/DP KO mice; dysplastic glands were observed in TFF1 KO mice (upper panel). Representative immune-histochemical staining of DARPP-32 and p-STAT3 in gastric tissues from wild-type, TFF1 KO, DP KO, and TFF1 KO/DP KO 2–4 month old mice (lower panel). C) Western blot analysis of IGFR-SRC pathway in 2–4 month old mice. D) Histological changes were examined in TFF1 KO and TFF1 KO/DP KO old mice. Wild-type mice (n=21), TFF1 KO (n=54), DP KO (n=11), and TFF1 KO/DP KO (n=37) mice were evaluated for chronic inflammation and dysplasia. Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test.', 'hash': '8ed9bb7537f90f1dac81b9754a9d446c1789719ac05292577bb6ebe60e98ad9c'}, {'image_id': 'nihms-1522657-f0001', 'image_file_name': 'nihms-1522657-f0001.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0001.jpg', 'caption': 'DARPP-32 enhances activation of p-IGF1R.A-B) p-IGF1R, p-STAT3, and DARPP-32 protein levels were determined by Western blot analysis in AGS cells with stable expression of DARPP-32 (DP32) or MKN45 cells with siRNA knockdown of DARPP-32. C-D) Luciferase reporter assay for STAT3-luc following siRNA knockdown of IGF1R in cells with stable expression of DARPP-32 (DP32) or DARPP-32 siRNA knockdown. Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test.', 'hash': '4c1fb62cac06955e9c4d8816b9607221a3fc60614a03cdc4fe54514ffa3d1d18'}, {'image_id': 'nihms-1522657-f0007', 'image_file_name': 'nihms-1522657-f0007.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0007.jpg', 'caption': 'Immunohistochemistry for DARPP-32 and p-STAT3 in human gastric tissuesA) Representative images of immune-histochemical staining of DARPP-32 and p-STAT3 in tissue sections from human gastric mucosa with normal histology (NG, n=108) and adenocarcinoma (AdCa, n=108); original magnification ×20. B-C) The graphs summarize the immunohistochemical staining results on gastric tissue microarrays. D) Survival analysis of DARPP-32 and STAT3 mRNA expression in gastric cancer patients by the Kaplan-Meier survival curve, n=882, following analysis of public data online (http://kmplot.com/analysis/index.php?p=service) [25]. E) A diagram depicting the role of DARPP-32 in activation of STAT3 in gastric cancer cells. In summary, DARPP-32 interacts with IGF1R and promotes IGF1R and SRC phosphorylation, allowing sustained IL6-mediated phosphorylation and activation of STAT3. The translocation of p-STAT3 into the nucleus initiates transcriptional regulation of downstream target genes that regulate cell proliferation, transformation.', 'hash': '2dbcba54c97143bbd0d9869468f37f06153b8f582cda89ee1bd1132e64594878'}, {'image_id': 'nihms-1522657-f0004', 'image_file_name': 'nihms-1522657-f0004.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0004.jpg', 'caption': 'DARPP-32 induces STAT3 activity through SRC signaling pathway.A) Western blots analysis of p-SRC, p-STAT3, and DARPP-32 in AGS cells stably expressing DARPP-32 (DP32), using pcDNA-DARPP-32, following knockdown of SRC by SRC siRNA. B) p-SRC, p-STAT3, and DARPP-32 protein levels were determined by Western blot analysis in AGS cells stably expressing DARPP-32 (DP32), following treatment with Dasatinib (10 μM) or vehicle. C) Luciferase reporter assays for STAT3-luc were performed following treatment with Dasatinib (10 μM) in AGS cells stably expressing DARPP-32 (DP32). Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test. D) p-SRC, p-STAT3, and DARPP-32 protein levels were determined by Western blot analysis in DARPP-32 siRNA knockdown MKN45 cells following treatment with Dasatinib (10 μM) or vehicle. E) Luciferase reporter assay for STAT3-luc were performed following Dasatinib (10 μM) treatment in DARPP-32 siRNA knockdown MKN45 cells. Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test. F) Immunofluorescence analysis using AGS cells, following overexpression of DARPP-32 (red), demonstrates an increase in expression of p-SRC (green) in DARPP-32-expressing AGS cells (left panel). Immunofluorescence analysis in MKN45 cells, following DARPP-32 siRNA knockdown, demonstrates a decrease of p-SRC in DARPP-32 siRNA/MKN45 cells; DARPP-32 (red), p-STAT3 (green) (right panel).', 'hash': 'dddd069300c6f633e74a0fc0861a91f21cb5d379fcbac8eb76e209e2801b04cd'}, {'image_id': 'nihms-1522657-f0003', 'image_file_name': 'nihms-1522657-f0003.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0003.jpg', 'caption': 'DARPP-32 regulates IGF1R-mediated STAT3 signaling pathway in gastric cancer cells:A) Western blots analysis of IGF1R, p-IGF1R, SRC, and p-SRC in AGS cells stably expressing DARPP-32 (DP32), following IGF1R siRNA knockdown. B) p-IGF1R, SRC, and p-SRC protein levels were determined by Western blot analysis in MKN45 cells, following DARPP-32 siRNA knockdown and treatment with OSI-096 (2 μg/ml) or vehicle. C-D) Luciferase reporter assay for STAT3-luc following treatment with OSI-096 (2 μg/ml) in AGS cells stably expressing DARPP-32 (DP32), using pcDNA-DARPP-32 or DARPP-32 siRNA knockdown in MKN45 cells. Statistical significance in all panels was calculated by 1-way ANOVA, followed by the student’s t test.', 'hash': 'fd6668a69eb7288205e4cd6d12d21da97b14faceb8ae777209d36c36c21503fb'}, {'image_id': 'nihms-1522657-f0002', 'image_file_name': 'nihms-1522657-f0002.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0002.jpg', 'caption': 'STAT3 targeted genes mRNA expression regulated by DARPP-32.A-D) The qRT-PCR analysis of IL6, c-MYC, CXCL3 and IL17 expression was performed in AGS cells, following transient expression of DARPP-32 (DP32), using pcDNA-DARPP-32 (AGS) or DARPP-32 shRNA knockdown in MKN45 cells. Statistical significance in all panels was calculated by 1-way ANOVA, followed by the student’s t test.', 'hash': '1cc9bb598c9ffddccf911c20f3beee7f32b3641f16921f277b6e97d66e9b5ba3'}, {'image_id': 'nihms-1522657-f0005', 'image_file_name': 'nihms-1522657-f0005.jpg', 'image_path': '../data/media_files/PMC6639157/nihms-1522657-f0005.jpg', 'caption': 'DARPP-32 interacts with IGF1R.A) Two-way co-immunoprecipitation and Western blot analysis demonstrates the interaction of DARPP-32 and IGF1R. B) in situ proximity ligation assay (PLA) showing co-localization of DARPP-32 with IGF1R (red dots, left panel, bottom row), no ligation signals were seen in controls. C) Using immunoprecipitation of p-SRC and IGF1R, the IGF1R-p-SRC interaction was examined following DARPP-32 siRNA knockdown in MKN45 cells.', 'hash': 'cad1fc66f3fa5833d249556c8ed783eca6ad6bd9506c1902de7d7600a0b2f9e9'}]
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{'nihms-1522657-f0001': ['Activation of STAT3 transcription network has been reported in early stages of both human and mouse gastric cancer8. Overexpression of DARPP-32 has been noted as an early step in gastric tumorigenesis25. Therefore, we carried out experiments to determine if DARPP-32 played a role in the activation of STAT3. Transient expression of DARPP-32 in AGS cells, with low endogenous levels, led to an increase in p-STAT3 (Y705) expression (<xref rid="nihms-1522657-f0001" ref-type="fig">Figure 1A</xref>). In contrast, knockdown of endogenous DARPP-32 expression in MKN-45 cells resulted in opposite effects (). In contrast, knockdown of endogenous DARPP-32 expression in MKN-45 cells resulted in opposite effects (<xref rid="nihms-1522657-f0001" ref-type="fig">Figure 1B</xref>). We also found that DARPP-32 expression leads to a remarkable increase in p-IGF1R, whereas its knockdown results in complete abrogation of p-IGF1R protein levels (). We also found that DARPP-32 expression leads to a remarkable increase in p-IGF1R, whereas its knockdown results in complete abrogation of p-IGF1R protein levels (<xref rid="nihms-1522657-f0001" ref-type="fig">Figure 1A&1B</xref>). Of note, knockdown of IGF1R abrogated DARPP-32-induced activation of STAT3 reporter (). Of note, knockdown of IGF1R abrogated DARPP-32-induced activation of STAT3 reporter (<xref rid="nihms-1522657-f0001" ref-type="fig">Figure 1C</xref>, p < 0.01). Knockdown of endogenous IGF1R or DARPP-32 resulted in a similar reduction of STAT3 reporter activity levels, as compared to controls (, p < 0.01). Knockdown of endogenous IGF1R or DARPP-32 resulted in a similar reduction of STAT3 reporter activity levels, as compared to controls (<xref rid="nihms-1522657-f0001" ref-type="fig">Figure 1D</xref>, p < 0.01)., p < 0.01).'], 'nihms-1522657-f0002': ['To confirm activation of STAT3, analysis of the mRNA expression levels of STAT3 target genes demonstrated a significant increase in mRNA levels of IL6 (p<0.01), c-MYC (p<0.01), CXCL3 (p<0.01) and IL17 (p<0.001) in the DARPP-32 overexpression AGS cells, as compared with control (<xref rid="nihms-1522657-f0002" ref-type="fig">Figure 2A-2D</xref>). In contrast, knockdown of endogenous DARPP-32 expression in MKN-45 cells resulted in opposite effects (). In contrast, knockdown of endogenous DARPP-32 expression in MKN-45 cells resulted in opposite effects (<xref rid="nihms-1522657-f0002" ref-type="fig">Figure 2A-2D</xref>), confirming that DARPP-32 was required for expression of these STAT3 target genes. Next, we ruled out GP130 as a downstream effector of DARPP-32 as Western blot data did not reveal notable changes in phospho- or total GP130, following DARPP-32 overexpression (), confirming that DARPP-32 was required for expression of these STAT3 target genes. Next, we ruled out GP130 as a downstream effector of DARPP-32 as Western blot data did not reveal notable changes in phospho- or total GP130, following DARPP-32 overexpression (Supplementary figure S1A-S1C). Furthermore, immunoprecipitation and Western blot suggested that DARPP-32 does not interact with GP130 (Supplementary figure S1D), implying that DARPP-32 regulates STAT3 downstream of the IL6R-GP130 complex.'], 'nihms-1522657-f0003': ['To confirm the role of DARPP-32-IGF1R axis on STAT3 phosphorylation, we performed knockdown of endogenous IGF1R in AGS cells stably expressing DARPP-32. This led to a notable reduction of IGF1R, p-SRC and p-STAT3, as compared to controls cells (<xref rid="nihms-1522657-f0003" ref-type="fig">Figure 3A</xref>), confirming the integrity of this axis in regulating STAT3. Similar results were obtained by using the IGF1R inhibitor OSI-096 (2 μg/ml) and knocking-down endogenous DARPP-32 in MKN45 cells (), confirming the integrity of this axis in regulating STAT3. Similar results were obtained by using the IGF1R inhibitor OSI-096 (2 μg/ml) and knocking-down endogenous DARPP-32 in MKN45 cells (<xref rid="nihms-1522657-f0003" ref-type="fig">Figure 3B</xref>). STAT3 luciferase reporter assay results, using the same conditions as in ). STAT3 luciferase reporter assay results, using the same conditions as in <xref rid="nihms-1522657-f0003" ref-type="fig">3A & 3B</xref>, were in agreement with the aforementioned data (, were in agreement with the aforementioned data (<xref rid="nihms-1522657-f0003" ref-type="fig">Figure 3C&3D</xref>).).'], 'nihms-1522657-f0004': ['Furthermore, we confirmed the role of SRC in regulating DARPP-32-mediated STAT3 activity. Knockdown of endogenous SRC in AGS-pcDNA or AGS-DARPP-32 cells resulted in a decrease in p-SRC and p-STAT3 levels, as compared to controls cells (<xref rid="nihms-1522657-f0004" ref-type="fig">Figure 4A</xref>). Similarly, the use of SRC inhibitor Dasatinib (10 μM) blocked the DARPP-32-induced p-SRC, and p-STAT3, as compared to controls cells (). Similarly, the use of SRC inhibitor Dasatinib (10 μM) blocked the DARPP-32-induced p-SRC, and p-STAT3, as compared to controls cells (<xref rid="nihms-1522657-f0004" ref-type="fig">Figure 4B & 4C</xref>, p<0.01). The use of Dasatinib alone or in combination with knockdown of DARPP-32 resulted in a significant decrease in p-STAT3 (Y705) and its reporter activity (, p<0.01). The use of Dasatinib alone or in combination with knockdown of DARPP-32 resulted in a significant decrease in p-STAT3 (Y705) and its reporter activity (<xref rid="nihms-1522657-f0004" ref-type="fig">Figure 4D&4E</xref>, p < 0.01). There were no significant changes in , p < 0.01). There were no significant changes in SRC and IGF1R mRNA levels following overexpression or knockdown of DARPP-32 (Supplementary figure S2). Immunofluorescence analysis confirmed these findings by showing high levels of p-SRC (Y416) expression in AGS-DARPP-32 cells that were markedly reduced following knockdown of endogenous DARPP-32 (<xref rid="nihms-1522657-f0004" ref-type="fig">Figure 4F</xref>).).'], 'nihms-1522657-f0005': ['We next utilized dual co-immunoprecipitation to investigate whether DARPP-32 enhances STAT3 activity through protein-protein interaction with IGF1R. We found that DARPP-32 co-exists with IGF1R (<xref rid="nihms-1522657-f0005" ref-type="fig">Figure 5A</xref>), but not with SRC (), but not with SRC (Supplementary figure S3A-S3B). We used in situ proximity ligation assay (PLA) assay to test interaction between endogenous DARPP-32 and IGF1R. We detected a positive ligation, visualized by red immunofluorescence signals, indicative of their close interaction in MKN-45 cells (<xref rid="nihms-1522657-f0005" ref-type="fig">Figure 5B</xref>). These signals were absent in the negative control as well as in PLA control using single DARPP-32 or IGF1R immunofluorescence. These results indicated that DARPP-32 and IGF1R bind to each other and co-exist in the same protein complex, a step that is essential for constitutive phosphorylation of IGF1R (Y1135). Because SRC is a downstream substrate for IGF1R). These signals were absent in the negative control as well as in PLA control using single DARPP-32 or IGF1R immunofluorescence. These results indicated that DARPP-32 and IGF1R bind to each other and co-exist in the same protein complex, a step that is essential for constitutive phosphorylation of IGF1R (Y1135). Because SRC is a downstream substrate for IGF1R9, we also investigated if DARPP-32 can also enhance the interaction between IGF1R and SRC to facilitate SRC phosphorylation. The co-immunoprecipitation experiments showed that knockdown DARPP-32 can not decrease IGF1R-SRC interaction (Supplementary Figure S3C), but decreases the interaction between IGF1R and p-SRC (<xref rid="nihms-1522657-f0005" ref-type="fig">Figure 5C</xref>), which is in agreement with the reduced IGF1R phosphorylation, upstream of SRC, following knockdown of DARPP-32 (), which is in agreement with the reduced IGF1R phosphorylation, upstream of SRC, following knockdown of DARPP-32 (<xref rid="nihms-1522657-f0003" ref-type="fig">Figure 3B</xref>).).'], 'nihms-1522657-f0006': ['Using organoid tissue cultures from antral stomach regions, we demonstrated that organoids from the TFF1 KO mice have higher p-IGF1R expression and p-STAT3 nuclear immunostaining than in the DP KO and TFF1 KO/DP KO groups (<xref rid="nihms-1522657-f0006" ref-type="fig">Figure 6A</xref>, , supplementary Figure 6). To confirm activation of STAT3, examination of the STAT3 target genes mRNA expression levels demonstrated a significant increase in mRNA levels of Il6 (p<0.01), c-Myc (p<0.01), Cxcl3 (p<0.01) and Il17 (p<0.001) in the TFF1 KO mice, as compared with wild-type mice. On the other hand, the TFF1 KO/DP KO gastric tissues demonstrated expression levels comparable to wild-type mice (Supplementary figure S4A-S4D), confirming that DARPP-32 was required for expression of these STAT3 target genes. Gross pathology of the stomach revealed nodular mucosa in the glandular antropyloric of the stomach in TFF1 KO mice at the age of 3–4 months, whereas wild-type, DP KO and TFF1 KO/DP KO mice had no visual lesions (Supplementary figure S5). H&E staining of representative histological features of gastric mucosa showed non-dysplastic gastric glands in wild-type, DP KO and TFF1 KO/DP KO mice, whereas dysplastic glands were observed in TFF1 KO mice (<xref rid="nihms-1522657-f0006" ref-type="fig">Figure 6B</xref>). This suggested that DARPP-32 was required for tumorigenesis at an early age in the TFF1 KO mice. Immunohistochemistry (IHC) staining of p-STAT3 (Y705) and DARPP-32 showed that gastric tissues from the TFF1 KO/DP KO mice had lower levels of p-STAT3 at the age of 2–4 months, as compared to the TFF1 KO mice (). This suggested that DARPP-32 was required for tumorigenesis at an early age in the TFF1 KO mice. Immunohistochemistry (IHC) staining of p-STAT3 (Y705) and DARPP-32 showed that gastric tissues from the TFF1 KO/DP KO mice had lower levels of p-STAT3 at the age of 2–4 months, as compared to the TFF1 KO mice (<xref rid="nihms-1522657-f0006" ref-type="fig">Figure 6B</xref>). Consistent with these findings, Western blot analysis of the glandular gastric tissue showed that TFF1 KO/DP KO mouse models had lower levels of p-IGF1R, p-SRC and p-STAT3 at the age of 2–4 months, as compared to the TFF1 KO mice (). Consistent with these findings, Western blot analysis of the glandular gastric tissue showed that TFF1 KO/DP KO mouse models had lower levels of p-IGF1R, p-SRC and p-STAT3 at the age of 2–4 months, as compared to the TFF1 KO mice (<xref rid="nihms-1522657-f0006" ref-type="fig">Figure 6C</xref>). This confirmed the role of DARPP-32 in regulating IGF1R and suggesting that phosphorylation of STAT3 in the TFF1 KO/DP KO. Furthermore, we found that the TFF1 KO/DP KO mouse model had a significantly lower incidence of low-grade dysplasia (LGD) at the age of 3 months, as compared to the TFF1 KO mouse model (). This confirmed the role of DARPP-32 in regulating IGF1R and suggesting that phosphorylation of STAT3 in the TFF1 KO/DP KO. Furthermore, we found that the TFF1 KO/DP KO mouse model had a significantly lower incidence of low-grade dysplasia (LGD) at the age of 3 months, as compared to the TFF1 KO mouse model (<xref rid="nihms-1522657-f0006" ref-type="fig">Figure 6D</xref>).).'], 'nihms-1522657-f0007': ['The results of immunohistochemistry (IHC) staining on human gastric tissue samples showed weak immunostaining of p–STAT3 (Y705) and DARPP-32 in normal gastric mucosa (<xref rid="nihms-1522657-f0007" ref-type="fig">Figure 7A</xref>). On the contrary, strong immunostaining of p–STAT3 and DARPP-32 were observed in adenocarcinomas (). On the contrary, strong immunostaining of p–STAT3 and DARPP-32 were observed in adenocarcinomas (<xref rid="nihms-1522657-f0007" ref-type="fig">Figure 7A</xref>). Using a composite expression score (CES) as described in the ). Using a composite expression score (CES) as described in the Methods section, the IHC data demonstrated a significant increase in expression of p-STAT3 and DARPP-32 (P<0.01) in gastric tumors (<xref rid="nihms-1522657-f0007" ref-type="fig">Figure 7B&7C</xref>). Survival analysis by Kaplan-Meier and log rank test, using public data (). Survival analysis by Kaplan-Meier and log rank test, using public data (http://kmplot.com/analysis/index.php?p=service)30, demonstrated that patients with low expression of DARPP-32 or STAT3 had an overall better survival than those with high expression (P = 0.05 and P = 0.004, respectively, <xref rid="nihms-1522657-f0007" ref-type="fig">Figure 7D</xref>). Our results from ). Our results from in vitro, mouse and human models support the link between p-STAT3 activation and DARPP-32 expression in gastric carcinogenesis. A diagram summarizing our findings is shown in <xref rid="nihms-1522657-f0007" ref-type="fig">Figure 7E</xref>..']}
|
Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells
|
[
"Darpp-32",
"organoids",
"mouse",
"human",
"cancer"
] |
Oncogene
|
1563951600
|
Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is frequently overexpressed in early stages of gastric cancers. We utilized in vitro assays, 3D gastric gland organoid cultures, mouse models, and human tissue samples to investigate the biological and molecular impact of DARPP-32 on activation of IGF1R and STAT3 signaling and gastric tumorigenesis. DARPP-32 enhanced phosphorylation of IGF1R (Y1135), a step that was critical for STAT3 phosphorylation at Y705, nuclear localization, and transcription activation. By using proximity ligation and co-immunoprecipitation assays, we found that IGF1R and DARPP-32 co-existed in the same protein complex. Binding of DARPP-32 to IGF1R promoted IGF1R phosphorylation with subsequent activation of downstream SRC and STAT3. Analysis of gastric tissues from the TFF1 knockout (KO) mouse model of gastric neoplasia, demonstrated phosphorylation of STAT3 in the early stages of gastric tumorigenesis. By crossing the TFF1 KO mice with DARPP-32 (DP) knockout (KO) mice, that have normal stomach, we obtained double knockout (TFF1 KO/DP KO). The gastric mucosa from the double KO mice did not show phosphorylation of IGF1R or STAT3. In addition, the TFF1 KO/DP KO mice had a significant delay in developing neoplastic gastric lesions. Analysis of human gastric cancer tissue microarrays, showed high levels of DARPP-32 and positive immunostaining for nuclear STAT3 in cancer tissues, as compared to non-cancer histologically normal tissues. In summary, the DARPP-32-IGF1R signaling axis plays a key role in regulating the STAT3 signaling, a critical step in gastric tumorigenesis.
|
[
"Animals",
"Carcinogenesis",
"Cell Line, Tumor",
"Dopamine and cAMP-Regulated Phosphoprotein 32",
"Humans",
"Mice",
"Mice, Knockout",
"Phosphorylation",
"Receptor, IGF Type 1",
"STAT3 Transcription Factor",
"Signal Transduction",
"Stomach Neoplasms",
"Trefoil Factor-1"
] |
other
|
PMC6639157
| null |
41
|
[
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] |
Oncogene. 2019 Jul 24; 38(29):5805-5816
|
NO-CC CODE
|
|
Lipid preparation on
CYTOP surface. (A) Schematic of SAM formation.
SUVs were incubated on the CYTOP surface, where they fuse to form
a homogeneous SAM. (B,C) Subsequent FRAP analysis shows fluorescence
recovery. (D) After coating the surface with SAM, the chambers can
be sealed by flushing in mineral oil containing lipids through a flow
cell. (E) Z-stacks of the sealed chambers were taken
with a confocal microscope. The lipids are labeled red (DOPE-Atto655)
and soluble Alexa 488 dye in the encapsulated volume (cyan).
|
am9b05073_0003
|
2
|
39d8cecc35f754c35502fbf04d63e2967cab0c933839e2dec795ebcb9662e225
|
am9b05073_0003.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
750,
1229
] |
[{'image_id': 'am9b05073_m002', 'image_file_name': 'am9b05073_m002.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_m002.jpg', 'caption': 'No caption found', 'hash': 'f8c2f4554d224ef8f8878adf8a2270e38bb421a593099179d28498205408b5b4'}, {'image_id': 'am9b05073_0006', 'image_file_name': 'am9b05073_0006.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_0006.jpg', 'caption': 'No caption found', 'hash': 'fef32ee280d95a29858f9c120c22eb466ddcdcb48e8272d9e16dda65edeaa751'}, {'image_id': 'am9b05073_0001', 'image_file_name': 'am9b05073_0001.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_0001.jpg', 'caption': 'CYTOP\nchambers microfabricated by (A) photolithography involving\nreactive ion etching (RIE) and (B) replica molding. Fabricated chambers\nwere imaged on a laser profiler (C, above): RIE; (C, below): replica\nmolded. Chambers were fabricated with height ∼3 μm and\nwidth ∼5 μm, and their height profiles are plotted in\n(D), which reveals that replica-molded chambers can be fabricated\nwith remarkable similarity to those made by RIE.', 'hash': '64606897d0519be352d651d08ca64be23a8ad64aec1094a0e49ce910125e1467'}, {'image_id': 'am9b05073_0002', 'image_file_name': 'am9b05073_0002.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_0002.jpg', 'caption': '(A) GUVs hemifuse with CYTOP to form SAM\npatches. (B) Intensity\nprofile of lipid membrane patches (taken through the diameter) formed\nby GUV fusion on glass (D) and on CYTOP (E). (C) Area of the membrane\npatch was plotted over time [from time lapse image, (E)]. From the\ninitial gradient, we calculated the surface energy of the material\n(21 ± 3 mN m–1).', 'hash': '50af07ae7e845bbdfc215cc94e4a93f069d8211be77b0f16a85c156ce11e520e'}, {'image_id': 'am9b05073_0005', 'image_file_name': 'am9b05073_0005.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_0005.jpg', 'caption': 'MinDE\nsystem dynamics reconstituted on CYTOP-SAM. (A) Min proteins\nform spiral waves on a planar surface. (B) As a control, MinD activity\non the surface was checked by FRAP with and without the SAM. (C) Min\nproteins can be encapsulated in rod-shaped chambers, with aspect ratios\nranging from 1:1–5. (D) Time lapse images of pole-to-pole oscillations\nin rod-shaped chambers and (E) “blinking” dynamics in\nsymmetrical compartments.', 'hash': 'a3037e05c74d05de97e4c97e0b0ee84a40c47682f442282e628f2534d8ba55dc'}, {'image_id': 'am9b05073_0004', 'image_file_name': 'am9b05073_0004.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_0004.jpg', 'caption': 'GFP expression by CFPS\nin sealed CYTOP chambers. (A) Time lapse\nimages of the chambers, with time intervals of 30 min. (B) Z-stack image of the sealed chambers. (C) GFP expression\nlevels against incubation time at 37 °C. Error bars represent\nstandard deviation of measurement values from n =\n5 independent experiments.', 'hash': '7085ce687a2f736ce1ef57b040d5e67fe02e21631f563a0162da32f49a90ae95'}, {'image_id': 'am9b05073_0003', 'image_file_name': 'am9b05073_0003.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_0003.jpg', 'caption': 'Lipid preparation on\nCYTOP surface. (A) Schematic of SAM formation.\nSUVs were incubated on the CYTOP surface, where they fuse to form\na homogeneous SAM. (B,C) Subsequent FRAP analysis shows fluorescence\nrecovery. (D) After coating the surface with SAM, the chambers can\nbe sealed by flushing in mineral oil containing lipids through a flow\ncell. (E) Z-stacks of the sealed chambers were taken\nwith a confocal microscope. The lipids are labeled red (DOPE-Atto655)\nand soluble Alexa 488 dye in the encapsulated volume (cyan).', 'hash': '39d8cecc35f754c35502fbf04d63e2967cab0c933839e2dec795ebcb9662e225'}, {'image_id': 'am9b05073_m001', 'image_file_name': 'am9b05073_m001.jpg', 'image_path': '../data/media_files/PMC6750829/am9b05073_m001.jpg', 'caption': 'No caption found', 'hash': '8f3a78429b36b71e38828440d43326f342c9bd3d33cc2d261e35a283829d7779'}]
|
{'am9b05073_0001': ['Photolithography is a commonly used method to pattern\nmaterials, including CYTOP, that are themselves not photo-reactive\n(<xref rid="am9b05073_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>A). A photoresist\nis first deposited on top of the material and patterned by UV light,\nwhich then becomes a mask for the subsequent chemical or plasma treatment\nthat etches away the material in the regions that are exposed. The\nphotoresist is then lifted off to reveal the patterned structures.<xref rid="am9b05073_0001" ref-type="fig">1</xref>A). A photoresist\nis first deposited on top of the material and patterned by UV light,\nwhich then becomes a mask for the subsequent chemical or plasma treatment\nthat etches away the material in the regions that are exposed. The\nphotoresist is then lifted off to reveal the patterned structures.', 'With CYTOP, the etching step is done with a high-power oxygen\nplasma,\nwhich requires a reactive ion etcher (RIE). Although this is a reliable\nand effective technique, the equipment is costly and many cleanroom\nfacilities do not offer respective procedures. We therefore present\nan alternative fabrication method by replica molding (<xref rid="am9b05073_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>B), which requires significantly\nless technical expertise and lowers equipment costs. In replica molding,<xref rid="am9b05073_0001" ref-type="fig">1</xref>B), which requires significantly\nless technical expertise and lowers equipment costs. In replica molding,18 we first require a master or a template. The\nmaster can be either purchased or fabricated by patterning a photoresist,\nsuch as SU8, by UV exposure. Then, a PDMS stamp is cast out of the\nmaster mold and pressed down on uncured CYTOP. The CYTOP is cured\nwith the stamp in place, taking its form. Once the stamp is lifted\noff, a high-fidelity replica of the master remains.', 'The fabricated\nchambers by both methods were imaged on a profiler\n(<xref rid="am9b05073_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>C, top: RIE,\nbottom: replica molding), and the height profile (<xref rid="am9b05073_0001" ref-type="fig">1</xref>C, top: RIE,\nbottom: replica molding), and the height profile (<xref rid="am9b05073_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>D) shows little difference\nbetween the two methods. In the subsequent encapsulation experiments,\nwe used chambers produced with both methods and compared the obtained\nresults. Replica molding is a low-cost alternative to the highly reliable\nphotolithography method using RIE. It can, however, lead to small\nvariations across samples. For example, there can be small damages\nin parts when the stamp is peeled off, and variations in the way the\nstamp is pressed down onto the uncured CYTOP can sometimes cause deformed\nstructures. A rigid frame around the PDMS stamp<xref rid="am9b05073_0001" ref-type="fig">1</xref>D) shows little difference\nbetween the two methods. In the subsequent encapsulation experiments,\nwe used chambers produced with both methods and compared the obtained\nresults. Replica molding is a low-cost alternative to the highly reliable\nphotolithography method using RIE. It can, however, lead to small\nvariations across samples. For example, there can be small damages\nin parts when the stamp is peeled off, and variations in the way the\nstamp is pressed down onto the uncured CYTOP can sometimes cause deformed\nstructures. A rigid frame around the PDMS stamp28 would reduce deformations and increase the fidelity of\nthe replica, and thus should be attempted in the next iteration to\nimprove this technique.'], 'am9b05073_0002': ['To test the hypothesis that\nvesicles form a lipid monolayer upon hemifusion on the CYTOP surface,\nwe utilized GUVs (<xref rid="am9b05073_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>A). Typically with diameters 5–20 μm, these membrane\nstructures are large enough to be well resolved by optical microscopy,\nmaking them ideal test substrates for detailed analyses of the fusion\nprocess and the formed lipid patch.<xref rid="am9b05073_0002" ref-type="fig">2</xref>A). Typically with diameters 5–20 μm, these membrane\nstructures are large enough to be well resolved by optical microscopy,\nmaking them ideal test substrates for detailed analyses of the fusion\nprocess and the formed lipid patch.', 'We prepared a flat spin-coated CYTOP and deposited a solution\ncontaining\nGUVs on top of the CYTOP surface. We then observed the hemifusion\nprocess by TIRF microscopy. The SAM patch formed through GUV hemifusion\nresulted in a unique intensity profile on the CYTOP surface (<xref rid="am9b05073_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>B) that is markedly\ndifferent from that of a supported lipid bilayer (SLB) formed on glass\n(<xref rid="am9b05073_0002" ref-type="fig">2</xref>B) that is markedly\ndifferent from that of a supported lipid bilayer (SLB) formed on glass\n(<xref rid="am9b05073_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>D). The formed\nSAM patch appeared fuzzy, with a less-defined edge because of a gradual\nintensity fall-off that implies a sparser lipid density away from\nthe center of the fusion site. In contrast, an SLB patch had a very\ndefined edge with a sharp intensity fall-off, and the lipids formed\na homogeneous coverage. A previous study on the hemifusion of GUVs\non a hydrophobically functionalized glass surface showed that GUV-fused\nSAM patches have a characteristic morphology and behavior, from which\nwe could draw close comparisons with our observations.<xref rid="am9b05073_0002" ref-type="fig">2</xref>D). The formed\nSAM patch appeared fuzzy, with a less-defined edge because of a gradual\nintensity fall-off that implies a sparser lipid density away from\nthe center of the fusion site. In contrast, an SLB patch had a very\ndefined edge with a sharp intensity fall-off, and the lipids formed\na homogeneous coverage. A previous study on the hemifusion of GUVs\non a hydrophobically functionalized glass surface showed that GUV-fused\nSAM patches have a characteristic morphology and behavior, from which\nwe could draw close comparisons with our observations.29'], 'am9b05073_0003': ['We observed the hemifusion of a GUV in\nhigh time resolution (30\nms time intervals) by TIRF microscopy; we can see the vesicle coming\ninto contact, fusing, and spreading on the CYTOP surface (<xref rid="am9b05073_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>E) and plotted the\narea increase over time (<xref rid="am9b05073_0003" ref-type="fig">3</xref>E) and plotted the\narea increase over time (<xref rid="am9b05073_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>C). We also observe the same by spinning disk confocal\nmicroscopy (<xref rid="am9b05073_0003" ref-type="fig">3</xref>C). We also observe the same by spinning disk confocal\nmicroscopy (Figure S1), where we can more\nclearly see the vesicle outline during fusion, which shrinks as lipids\nare reorganized and the SAM spreads over the surface. A physical model\nwas developed by Zan et al.,29 in which\nthe instantaneous free energy gain because of the lipids spreading\nand the covering of the hydrophobic surface by the lipid tails is\nbalanced by the frictional losses of the lipids flowing from the center\nof the hemifusion toward the edge of the covered area.', 'Having obtained strong evidence for the formation\nof SAMs on CYTOP, we attempted to form a uniform coating of lipids\non the fabricated chamber surface. For this purpose, we deposited\na solution containing a high concentration of SUVs on top of the CYTOP\nsurface (<xref rid="am9b05073_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>A).\nWe prepared the SUVs with two lipid compositions: one containing DOPC\nand another with a DOPC/DOPG mixture (7:3). The latter has a net negative\ncharge, which is crucial for the function of many membrane-interacting\nproteins, such as <xref rid="am9b05073_0003" ref-type="fig">3</xref>A).\nWe prepared the SUVs with two lipid compositions: one containing DOPC\nand another with a DOPC/DOPG mixture (7:3). The latter has a net negative\ncharge, which is crucial for the function of many membrane-interacting\nproteins, such as E. coli MinDE.', 'Both RIE-prepared and replica-molded structures were successfully\ncoated with a homogeneous SAM (Figure S2), whose membrane mobility was assessed by FRAP (<xref rid="am9b05073_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>B,C). In all cases, the obtained\nlipid diffusion coefficients were between 1.0 and 1.5 μm<xref rid="am9b05073_0003" ref-type="fig">3</xref>B,C). In all cases, the obtained\nlipid diffusion coefficients were between 1.0 and 1.5 μm2 s–1 (Table 1), which are comparable to those of other SAM systems\n(0.2–2 μm2 s–1),\\30,31 and still in the range of those measured for SLBs (1–6 μm2 s–1).32,33 More importantly,\nthe immobile fraction was negligible, indicating the lack of major\nlipid aggregates and other surface artifacts.', 'Having fabricated the chambers\nand coated their surfaces with lipid\nmembranes, we encapsulated biochemical reactions. The loading of the\nchambers was done in a flow cell, as shown in the schematic (<xref rid="am9b05073_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>D). The confocal\ncross-sectional image (<xref rid="am9b05073_0003" ref-type="fig">3</xref>D). The confocal\ncross-sectional image (<xref rid="am9b05073_0003" ref-type="fig">Figures <xref rid="fig3" ref-type="fig">3</xref></xref>E and <xref rid="am9b05073_0003" ref-type="fig">3</xref>E and S3) shows Alexa 488 dye successfully\nencapsulated in these lipid-coated chambers.'], 'am9b05073_0004': ['The chambers were fabricated by the two\nalternative methods outlined\nbefore (photolithography and replica molding). The SAM was formed\non the surface, and the chambers were subsequently loaded with the\ncell-free extract. After sealing the chambers, we incubated them at\n37 °C over 5 h, monitoring the GFP expression levels by measuring\nthe fluorescence intensity in the fabricated wells every 30 min (<xref rid="am9b05073_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>).<xref rid="am9b05073_0004" ref-type="fig">4</xref>).'], 'am9b05073_0005': ['First,\nwe prepared a planar CYTOP surface by spin-coating and coated\nthe surface by SUV hemifusion (lipid composition DOPC/DOPG in 7:3\nratio). We then incubated purified MinDE proteins and observed their\ndynamics. Dynamic spiral patterns were observed on the surface (<xref rid="am9b05073_0005" ref-type="fig">Figures <xref rid="fig5" ref-type="fig">5</xref></xref>A and <xref rid="am9b05073_0005" ref-type="fig">5</xref>A and S5), in good qualitative agreement with observations\nmade in previous studies on supported bilayers.39 The measured period (1–2 min) and wavelength (∼30\nμm) compare well with previous studies on bilayers (period 0.6–2\nmin, wavelength 50–110 μm)39,40,43 as well as on monolayer surfaces at the air–water\ninterface (period 0.5–1 min, wavelength 30–60 μm).44 The Min wavelength is known to vary according\nto the substrate and its preparation,43 and a different wavelength on CYTOP can therefore be expected.', 'As a control, we checked that MinD is indeed adhering to\nand denaturing\non CYTOP without the SAM (<xref rid="am9b05073_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>B). Without MinE and in the presence of ATP, MinD still\nhas a basal on/off rate from the membrane that is not catalyzed by\nMinE binding. Without the SAM, FRAP analysis on this system shows\nonly partial recovery, which suggests that most of the MinD is permanently\nbound to the CYTOP surface. With the SAM, MinD fluorescence almost\nfully recovers, which indicates a healthy cycling of MinD from the\nmembrane into the bulk volume.<xref rid="am9b05073_0005" ref-type="fig">5</xref>B). Without MinE and in the presence of ATP, MinD still\nhas a basal on/off rate from the membrane that is not catalyzed by\nMinE binding. Without the SAM, FRAP analysis on this system shows\nonly partial recovery, which suggests that most of the MinD is permanently\nbound to the CYTOP surface. With the SAM, MinD fluorescence almost\nfully recovers, which indicates a healthy cycling of MinD from the\nmembrane into the bulk volume.', 'We fabricated chambers as bacteria\nmimicries—in elongated\nchambers with aspect ratios (width/length) ranging from 1:1 to 1:5.\nBoth photolithography (<xref rid="am9b05073_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>) and replica molding (<xref rid="am9b05073_0005" ref-type="fig">5</xref>) and replica molding (Figure S6) methods were tested, and we confined the MinDE proteins within\nthese chambers and observed their dynamics.']}
|
Design
of Sealable Custom-Shaped Cell Mimicries Based
on Self-Assembled Monolayers on CYTOP Polymer
|
[
"micropatterning",
"bottom-up biology",
"lab-on-a-chip",
"synthetic\nbiology",
"self-assembled monolayers",
"lipids",
"polymer",
"CYTOP"
] |
ACS Appl Mater Interfaces
|
1560927600
|
In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.
|
[
"Escherichia coli",
"Hydrophobic and Hydrophilic Interactions",
"Microscopy, Fluorescence",
"Phospholipids",
"Photobleaching",
"Polymers",
"Unilamellar Liposomes"
] |
other
|
PMC6750829
| null |
46
|
[
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] |
ACS Appl Mater Interfaces. 2019 Jun 19; 11(24):21372-21380
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NO-CC CODE
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Depletion of Mob1 disrupts CPC localization to the spindle midzone during early anaphase. (A) RPE1 cells were synchronized by double thymidine block and transfected with either nontargeting siRNA (control, left) or siRNA targeting both Mob1A and Mob1B (Mob1A/1B, right). In contrast to controls where the CPC rapidly relocated to the forming spindle midzone (a–c and g–i), Aurora B and INCENP was found throughout the spindle as associated with chromatin in Mob1A-depleted cells (d–f and j–l). However, by late anaphase, the CPC could be seen in a narrow band at the cell equator in both controls (m-o) and Mob1A-depleted cells (p–r). Bar, 10 μm. (B) Quantification of the CPC phenotype indicated that during early anaphase, 66.8% of cells depleted of Mob1A and Mob1B in early anaphase (n = 482) displayed diffuse Aurora B and INCENP localization along the spindle, whereas by late anaphase, CPC localization to the midzone was normal in 100% Mob1A/B-depleted cells (n = 270).
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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"fluorescence microscopy",
"bar plot",
"immunoblot"
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[{'image_id': 'zmk0031093370001', 'image_file_name': 'zmk0031093370001.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370001.jpg', 'caption': "Phylogenetic analysis of the Mob1 family. (A) A dendogram was constructed using Mob1 protein sequences from across multiple phyla (Supplemental Table 3), rooted to the basal eukaryote Giardia lamblia, with bootstrap values provided for each node. Based on this analysis and the nomenclature of Stavridi (Stavridi et al., 2003), vertebrate Mob1's were given the assignations Mob1A–E. Branches noted by the asterisks indicate possible gene duplications. (B) Amino acid alignment of the five human Mob1 isoforms. Identical residues are shaded in black, similar residues are shaded in gray and nonconserved residues are not shaded.", 'hash': '45f7a1b1248bc154de844790bd317464240c9be44a4f27220d0837c35740ec3d'}, {'image_id': 'zmk0031093370006', 'image_file_name': 'zmk0031093370006.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370006.jpg', 'caption': 'Mob1A localization at kinetochore is lost in the absence of Aurora B activity. GFP-Mob1A–expressing HeLa cells were treated with 200 nM nocodazole for 3 h and then released into DMSO (A–D) or 5 μM ZM477439 (E–H) for an additional 30 min. Cells were fixed and counterstained with phospho- (Ser10) histone H3 (B and F). Note that although spindle pole localization was maintained in cells with compromised Aurora B activity (A and E), kinetochore localization was lost (E). Bar, 10 μm.', 'hash': '0fcf1e60408799c4be2d7082a8e6cc51e5ed81e698a739e6b93bba1e7f8e24f4'}, {'image_id': 'zmk0031093370008', 'image_file_name': 'zmk0031093370008.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370008.jpg', 'caption': 'Depletion of Mob1 disrupts CPC localization to the spindle midzone during early anaphase. (A) RPE1 cells were synchronized by double thymidine block and transfected with either nontargeting siRNA (control, left) or siRNA targeting both Mob1A and Mob1B (Mob1A/1B, right). In contrast to controls where the CPC rapidly relocated to the forming spindle midzone (a–c and g–i), Aurora B and INCENP was found throughout the spindle as associated with chromatin in Mob1A-depleted cells (d–f and j–l). However, by late anaphase, the CPC could be seen in a narrow band at the cell equator in both controls (m-o) and Mob1A-depleted cells (p–r). Bar, 10 μm. (B) Quantification of the CPC phenotype indicated that during early anaphase, 66.8% of cells depleted of Mob1A and Mob1B in early anaphase (n = 482) displayed diffuse Aurora B and INCENP localization along the spindle, whereas by late anaphase, CPC localization to the midzone was normal in 100% Mob1A/B-depleted cells (n = 270).', 'hash': 'f7bf2bcf02cda510e776e4fdb866f798631e14bd9d8286d0fcc458dd05429d25'}, {'image_id': 'zmk0031093370010', 'image_file_name': 'zmk0031093370010.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370010.jpg', 'caption': "MKLP2 localization is altered in Mob1A/Mob1B-depleted cells. (A) Thymidine-synchronized RPE1 cells were transfected with either nontargeting control (left) or Mob1A/Mob1B siRNA's (right), and probed for INCENP and MKLP2 localization. In contrast to controls (a–c), MKLP2 recruitment during early anaphase was disrupted in Mob1A/B-depleted cells (d–f), but by mid-late anaphase (j–l), MKLP2 recruitment to the midzone resembled controls (g–i). Bar, 10 μm. (B) Disruption of MKLP2 localization to the central spindle occurred in 63.7% of cell depleted of Mob1A/1B during early anaphase (n = 92 cells, SE = 0.0358), but by mid-late anaphase, MKLP2 localization matched controls (n = 102).", 'hash': 'f6a2946c0c1bf1fb6e7b21514ab390a78a015215c64d4129faf784fb1eb2889e'}, {'image_id': 'zmk0031093370009', 'image_file_name': 'zmk0031093370009.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370009.jpg', 'caption': 'Histone H3 phosphorylation is maintained on chromatid arms during anaphase in Mob1A/B-depleted cells. Thymidine-synchronized RPE1 cells were transfected with either nontargeting control (left) or Mob1A/Mob1B siRNAs (right), and probed for phospho-Ser10 histone H3 and Aurora B localization. Note that in contrast to cells in metaphase (A–F), anaphase phosphohistone reactivity in controls was limited to those regions of trailing chromatin arms closest to the spindle midzone (G–I and M–O). In Mob1A/Mob1B-depleted cells where Aurora B localization was spread throughout the anaphase spindle, phosphohistone reactivity was found along the entire length of the sister chromatid arms (J–L and P–R). Bar, 10 μm.', 'hash': 'a88af035607b7a3ec53cc1a515e666337df1b1a30fe2f271b6d34677034b1972'}, {'image_id': 'zmk0031093370007', 'image_file_name': 'zmk0031093370007.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370007.jpg', 'caption': 'Expression profile and depletion of Mob1 in HeLa and hTERT-RPE1 cells. (A) Quantitative PCR was performed using cDNA from HeLa and RPE1 cells. The graph shows the reciprocal values of Ct on y-axis to represent relative expression of Mob1 isoforms in HeLa and RPE1 cells. Error bars, SD. (B) RPE1 cells were transfected with Mob1A and Mob1B siRNA singly or in combination, and RNA was harvested 48h after transfection. qPCR was performed to determine the transcript levels for Mob1A, -B, and -C (which was not targeted). The -fold changes shown are the means of at least two different experiments done in triplicate. Values between 0.5 and 2.0 were not considered as significant change in gene expression. Error bars, SD. (C) Confirmation of protein depletion was determined by Western blot using an antibody specific for both Mob1A and Mob1B.', 'hash': '5e3d4e80b2377f5fc429212c2d9cdd381a84bf9a4ea65febcb4ffbf046855de1'}, {'image_id': 'zmk0031093370011', 'image_file_name': 'zmk0031093370011.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370011.jpg', 'caption': 'Depletion of Mob1A affects the organization of centralspindlin during anaphase. (A) RPE1 cells transfected with control- or Mob1A/1B siRNAs were probed for MKLP1 or MgcRacGAP and counterstained with INCENP or Aurora B, respectively. Bar, 10 μm. (B) Control- or Mob1A/1B-depleted cells were processed for tubulin and MKLP1 localization, and the zone of MKLP1 was measured as a function of spindle length for 200 cells per condition. For each cell, the width of the MKLP1 signal at the midzone was measured, along with the length of the spindle and width of central spindle. Lines of correlation that best fit the scatter plot data are depicted for control (black line) and Mob1A/1B-depleted cells (red line). As shown in the graph, the MKLP1 zone narrowed as anaphase progressed for both control- and Mob1-depleted cells. However, in contrast to controls, the zone of MKLP1 was wider in Mob1-depleted cells both early and late in anaphase. (C) Analysis of covariance was performed on the two data sets, and although there was no statistical significance in the rate of MKLP1 narrowing between control and Mob1-depleted cells (p = 0.537957) (measured by comparing the 2 slopes), there was a significant difference between the widths of the two zones as cells progressed through anaphase (p = 0.00001).', 'hash': '7c469fbff91b6252d10ae414a55871370d86ee7dcb7e4fe75bd917212dd9279e'}, {'image_id': 'zmk0031093370003', 'image_file_name': 'zmk0031093370003.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370003.jpg', 'caption': 'GFP-Mob1 colocalization with prominent kinetochore components. GFP-Mob1A was transiently transfected into HeLa cells, processed for Aurora B (A–C), Plk1 (D–F), Hec1 (G–I), and BubR1 (J–L) localization. Note that although Mob1 could be found on either side of the Aurora B centromeric signal (C), Mob1 localization overlapped with Plk1, Hec1, and BubR1 (D–L). Bar, 5 μm.', 'hash': 'd36b559d0f8c37f0a072bbef5143a5be110d4318542e5dcf74815f041d126faa'}, {'image_id': 'zmk0031093370004', 'image_file_name': 'zmk0031093370004.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370004.jpg', 'caption': 'Plk1 is required for Mob1 recruitment to the spindle poles. GFP-Mob1A–expressing HeLa cells were transfected with nontargeting control and Plk1 siRNA (for Western blot confirmation, see Supplemental Figure 4). Twenty-four hours post-siRNA transfection, cells were processed for localization of γ tubulin (A–F) and Plk1 (G–L). In the absence of Plk1, cells formed a monopolar spindle, and although Mob1 was lost from the centrally located spindle pole (D–F), kinetochore localization was unaffected (D–F, J–L, and M–O). Bar, 10 μm.', 'hash': '0316c997136f879828acca023d5294605ba7aac6630916b9bfa78245bdc845bf'}, {'image_id': 'zmk0031093370005', 'image_file_name': 'zmk0031093370005.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370005.jpg', 'caption': 'The chromosomal passenger complex is required for Mob1A recruitment to the kinetochore. GFP-Mob1A–expressing HeLa cells were transfected with nontargeting control (A–C, G–I) and INCENP (D–F, J–L) siRNA (for Western blot confirmation, see Supplemental Figure 4), and 24 h post-siRNA transfection, cells were processed for colocalization of Hec1 (B and E) and INCENP (H and K). When INCENP was depleted, Mob1A no longer localized on the kinetochore, but spindle pole localization was unaffected (D and J) (only a single pole was visible in the optical sections shown in D and G). Bar, 10 μm.', 'hash': '793cc8c8e39e889ab71fddd20b3c856e43415063c2c3d3f261f7e354098d793d'}, {'image_id': 'zmk0031093370002', 'image_file_name': 'zmk0031093370002.jpg', 'image_path': '../data/media_files/PMC2814784/zmk0031093370002.jpg', 'caption': 'GFP-Mob1 localization in dividing HeLa cells. Optical sections of HeLa cells transfected with GFP-Mob1A revealed that Mob1 recruitment to centrosomes began in late G2 phase and was maintained through cytokinesis (A–F). Kinetochore localization was most prominent during prometaphase (B) but lost during anaphase and cytokinesis (D and E). Mob1 localized to the spindle midzone during anaphase and was enriched in the midbody late in cytokinesis (D and E). Treatment with 250 nM nocodazole failed to disrupt either spindle pole- or kinetochore localization (F). Bar, 10 μm.', 'hash': '3aade60191ee7a624b4b0669bf5d62cafbb7faebbd3843053e0e7c0e0a23901f'}]
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{'zmk0031093370001': ['In budding and fission yeast, Mob1 facilitates mitotic exit and cytokinesis by acting as a regulatory subunit for Dbf2/Sid2 kinase (Komarnitsky et al., 1998; Luca et al., 2001; Devroe et al., 2004; Hou et al., 2004). Mob1 and its effector kinase localize first to the spindle pole bodies until anaphase onset, at which time the complex relocalizes to the bud neck/actin ring (Frenz et al., 2000; Salimova et al., 2000; Luca et al., 2001; Yoshida and Toh-e, 2001; Stoepel et al., 2005). As an initial effort toward understanding how the MEN might function in animal cells, we sought to follow Mob1 localization dynamics through mitosis in cultured human cells. In contrast to unicellular fungi where there is a single Mob1 gene, there are five Mob1 genes in the human genome (<xref ref-type="fig" rid="zmk0031093370001">Figure 1</xref>). Moreover, the nomenclature for these genes varies greatly (Supplemental Table 3), creating confusion in both the literature and in the National Center for Biotechnology Information Database. A dendogram of Mob1 sequences from multiple model organisms rooted to the basal eukaryote ). Moreover, the nomenclature for these genes varies greatly (Supplemental Table 3), creating confusion in both the literature and in the National Center for Biotechnology Information Database. A dendogram of Mob1 sequences from multiple model organisms rooted to the basal eukaryote Giardia lamblia (<xref ref-type="fig" rid="zmk0031093370001">Figure 1</xref>A) resolved Mob1 into two monophyletic groups. The first clade includes a single lineage of plant Mob1 proteins, single copy A) resolved Mob1 into two monophyletic groups. The first clade includes a single lineage of plant Mob1 proteins, single copy Drosophila- and sea urchin Mob1 proteins, and two vertebrate Mob1 proteins (MobKL1B and MobKL1A), arising from a likely gene duplication. Following the nomenclature used by Stavridi et al. (2003), we designated these proteins as Mob1A and Mob1B, respectively. Mob1A and B are 96% identical to each other at the amino acid level (<xref ref-type="fig" rid="zmk0031093370001">Figure 1</xref>B) and are known to bind the NDR1/2- and Lats1/2 kinases (B) and are known to bind the NDR1/2- and Lats1/2 kinases (Bichsel et al., 2004; Devroe et al., 2004; Bothos et al., 2005; Hergovich et al., 2005, 2006a; Chow et al., 2009). The other major cluster includes protozoan Mob1\'s, a single Drosophila Mob1 isoform and three vertebrate Mob1 isoforms arising from two gene duplication events (denoted by asterisks, <xref ref-type="fig" rid="zmk0031093370001">Figure 1</xref>A). These three human Mob1\'s share ∼50% identity with Mob1A and Mob1B, and do not interact with Lats1 or Lats2 (A). These three human Mob1\'s share ∼50% identity with Mob1A and Mob1B, and do not interact with Lats1 or Lats2 (Chow et al., 2009). Otherwise, little is known of their biological activity. In keeping with the nomenclature used for the other isoforms, we therefore assigned- and hereafter refer to these noncanonical isoforms as Mob1C–E.', 'Mammals have five Mob1 genes (<xref ref-type="fig" rid="zmk0031093370001">Figure 1</xref>), and given the high degree of similarity between the different isoforms and lack of discriminating antibodies, we elected to express GFP fusions to follow isoform-specific localization dynamics. To our surprise, we found almost superimposable localization dynamics between the isoforms, beginning in late G2/prophase, where Mob1 could be detected at the forming spindle poles and kinetochores (), and given the high degree of similarity between the different isoforms and lack of discriminating antibodies, we elected to express GFP fusions to follow isoform-specific localization dynamics. To our surprise, we found almost superimposable localization dynamics between the isoforms, beginning in late G2/prophase, where Mob1 could be detected at the forming spindle poles and kinetochores (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref> and Supplemental Figure 1). Whereas spindle pole-associated Mob1 was maintained throughout mitosis and cytokinesis, kinetochore recruitment peaked during prometaphase and was lost by anaphase ( and Supplemental Figure 1). Whereas spindle pole-associated Mob1 was maintained throughout mitosis and cytokinesis, kinetochore recruitment peaked during prometaphase and was lost by anaphase (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>D and Supplemental Figure 1, C and G). Last, Mob1 could be detected at the spindle midzone and midbody (D and Supplemental Figure 1, C and G). Last, Mob1 could be detected at the spindle midzone and midbody (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>, D and E, and Supplemental Figure 1, D and H). And although localization of Mob1A to spindle poles and midbodies in mammalian cells has been reported previously (, D and E, and Supplemental Figure 1, D and H). And although localization of Mob1A to spindle poles and midbodies in mammalian cells has been reported previously (Bothos et al., 2005), the kinetochore localization of Mob1 isoforms has not been reported to date, and represents a novel and exciting finding. Drosophila Mob4, which most resembles Mob3/phocein in humans, localizes to the spindle poles and weakly to kinetochores, and seems to play a role in spindle pole integrity (Trammell et al., 2008).'], 'zmk0031093370002': ['Given the high degree of similarity between the Mob1 proteins (and lack of discriminating antibodies), we tagged four of the human isoforms (Mob1A, Mob1C, Mob1D, and Mob1E) with enhanced green fluorescent protein (EGFP) at their N termini to follow their localization dynamics during mitosis and cytokinesis. Transient expression in HeLa cells revealed redundant localization patterns for all isoforms examined, with Mob1E displaying the least consistent expression and localization patterns, and was discontinued for all subsequent experiments. GFP-tagged Mob1\'s localized to the centrosomes in late G2 and remained associated with the spindle poles throughout mitosis and cytokinesis (96% of 544 cells scored) (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>, A–E, and Supplemental Figure 1, A–H). Mob1 could also be observed at kinetochores beginning in early prophase before nuclear envelope breakdown (Supplemental Figure 1, A and E) with signal intensity peaking at prometaphase (, A–E, and Supplemental Figure 1, A–H). Mob1 could also be observed at kinetochores beginning in early prophase before nuclear envelope breakdown (Supplemental Figure 1, A and E) with signal intensity peaking at prometaphase (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>B and Supplemental Figure 1, A and E) (90%; n = 170 cells). However, by metaphase (B and Supplemental Figure 1, A and E) (90%; n = 170 cells). However, by metaphase (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>C), only 69% of Hec1-positive kinetochores were Mob1 positive (n = 55), and by anaphase no kinetochore localization could be detected (n = 45). Treatment of cells with nocodazole failed to alter GFP-Mob1 recruitment to spindle poles or kinetochores, suggesting that Mob1 recruitment to these structures was microtubule-independent (C), only 69% of Hec1-positive kinetochores were Mob1 positive (n = 55), and by anaphase no kinetochore localization could be detected (n = 45). Treatment of cells with nocodazole failed to alter GFP-Mob1 recruitment to spindle poles or kinetochores, suggesting that Mob1 recruitment to these structures was microtubule-independent (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>F). After anaphase onset, Mob1 localized weakly to the spindle midzone (F). After anaphase onset, Mob1 localized weakly to the spindle midzone (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>D and Supplemental Figures 1, C and G, and 2A, g and h; 64% of 45 cells counted) but was enriched at the midbody at the end of cytokinesis (D and Supplemental Figures 1, C and G, and 2A, g and h; 64% of 45 cells counted) but was enriched at the midbody at the end of cytokinesis (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref>E and Supplemental Figure 1, D and H, and 2J) (99%; n = 107). Although localization at kinetochores was only poorly visible using antibodies specific for Mob1A (Supplemental Figure 2, B and M), GFP-Mob1 localization was otherwise superimposable with the endogenous protein. Kinetochore localization could not be detected if EGFP was expressed alone (Supplemental Figure 1C, p–s), nor did expression of GFP-Mob1 effect the centrosomal localization of the Mob1-binding protein Lats1 or the MEN effector Cdc14A (data not shown), which together suggested that the observed localization patterns of the GFP-chimera were not an artifact of overexpression of the GFP-Mob1 chimeras or were disruptive to endogenous Mob1 function.E and Supplemental Figure 1, D and H, and 2J) (99%; n = 107). Although localization at kinetochores was only poorly visible using antibodies specific for Mob1A (Supplemental Figure 2, B and M), GFP-Mob1 localization was otherwise superimposable with the endogenous protein. Kinetochore localization could not be detected if EGFP was expressed alone (Supplemental Figure 1C, p–s), nor did expression of GFP-Mob1 effect the centrosomal localization of the Mob1-binding protein Lats1 or the MEN effector Cdc14A (data not shown), which together suggested that the observed localization patterns of the GFP-chimera were not an artifact of overexpression of the GFP-Mob1 chimeras or were disruptive to endogenous Mob1 function.', 'The CPC localizes to the inner centromere early in mitosis and functions in organizing the spindle as well as monitoring tension at the kinetochore (May and Hardwick, 2006; Ruchaud et al., 2007b). In addition, upon anaphase onset the CPC plays a critical role in organizing the spindle midzone and facilitating cytokinesis (Vader et al., 2008; Glotzer, 2009). Because Mob1 was also found at the kinetochores and spindle midzone (<xref ref-type="fig" rid="zmk0031093370002">Figure 2</xref> and Supplemental Figures 1 and 2), we wanted to determine whether Mob1 localization at the kinetochores was dependent on the CPC in mammalian cells. When INCENP was depleted in HeLa cells expressing GFP-Mob1A, -C, or -D, kinetochore localization of Mob1A was partially lost in 42% and completely lost 43% in cells scored (n = 834; and Supplemental Figures 1 and 2), we wanted to determine whether Mob1 localization at the kinetochores was dependent on the CPC in mammalian cells. When INCENP was depleted in HeLa cells expressing GFP-Mob1A, -C, or -D, kinetochore localization of Mob1A was partially lost in 42% and completely lost 43% in cells scored (n = 834; <xref ref-type="fig" rid="zmk0031093370005">Figure 5</xref> and Supplemental Figure 5). In contrast, spindle pole localization was unperturbed in all cells observed. and Supplemental Figure 5). In contrast, spindle pole localization was unperturbed in all cells observed.', 'Examination of four of the five mammalian isoforms of Mob1 revealed redundant patterns (<xref ref-type="fig" rid="zmk0031093370002">Figures 2</xref> and and <xref ref-type="fig" rid="zmk0031093370003">3</xref> and Supplemental Figures 1 and 2), raising the issue of whether these isoforms are functionally redundant. Quantitative PCR analyses of HeLa- and RPE1 cells indicated that different cell lines express slightly different Mob1 isoform profiles, and differential expression patterns have been described for the Mob1 isoforms in human tissues ( and Supplemental Figures 1 and 2), raising the issue of whether these isoforms are functionally redundant. Quantitative PCR analyses of HeLa- and RPE1 cells indicated that different cell lines express slightly different Mob1 isoform profiles, and differential expression patterns have been described for the Mob1 isoforms in human tissues (Chow et al., 2009). Mob1\'s C–E do not bind the Lats1/2 kinases (Chow et al., 2009), suggesting that these isoforms perform nonredundant functions. To date, this is the first report to describe the localization patterns of the different Mob1 isoforms, but at this juncture we cannot say whether Mob1C–E can compensate for the loss of other Mob1\'s during mitosis. Further experimentation will determine whether this is, indeed, the case.'], 'zmk0031093370003': ['To more closely examine Mob1 localization at the kinetochore, we processed GFP-Mob1A–transfected HeLa cells for colocalization with key kinetochore markers. Aurora B has a broad signal, spanning the inner centromere (Carmena and Earnshaw, 2003) and when colocalized with Mob1A, Mob1A could be detected at both ends of the Aurora B signal (<xref ref-type="fig" rid="zmk0031093370003">Figure 3</xref>, A–C). Compared with Plk1, Hec1 and BubR1, all of which reside within the outer two layers of the kinetochore, the Mob1A signal was superimposable with each marker (, A–C). Compared with Plk1, Hec1 and BubR1, all of which reside within the outer two layers of the kinetochore, the Mob1A signal was superimposable with each marker (<xref ref-type="fig" rid="zmk0031093370003">Figure 3</xref>, D–L). Examination of GFP-Mob1C and Mob1D revealed similar patterns, suggesting that the observed kinetochore localization was not isoform specific., D–L). Examination of GFP-Mob1C and Mob1D revealed similar patterns, suggesting that the observed kinetochore localization was not isoform specific.', 'The localization of Mob1 during mitosis was highly reminiscent of the key mitotic kinases Plk1 (Golsteyn et al., 1995; van de Weerdt and Medema, 2006) and Aurora B kinase (Schumacher et al., 1998; Adams et al., 2001b; Giet and Glover, 2001; Murata-Hori et al., 2002), and we sought to determine whether Mob1\'s localization dynamics was dependent on these kinases. Plk1 is a major regulator throughout mitosis, playing roles in the transition G2/M, centrosomal maturation, spindle bipolarity and cytokinesis (Glover et al., 1998; van de Weerdt and Medema, 2006). Plk1 genetically interacts with Mob1 in yeast (Luca and Winey, 1998; Lee et al., 2001; Tanaka et al., 2001) and in animal cells is recruited to the spindle poles, kinetochores and spindle midzone (Golsteyn et al., 1995; Glover et al., 1998; Barr et al., 2004; van de Weerdt and Medema, 2006; Petronczki et al., 2008). Mob1 and Plk1 colocalized in mitotic cells (<xref ref-type="fig" rid="zmk0031093370003">Figure 3</xref>, D–F), and to determine whether Plk1 influenced Mob1 localization in dividing cells, HeLa cells transiently expressing GFP-Mob1A, -C, and -D were depleted of Plk1 by RNA interference. Consistent with previous reports, Plk1-depleted cells arrested in mitosis with a characteristic monopolar spindle with diffuse γ-tubulin localization (, D–F), and to determine whether Plk1 influenced Mob1 localization in dividing cells, HeLa cells transiently expressing GFP-Mob1A, -C, and -D were depleted of Plk1 by RNA interference. Consistent with previous reports, Plk1-depleted cells arrested in mitosis with a characteristic monopolar spindle with diffuse γ-tubulin localization (<xref ref-type="fig" rid="zmk0031093370004">Figure 4</xref>E; E; Lane and Nigg, 1996; Donaldson et al., 2001). In Plk1-depeleted cells, Mob1A was lost from the spindle poles (66%; n = 372 cells) but kinetochore localization was unaffected (<xref ref-type="fig" rid="zmk0031093370004">Figure 4</xref>D, J and M). Identical results were obtained when GFP-Mob1C and Mob1D were expressed in Plk1-depleted cells (Supplemental Figure 3, D, J, P, and V).D, J and M). Identical results were obtained when GFP-Mob1C and Mob1D were expressed in Plk1-depleted cells (Supplemental Figure 3, D, J, P, and V).'], 'zmk0031093370006': ['INCENP is required for Aurora B activation and function (Adams et al., 2000; Terada, 2001; Bishop and Schumacher, 2002; Honda et al., 2003) and acts as a binding scaffold for the entire CPC complex (Sessa et al., 2005; Jeyaprakash et al., 2007). To determine whether the catalytic activity of the CPC (Aurora B) is required for Mob1 recruitment to kinetochores, GFP-Mob1A–, -C–, and -D–expressing HeLa cells were synchronized in prometaphase with nocodazole and then released into either dimethyl sulfoxide (DMSO) carrier control (<xref ref-type="fig" rid="zmk0031093370006">Figure 6</xref>, A–D, and Supplemental Figure 6, A–D and I–L) or the Aurora B inhibitor ZM477439 (, A–D, and Supplemental Figure 6, A–D and I–L) or the Aurora B inhibitor ZM477439 (<xref ref-type="fig" rid="zmk0031093370006">Figure 6</xref>, E–H, and Supplemental Figure 6, E–H and M–P). Kinetochore localization of Mob1A, -C, and -D was lost (93%; n = 148 cells) in the absence of Aurora B activity (, E–H, and Supplemental Figure 6, E–H and M–P). Kinetochore localization of Mob1A, -C, and -D was lost (93%; n = 148 cells) in the absence of Aurora B activity (<xref ref-type="fig" rid="zmk0031093370006">Figure 6</xref>E and Supplemental Figure 6, E and M) as was Ser10 phosphorylation of histone H3 (E and Supplemental Figure 6, E and M) as was Ser10 phosphorylation of histone H3 (<xref ref-type="fig" rid="zmk0031093370006">Figure 6</xref>F and Supplemental Figure 6, F and N), an established marker for Aurora B activity (F and Supplemental Figure 6, F and N), an established marker for Aurora B activity (Hsu et al., 2000; Adams et al., 2001a; Giet and Glover, 2001; George et al., 2006). Thus, either siRNA depletion or pharmacologic inhibition of the CPC blocked Mob1 recruitment to the kinetochores.'], 'zmk0031093370007': ['Alteration of Mob1 activity in yeast compromises Dbf2/Sid2- as well as Cdc14/Clp1 function (Mah et al., 2001) and alters CPC relocation to the spindle midzone (Stoepel et al., 2005). To identify the possible functions of Mob1 at the kinetochore or spindle midzone, we depleted Mob1 isoforms in cultured human cells. For these experiments, we used hTERT-immortalized retinal pigmented (RPE1) cells, which in comparison with HeLa cells display low rates of mitotic errors (Kigasawa et al., 1994; Jiang et al., 1999; Thompson and Compton, 2008) and express only Mob1A–D as detected by quantitative PCR (<xref ref-type="fig" rid="zmk0031093370007">Figure 7</xref>A). Because Mob1C and -D do not bind the mammalian NDR family kinases (A). Because Mob1C and -D do not bind the mammalian NDR family kinases (Chow et al., 2009), Mob1A and -B were depleted by transfection with siRNAs targeting each isoform and analyzed by quantitative PCR and Western blotting (<xref ref-type="fig" rid="zmk0031093370007">Figure 7</xref>, B and C). Although simultaneous depletion of Mob1A and -B drastically reduced both transcript levels without affecting Mob1C expression (, B and C). Although simultaneous depletion of Mob1A and -B drastically reduced both transcript levels without affecting Mob1C expression (<xref ref-type="fig" rid="zmk0031093370007">Figure 7</xref>B), transfection with siRNA targeting Mob1A and Mob1B isoforms resulted in only a partial reduction (30–50%) in Mob1A/B protein levels 48 h after transfection (B), transfection with siRNA targeting Mob1A and Mob1B isoforms resulted in only a partial reduction (30–50%) in Mob1A/B protein levels 48 h after transfection (<xref ref-type="fig" rid="zmk0031093370007">Figure 7</xref>C), suggesting that Mob1 may be a relatively long-lived protein.C), suggesting that Mob1 may be a relatively long-lived protein.'], 'zmk0031093370005': ['In addition to its role in regulating mitotic exit, functional interactions between Mob1 and the CPC have been described in yeast (Stoepel et al., 2005). Our own examination of Mob1 in human cells indicated that Mob1A localization to the kinetochore required INCENP and Aurora B (<xref ref-type="fig" rid="zmk0031093370005">Figures 5</xref> and and <xref ref-type="fig" rid="zmk0031093370006">6</xref> and Supplemental Figures 5 and 6) and to determine whether Mob1A was important for the CPC localization, we examined the organization of Aurora B and its downstream effectors in the presence or absence of Mob1A and 1B. In control cells, Aurora B displayed a well-defined, broad centromeric distribution early in mitosis up until anaphase onset at which time Aurora B and the CPC localize to the spindle midzone during anaphase ( and Supplemental Figures 5 and 6) and to determine whether Mob1A was important for the CPC localization, we examined the organization of Aurora B and its downstream effectors in the presence or absence of Mob1A and 1B. In control cells, Aurora B displayed a well-defined, broad centromeric distribution early in mitosis up until anaphase onset at which time Aurora B and the CPC localize to the spindle midzone during anaphase (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, g–h and m–o), where the CPC influences the organization of the spindle midzone and factors required for the execution of cytokinesis (A, g–h and m–o), where the CPC influences the organization of the spindle midzone and factors required for the execution of cytokinesis (Glotzer, 2009). However in Mob1A/B-depleted cells, we observed Aurora B and INCENP spread throughout the spindle midzone during early anaphase in 66.8% of cells scored (n = 482) with a fraction of the CPC remaining associated with the segregating sister chromatids (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, j–l). This stood in sharp contrast to controls where Aurora B and INCENP were cleared from the centromeres by early anaphase and were confined within a narrow zone at the spindle midzone by mid-anaphase (A, j–l). This stood in sharp contrast to controls where Aurora B and INCENP were cleared from the centromeres by early anaphase and were confined within a narrow zone at the spindle midzone by mid-anaphase (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, g–i). However, later in anaphase, no significant difference could be seen in the CPC localization between Mob1-depleted cells (A, g–i). However, later in anaphase, no significant difference could be seen in the CPC localization between Mob1-depleted cells (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, p–r; and B) and controls (A, p–r; and B) and controls (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, m–o; and B), suggesting that CPC localization recovered as mitotic exit progressed. Because all four components of the CPC function as a single functional unit (A, m–o; and B), suggesting that CPC localization recovered as mitotic exit progressed. Because all four components of the CPC function as a single functional unit (Jeyaprakash et al., 2007), the fact that both Aurora B and INCENP displayed this phenotype suggested that the mobilization of the entire CPC was being affected.'], 'zmk0031093370008': ['In Mob1A/Mob1B-depleted cells, Aurora B was spread out throughout the early anaphase spindle (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, j–l), but there were none of the dramatic phenotypes normally associated with compromised Aurora B function (A, j–l), but there were none of the dramatic phenotypes normally associated with compromised Aurora B function (Kallio et al., 2002; Hauf et al., 2003). To determine whether there are any functional consequences to this altered localization we probed control- and Mob1A/Mob1B-depleted cells for histone H3 phosphorylation, a convenient marker for Aurora B activity. In control cells, phosphohistone reactivity during anaphase is normally restricted to the trailing arms of segregating chromatids (<xref ref-type="fig" rid="zmk0031093370009">Figure 9</xref>, G and M). However, in 55% of Mob1A/Mob1B-depleted cells (n = 70), phosphohistone reactivity was maintained along the entire length of the chromatid arms (, G and M). However, in 55% of Mob1A/Mob1B-depleted cells (n = 70), phosphohistone reactivity was maintained along the entire length of the chromatid arms (<xref ref-type="fig" rid="zmk0031093370009">Figure 9</xref>, J and P), suggesting that although the CPC was not localizing normally to a tight zone at the cell equator, its catalytic activity was normal., J and P), suggesting that although the CPC was not localizing normally to a tight zone at the cell equator, its catalytic activity was normal.', 'Later in anaphase, Aurora B and INCENP (<xref ref-type="fig" rid="zmk0031093370008">Figure 8</xref>A, m–r) could be found enriched at the spindle midzone in both control- and Mob1A/Mob1B-depleted cells, suggesting that CPC recruitment to the midzone recovered in the absence of Mob1A/Mob1B. The relocalization of the CPC from the centromeres to the spindle midzone requires the action of the kinesin-like motor MKLP2 (A, m–r) could be found enriched at the spindle midzone in both control- and Mob1A/Mob1B-depleted cells, suggesting that CPC recruitment to the midzone recovered in the absence of Mob1A/Mob1B. The relocalization of the CPC from the centromeres to the spindle midzone requires the action of the kinesin-like motor MKLP2 (Gruneberg et al., 2004), and to determine whether the effect of Mob1A/Mob1B on CPC localization involved this motor, cells were depleted of Mob1A/Mob1B and probed for MKLP2. In early anaphase, MKLP2 begins to localize at the spindle midzone (<xref ref-type="fig" rid="zmk0031093370010">Figure 10</xref>A, a–c), but in Mob1A/Mob1B-depleted cells, MKLP2 recruitment was reduced or absent in >63% of cells observed (n = 92) (A, a–c), but in Mob1A/Mob1B-depleted cells, MKLP2 recruitment was reduced or absent in >63% of cells observed (n = 92) (<xref ref-type="fig" rid="zmk0031093370010">Figure 10</xref>A, d–f; and B). However, as was the case with the CPC, by late anaphase MKLP2 recruitment to the midzone in Mob1-depleted cells (A, d–f; and B). However, as was the case with the CPC, by late anaphase MKLP2 recruitment to the midzone in Mob1-depleted cells (<xref ref-type="fig" rid="zmk0031093370010">Figure 10</xref>A, j–l; and B) was indistinguishable from controls (A, j–l; and B) was indistinguishable from controls (<xref ref-type="fig" rid="zmk0031093370010">Figure 10</xref>A, g–i; and B).A, g–i; and B).', 'The CPC functions in chromosomal organization and alignment, kinetochore assembly, microtubule attachment, spindle midzone assembly, and cytokinesis (Ruchaud et al., 2007a; Vader et al., 2007). Work in budding yeast found that along with Cdc14, Mob1/Dbf2 partially localizes with kinetochores and is required for mobilization of the CPC from the centromere to the anaphase spindle (Stoepel et al., 2005). We found that in Mob1A/Mob1B-depleted RPE1 cells, the CPC was spread throughout the spindle, with some residual localization on the chromosomes, suggesting that Mob1A was important for the correct redistribution of the CPC to the spindle midzone in a manner analogous for what has been reported in yeast (<xref ref-type="fig" rid="zmk0031093370008">Figures 8</xref><xref ref-type="fig" rid="zmk0031093370009" />––<xref ref-type="fig" rid="zmk0031093370010">10</xref>). However, there were no indications that CPC function was compromised. Indeed, when we probed cells for known targets of Aurora B (such as phosphohistone H3 and MKLP1), we saw that histone H3 was phosphorylated normally early in mitosis (). However, there were no indications that CPC function was compromised. Indeed, when we probed cells for known targets of Aurora B (such as phosphohistone H3 and MKLP1), we saw that histone H3 was phosphorylated normally early in mitosis (<xref ref-type="fig" rid="zmk0031093370009">Figure 9</xref>) and that MKLP1 was recruited properly to the midzone during anaphase () and that MKLP1 was recruited properly to the midzone during anaphase (<xref ref-type="fig" rid="zmk0031093370011">Figures 11</xref>A). However, in both cases, the diffuse Aurora B localization along the early anaphase spindle seemed to affect the normal dynamics of histone phosphorylation and MKLP1 localization. In Mob1A/Mob1B-depleted cells, the phosphohistone reactivity could be found along the entire length of the segregating sister chromatids (A). However, in both cases, the diffuse Aurora B localization along the early anaphase spindle seemed to affect the normal dynamics of histone phosphorylation and MKLP1 localization. In Mob1A/Mob1B-depleted cells, the phosphohistone reactivity could be found along the entire length of the segregating sister chromatids (<xref ref-type="fig" rid="zmk0031093370009">Figure 9</xref>, J and P), in contrast to controls where phosphohistone reactivity was limited to those regions of chromatin most proximal to the midzone (, J and P), in contrast to controls where phosphohistone reactivity was limited to those regions of chromatin most proximal to the midzone (<xref ref-type="fig" rid="zmk0031093370009">Figure 9</xref>, G and M). Similarly, although MKLP1 could be found at the midzone of both control- and Mob1A/Mob1B-depleted cells, the zone of MKLP1 was broader in those cells lacking Mob1A/1B (, G and M). Similarly, although MKLP1 could be found at the midzone of both control- and Mob1A/Mob1B-depleted cells, the zone of MKLP1 was broader in those cells lacking Mob1A/1B (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>). It has been proposed that during anaphase there is a gradient of Aurora B activity at the spindle midzone that acts to influence the late events of mitosis (). It has been proposed that during anaphase there is a gradient of Aurora B activity at the spindle midzone that acts to influence the late events of mitosis (Fuller et al., 2008). It is possible then that in the Mob1-depleted cells, the diffuse distribution of Aurora B along the entire spindle effectively broadens the activity gradient and thus maintains phosphohistone reactivity and the widened distribution of MKLP1 at the midzone.'], 'zmk0031093370011': ['The principle target of the CPC during anaphase is the centralspindlin complex of MKLP1 and male germ cell Rac GTPase activating protein (MgcRacGap), which helps organize the spindle midzone and recruit the RhoGEF Ect2 to the cell equator (D\'Avino et al., 2005; Glotzer, 2005). Examination of both centralspindlin components in Mob1A/Mob1B-depleted cells revealed a similar phenotype in cells in early anaphase. In contrast to controls (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>A, b and h), the zone of both MKLP1 and MgcRacGap was slightly wider in Mob1-depleted cells (A, b and h), the zone of both MKLP1 and MgcRacGap was slightly wider in Mob1-depleted cells (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>A, e and k). However, because the zone of MKLP1 at the midzone gradually narrows as anaphase and cytokinesis progresses in control cells, we needed to compare the MKLP1 zones between control- and Mob1A/Mob1B-depleted cells as a function of spindle length to determine whether the observed differences were consistent throughout anaphase (A, e and k). However, because the zone of MKLP1 at the midzone gradually narrows as anaphase and cytokinesis progresses in control cells, we needed to compare the MKLP1 zones between control- and Mob1A/Mob1B-depleted cells as a function of spindle length to determine whether the observed differences were consistent throughout anaphase (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>A). Measurements of the MKLP1 zone and spindle length were made on cells (stained for tubulin and MKLP1) for 200 cells per condition, and examination of the two data sets revealed that the MKLP1 zone narrowed as anaphase progressed in both control (A). Measurements of the MKLP1 zone and spindle length were made on cells (stained for tubulin and MKLP1) for 200 cells per condition, and examination of the two data sets revealed that the MKLP1 zone narrowed as anaphase progressed in both control (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>B, black line)- and Mob1A/Mob1B (B, black line)- and Mob1A/Mob1B (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>A, red line)-depleted cells. However, in Mob1A/Mob1B-depleted cells the MKLP1 zone was wider than controls throughout anaphase (A, red line)-depleted cells. However, in Mob1A/Mob1B-depleted cells the MKLP1 zone was wider than controls throughout anaphase (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>B, red line). ANCOVA determined that there was no difference between the slopes of the two curves (p = 0.537957), indicating that the rate by which the MKLP1 zone narrowed was not significantly different between control- and Mob1A/Mob1B-depleted cells (B, red line). ANCOVA determined that there was no difference between the slopes of the two curves (p = 0.537957), indicating that the rate by which the MKLP1 zone narrowed was not significantly different between control- and Mob1A/Mob1B-depleted cells (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>C). However, the difference in the width of the MKLP1 zone between control- and Mob1A/Mob1B-depleted cells was significant (p = 0.00001) (C). However, the difference in the width of the MKLP1 zone between control- and Mob1A/Mob1B-depleted cells was significant (p = 0.00001) (<xref ref-type="fig" rid="zmk0031093370011">Figure 11</xref>C), with the MKLP1 zone in Mob1A/Mob1B-depleted cells slightly wider both early and late in anaphase. However, cells in both conditions were capable of initiating cytokinesis, suggesting that the broaden zone of centralspindlin was not a strong enough defect to inhibit RhoA activation and contractile ring assembly (data not shown). Thus, although the absence of Mob1A/Mob1B resulted in only subtle defects in spindle midzone organization, Mob1A and Mob1B did seem to play a role in restricting the zone by which the CPC defined the cell equator in dividing cells.C), with the MKLP1 zone in Mob1A/Mob1B-depleted cells slightly wider both early and late in anaphase. However, cells in both conditions were capable of initiating cytokinesis, suggesting that the broaden zone of centralspindlin was not a strong enough defect to inhibit RhoA activation and contractile ring assembly (data not shown). Thus, although the absence of Mob1A/Mob1B resulted in only subtle defects in spindle midzone organization, Mob1A and Mob1B did seem to play a role in restricting the zone by which the CPC defined the cell equator in dividing cells.'], 'zmk0031093370010': ['We found that in Mob1A/Mob1B-depleted cells, the mitotic kinesin MKLP2 was also late to arrive at the spindle midzone (<xref ref-type="fig" rid="zmk0031093370010">Figure 10</xref>). MKLP2 is required for the CPC to correctly load onto the spindle midzone during anaphase, as well as for the midzone targeting of the MEN effector Cdc14A (). MKLP2 is required for the CPC to correctly load onto the spindle midzone during anaphase, as well as for the midzone targeting of the MEN effector Cdc14A (Gruneberg et al., 2004). For both the CPC and MKLP2, the absence of Mob1A/B only affected midzone localization early in anaphase (<xref ref-type="fig" rid="zmk0031093370008">Figures 8</xref> and and <xref ref-type="fig" rid="zmk0031093370010">10</xref>), and that as anaphase and mitotic exit progressed MKLP2/CPC recruitment to the midzone recovered. MKLP2 is antagonized by Cdk1 (), and that as anaphase and mitotic exit progressed MKLP2/CPC recruitment to the midzone recovered. MKLP2 is antagonized by Cdk1 (Hummer and Mayer, 2009), raising the possibility that MKLP2 is a principle target by which the mitotic exit network regulates the CPC. If Mob1 functions through Cdc14A during mitotic progression (as it does in yeast), then one might predict as CDK1 activity continued to decline through anaphase, those factors negatively regulated by CDK1 (such as MKLP2 and the CPC) would eventually become fully active and function normally. Identifying the responsible NDR kinase (among the four human orthologues of Dbf2) through which Mob1 modulates Cdc14A activity, as well as the direct targets of Cdc14A will reveal exactly how mammalian homologues of the mitotic exit network modulate the final events of cell division.']}
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Mutual Dependence of Mob1 and the Chromosomal Passenger Complex for Localization during Mitosis
| null |
Mol Biol Cell
|
1265011200
|
Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a motility cycle driven by actin polymerization and actomyosin contraction. Using new analytical tools and statistical methods, we provide, for the first time, a statistically significant quantification of the spatial distribution of the traction forces generated at each phase of the cycle (protrusion, contraction, retraction, and relaxation). We show that cells are constantly under tensional stress and that wild-type cells develop two opposing "pole" forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that nonmuscular myosin II complex (MyoII) cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces, the changes in cell shape, and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII motor function, demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII actin cross-linking leads to a force redistribution throughout the cell perimeter pulling inward toward the center. However, it does not reduce significantly the magnitude of the traction forces, uncovering a non-MyoII-mediated mechanism for the contractility of the cell.
|
[
"Actins",
"Animals",
"Cell Movement",
"Cytoskeleton",
"Dictyostelium",
"Mathematics",
"Microscopy, Fluorescence",
"Models, Biological",
"Myosin Type II",
"Periodicity",
"Stress, Mechanical"
] |
other
|
PMC2814784
| null |
66
|
[
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] |
Mol Biol Cell. 2010 Feb 1; 21(3):380-392
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NO-CC CODE
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Distribution of CaV1.3 and CtBP2/RIBEYE in immature mouse IHCsA–C, an apical IHC from a P7 mouse immunostained for the CaV1.3 Ca2+ channel (A, red) and ribbon marker CtBP2/RIBEYE (B, green), merged image shown in C. Sparse dotted lines delineate IHCs. The region below the horizontal dotted line at the IHC nuclear level indicates the position for all single Ca2+ channel recordings shown in the following figures. Note that CaV1.3 is distributed over the whole surface area of the IHC (A), whereas CtBP2 is exclusively localized at its basal pole (B). D, single layer image from a P7 IHC showing that most Ca2+ channel spots delineate the boundaries (plasma membrane). E, magnification of the boxed area in C (examples of co-localization in yellow is indicated by filled arrowheads). F, basal region of an adult (P30) IHC showing co-localization between CtBP2/RIBEYE and Ca2+ channel immunopositive spots. Images in panels C, E and F represent the maximum intensity projection over all layers of the z-stack. D represents a single layer image at the nuclear level from z-stack deconvoluted images. Nuclei in A–F were stained with DAPI (blue); note that after deconvolution mainly nucleoli are visible. Scale bar in A–D and F indicates 10 μm; in E 5 μm. G, total number of immunopositive spots for CaV1.3 (red bar), total number of CaV1.3 positive spots below the IHC's nuclei (white bar) and CtBP2/RIBEYE (green bar) measured from eight P7 immature IHCs. Note that 70% of these Ca2+ spots were associated with the plasma membrane and that some CtBP2/RIBEYE spots were seen to co-localize (yellow bar) with CaV1.3 immunospots.
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tjp0588-0187-f1
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2
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f40d758d04829ec4e1df0341810d5b4176a0dd694261fc975df70b77faaf9cfe
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tjp0588-0187-f1.jpg
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multiple
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multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
454,
383
] |
[{'image_id': 'tjp0588-0187-f4', 'image_file_name': 'tjp0588-0187-f4.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f4.jpg', 'caption': 'Closed and open time constants of unitary CaV1.3 Ca2+ currentsA and B, closed (τc1, τc2 and τc3) and open (τo1 and τo2) time constants, respectively, as a function of membrane patch potential derived from fitting the dwell-time distributions using the sum of two or three exponentials (eqn (3); 1 ≤n≤ 3 patches for each voltage; n= 6). C and D, examples of closed and open time distributions, respectively, calculated at membrane potentials of −27 mV and −47 mV. Data are displayed on a log/linear scale (20 bins/decade) and fitted using eqn (3) with three (C) or two (D) exponentials. E, voltage dependence of the Ca2+ channel opening probability obtained by applying eqns (4) and (5) (see Methods), and using time constants from the above open- and closed-time distributions (see also Table 1). Data were fitted with a Boltzmann equation with parameters of: Po(min)= 0.03; Po(max)= 0.27; V1/2=−33.9 mV; S= 5.2 mV.', 'hash': '963cbecf2d825297978f9aafd91e95ed02ed0197f62c9e174127fe24f731f1d1'}, {'image_id': 'tjp0588-0187-f3', 'image_file_name': 'tjp0588-0187-f3.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f3.jpg', 'caption': 'Unitary CaV1.3 L-type Ca2+ channel currentsA, representative unitary currents (500 ms recordings) from IHCs at membrane patch potentials shown next to the traces using 5 mm Ca2+ and BayK 8644. Grey lines indicate the channel closed state. B, current recordings indicating the presence of a cluster of two Ca2+ channels. Dashed lines indicate the main open current levels. C, ensemble-averaged current at −17 mV derived from 130 active sweeps from 6 IHCs. Holding potential =−67 mV. The inset shows a fit to the current onset using eqn (8) and for clarity, one of every two data points is shown. D, average I–V from single-channel currents (2 ≤n≤ 6 patches for each voltage; n= 9, P5–P8). Shaded area represents the resting membrane potential of immature IHCs (Marcotti et al. 2003a). The red dot indicates the elementary current size recorded in normal Na+ extracellular solution at the cell resting potential (−1.1 ± 0.1 pA, n= 3). E, average I–V of the macroscopic Ca2+ current in P7 IHCs (n= 8) elicited using depolarizing voltage steps in 10 mV increments (500 ms in duration) from −81 mV. F, voltage-dependent activation of the macroscopic ICa (grey triangles) obtained by plotting the normalized chord conductance against the different membrane potentials. The continuous line is the fit obtained using a first-order Boltzmann equation (see Methods): gmax= 9.0 nS, V1/2=−34.0 mV, S= 4.5 mV. Black circles show the mean open probability (Po) of single Ca2+ channels at different membrane potentials (1 ≤n≤ 4 patches, n= 5, P5–P8). Data were fitted with a Boltzmann equation with parameters of: Po(min)= 0.007; Po(max)= 0.154; V1/2=−33.9 mV; S= 6.1 mV. Note that the maximal individual Po value measured at depolarized membrane potentials was 0.23.', 'hash': '81d10111661d4d6581e312115a54b5d62bfb398cf8dc4d4fa2285f8b09f002b4'}, {'image_id': 'tjp0588-0187-m8', 'image_file_name': 'tjp0588-0187-m8.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m8.jpg', 'caption': 'No caption found', 'hash': 'e41b6b869540db9cce91dddfa91e7b72f10d05bf3ca2cbed804af3cbd29caae2'}, {'image_id': 'tjp0588-0187-m6', 'image_file_name': 'tjp0588-0187-m6.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m6.jpg', 'caption': 'No caption found', 'hash': '8c83fb74c79efd81d02c6ae9ac45258faba19b7485a993ef03931ef0abc5fae7'}, {'image_id': 'tjp0588-0187-m1', 'image_file_name': 'tjp0588-0187-m1.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m1.jpg', 'caption': 'No caption found', 'hash': '6412905d0b60214e4cd99ee58d38a34def445174d5583eee4a6a3329321da4a1'}, {'image_id': 'tjp0588-0187-f2', 'image_file_name': 'tjp0588-0187-f2.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f2.jpg', 'caption': 'Effect of BayK 8644 on the unitary Ca2+ channel currentA and B, representative unitary currents recorded in immature IHCs in the presence of 70 mm Ba2+ with (A) and without (B) BayK 8644 in the pipette solution. C, I–V from single-channel experiments in 70 mm Ba2+ with (between 1 and 7 patches were used for each voltage step, P8–P9) and without (1–5 patches; P7–P10) BayK 8644. In this and the following figures the number of patches corresponds to the number of IHCs investigated and recordings were performed at 35–37°C.', 'hash': '09f7c00bf300ccb0be83064a1fe38dcd36d9d42da7bae5883ee81a5029fa344b'}, {'image_id': 'tjp0588-0187-f5', 'image_file_name': 'tjp0588-0187-f5.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f5.jpg', 'caption': 'First latency distribution in single-channel Ca2+ currentFirst latency distribution obtained by plotting the natural logarithm of the numbers of observations per millisecond [ln(n/ms)] as a function of time. The continuous curved line is the third-order exponential function obtained using eqn (6). Dashed lines are the single exponential components shown separately. Fitting parameters were: W1= 64.8%; τ1= 0.7 ms, W2= 41%, τ2= 10.5 ms, W3= 68.5%, τ3= 170.5 ms.', 'hash': '68a0757fcadabe2c8da9209296e2081f354e7366738b2876c85a8c22296fcd63'}, {'image_id': 'tjp0588-0187-m7', 'image_file_name': 'tjp0588-0187-m7.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m7.jpg', 'caption': 'No caption found', 'hash': 'a536c2ed05d9af5935a1fdfccb30b04db90d2b0a9d1e98aa305cb884b1fe9e0a'}, {'image_id': 'tjp0588-0187-m4', 'image_file_name': 'tjp0588-0187-m4.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m4.jpg', 'caption': 'No caption found', 'hash': 'c6febe69d6e57df18d59baa67ad020898fb5e8b7e03af2e2b5aa489fa83d9edc'}, {'image_id': 'tjp0588-0187-m3', 'image_file_name': 'tjp0588-0187-m3.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m3.jpg', 'caption': 'No caption found', 'hash': 'e96d49d0d83966ace485862f8dd615559c9b06b6801a47402e0c5f7e7837a27e'}, {'image_id': 'tjp0588-0187-f6', 'image_file_name': 'tjp0588-0187-f6.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f6.jpg', 'caption': 'Biophysics of the macroscopic Ca2+ current in immature mouse IHCsA, average I–V curves for the macroscopic ICa from P5–P7 IHCs in the presence of 1.3 mm Ca2+ (n= 11), 5 mm Ca2+ (n= 6) and 5 mm Ca2+ with BayK 8644 (n= 4). Currents were elicited by depolarizing voltage steps of 10 mV increments (10 ms in duration) from −81 mV. B, average maximal size (left panel), half-maximal activation (V1/2: middle) and voltage sensitivity (S, right) of ICa derived from A using eqn (7). C, ICa recorded using the conditions described in A (first 1.2 ms). Fits to ICa activation are according to eqn (8). D, average ICa activation time constant (τ). Asterisks indicate significant difference when 1.3 mm Ca2+ was compared to 5 mm Ca2+ or 5 mm Ca2++ BayK (*P < 0.05; **P < 0.01; ***P < 0.001, defined by the Tukey or Bonferroni post tests for panels B and D, respectively).', 'hash': '7347c102d10a4aac0b037945d9f04c5b3785ae1e4527699f2989ddf4fe091657'}, {'image_id': 'tjp0588-0187-f1', 'image_file_name': 'tjp0588-0187-f1.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f1.jpg', 'caption': "Distribution of CaV1.3 and CtBP2/RIBEYE in immature mouse IHCsA–C, an apical IHC from a P7 mouse immunostained for the CaV1.3 Ca2+ channel (A, red) and ribbon marker CtBP2/RIBEYE (B, green), merged image shown in C. Sparse dotted lines delineate IHCs. The region below the horizontal dotted line at the IHC nuclear level indicates the position for all single Ca2+ channel recordings shown in the following figures. Note that CaV1.3 is distributed over the whole surface area of the IHC (A), whereas CtBP2 is exclusively localized at its basal pole (B). D, single layer image from a P7 IHC showing that most Ca2+ channel spots delineate the boundaries (plasma membrane). E, magnification of the boxed area in C (examples of co-localization in yellow is indicated by filled arrowheads). F, basal region of an adult (P30) IHC showing co-localization between CtBP2/RIBEYE and Ca2+ channel immunopositive spots. Images in panels C, E and F represent the maximum intensity projection over all layers of the z-stack. D represents a single layer image at the nuclear level from z-stack deconvoluted images. Nuclei in A–F were stained with DAPI (blue); note that after deconvolution mainly nucleoli are visible. Scale bar in A–D and F indicates 10 μm; in E 5 μm. G, total number of immunopositive spots for CaV1.3 (red bar), total number of CaV1.3 positive spots below the IHC's nuclei (white bar) and CtBP2/RIBEYE (green bar) measured from eight P7 immature IHCs. Note that 70% of these Ca2+ spots were associated with the plasma membrane and that some CtBP2/RIBEYE spots were seen to co-localize (yellow bar) with CaV1.3 immunospots.", 'hash': 'f40d758d04829ec4e1df0341810d5b4176a0dd694261fc975df70b77faaf9cfe'}, {'image_id': 'tjp0588-0187-m2', 'image_file_name': 'tjp0588-0187-m2.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m2.jpg', 'caption': 'No caption found', 'hash': '14f9741007087e5d7f9044746d9d91bb42cdfecf9f6a4a3f6c5cb43ec87f7d8b'}, {'image_id': 'tjp0588-0187-m5', 'image_file_name': 'tjp0588-0187-m5.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-m5.jpg', 'caption': 'No caption found', 'hash': 'bfb5e23c5437d9a9f02e373f2a15a1f621784c490b28417f8a23d0df18df1e8f'}, {'image_id': 'tjp0588-0187-mu1', 'image_file_name': 'tjp0588-0187-mu1.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-mu1.jpg', 'caption': None, 'hash': '1b54822792dd0cb8fe255fd175566f9c3a47e69c18b57bffdc9016c7783807b9'}, {'image_id': 'tjp0588-0187-f7', 'image_file_name': 'tjp0588-0187-f7.jpg', 'image_path': '../data/media_files/PMC2817446/tjp0588-0187-f7.jpg', 'caption': 'Mean open Ca2+ channels and RRP vesicles released at each IHC active zone as a function of membrane potentialThe left axis indicates the mean number of open Ca2+ channels as a function of membrane potential obtained from N×Po, where N= 180 Ca2+ channels per ribbon synapse and Po is that estimated in 1.3 mm extracellular Ca2+ from the fit in Fig. 3F (maximal Po at −20 mV = 0.05: see Discussion). The right axis shows the RRP measured from ΔCm responses in early postnatal IHCs in response to 100 ms depolarization (from Fig. 5A in Johnson et al. 2005), using a conversion factor of 37 aF per vesicle (Lenzi et al. 1999). The number of fused synaptic vesicles per ribbon was estimated assuming 15 active zones (see Fig. 1G). Note that on average, due to the very low Po of Ca2+ channels, only a few of them will be open at any instant during the 100 ms depolarization (e.g. 9 Ca2+ channels out of 180 at −10 mV). Due to stochastic channel gating, the [Ca2+]i required to release the RRP/ribbon is provided by a large number (180) of Ca2+ channels, each randomly opening for a small fraction of time over the 100 ms. The [Ca2+]i provided by a total number of 9 Ca2+ channels per ribbon would be sufficient to control the RRP, instead of 180, if Po was near to a hypothetical value of 1 (i.e. open 100% of the time).', 'hash': '98b1f5ce5d380f617a59ba750dd6bab27d380d535a2c622d9a4e88367b379549'}]
|
{'tjp0588-0187-f3': ['For single Ca2+ channel recordings, patch pipettes were made from borosilicate glass capillaries (Harvard Apparatus Ltd, UK), fire-polished (resistance in the bath: 7–12 MΩ; seal resistance > 20 GΩ) and coated with surf wax (Mr Zoggs SexWax, USA) to minimize the fast electrode capacitative transient. Patch pipettes contained the following solution (in mm): 5 CaCl2, 102 CsCl, 10 Hepes-KOH, 15 4-aminopyridine and 40 TEA (pH 7.5). In some experiments 5 or 70 mm Ba2+ was used instead of 5 mm Ca2+. Apamin (300 nm: Merck Biosciences, UK), niflumic acid (50 μm: Sigma, UK) and BayK 8644 (5 μm: Sigma) were added to the pipette solution. Stock solutions of niflumic acid and BayK 8644 were prepared in DMSO and stored at −20°C (final dilution 1:2000). During the majority of recordings, the membrane potential of IHCs was zeroed by superfusing a high-K+ extracellular solution (Zampini et al. 2006) containing (in mm): 140 KCl, 0.2 CaCl2, 6.2 MgCl2, 0.7 NaH2PO4, 5.6 d-glucose, 15 Hepes-KOH (pH = 7.5). In three experiments, normal high-Na+ extracellular solution was used in order to measure the single Ca2+ channel current at the IHCs resting membrane potential (see red dot in <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>D</italic></xref>). Data were filtered at 2 or 5 kHz (4-pole Bessel) and sampled at 20 or 50 kHz. In a very few cases, current traces were additionally filtered offline at 1 kHz (8-pole Bessel). Membrane potentials were corrected for the liquid junction potential (LJP: +3 mV in 5 mD). Data were filtered at 2 or 5 kHz (4-pole Bessel) and sampled at 20 or 50 kHz. In a very few cases, current traces were additionally filtered offline at 1 kHz (8-pole Bessel). Membrane potentials were corrected for the liquid junction potential (LJP: +3 mV in 5 mm Ca2+ or 5 mm Ba2+; −5 mV in 70 mm Ba2+).', 'Single Ca2+ channel analysis was performed as previously described (Zampini et al. 2006) using Clampfit (Molecular Devices) and Origin (OriginLab, USA). Briefly, leak and uncompensated capacitive currents were corrected by subtracting average episodes without channel activity (null sweeps) from the active sweeps. Event detection was performed with the 50% threshold detection method with each transition visually inspected before being accepted. Idealized traces were used to calculate single-channel amplitude distribution (event duration > 0.34 ms), open probability (Po) and open and closed time histograms. Distributions were fitted with a single or double Gaussian function (current amplitude) or multiple exponentials (dwell times). The Po of Ca2+ channels as a function of voltage (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref>) was fitted using a first-order Boltzmann equation:\nF) was fitted using a first-order Boltzmann equation:\n(1)where Po represents the mean open probability, Po(min) and Po(max) are the minimum and maximum Po, V is voltage, V1/2 is the voltage at which Po is half-maximum and S is the voltage sensitivity. Po was corrected for the number of channels present in the patch.', 'The current–voltage curve shown in <xref ref-type="fig" rid="tjp0588-0187-f3">Figs 3<italic>E</italic></xref> and E and <xref ref-type="fig" rid="tjp0588-0187-f6">6<italic>A</italic></xref> was obtained using the following equation:\nA was obtained using the following equation:\n(7)where I is the current, Vrev is the reversal potential, gmax and gmin are the maximum and minimum chord conductance and the other parameters are as in eqn (1).', 'The activation curves of the macroscopic Ca2+ current (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref>) were obtained from the normalized chord conductance (F) were obtained from the normalized chord conductance (Zidanic & Fuchs, 1995; Johnson et al. 2005) using the reversal potential of +48 mV (Johnson et al. 2005), and approximated by a first-order Boltzmann equation (see eqn (1)). The activation kinetics of the macroscopic ICa (<xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6<italic>C</italic></xref>) were approximated using the following equation:\nC) were approximated using the following equation:\n(8)where I(t) is the current at time t, Imax is the peak ICa, τ is the time constant of activation and α was fixed at 2, which gave a better fit than a power of 3 (as previously described: Johnson et al. 2005; Johnson & Marcotti, 2008), consistent with a Hodgkin–Huxley model with two opening gating particles (Hodgkin & Huxley, 1952).', 'Following the brief topical enzymatic treatment of the IHC to be patched, the majority of the successful patches (77%) contained one (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>A</italic></xref>) or two CaA) or two Ca2+ channels (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>B</italic></xref>) and the rest contained three or four. We never recorded from membrane patches containing a large number of CaB) and the rest contained three or four. We never recorded from membrane patches containing a large number of Ca2+ channels such as those previously described in bullfrog auditory hair cells (Rodriguez-Contreras & Yamoah, 2001). One possible explanation for this discrepancy is that the mild enzymatic treatment used for the experiments was not sufficient to remove the afferent terminals completely and unmask all Ca2+ channels.', 'Remarkably, single Ca2+ channel activity was detectable at very negative potentials (from about −70 mV), which corresponds to the resting membrane potential for these immature IHCs (from −50 mV to −70 mV: Marcotti et al. 2003a). At around this potential the single-channel current size was about 1.1 pA in both high K+ and normal high Na+ (red filled circle: <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>D</italic></xref>) extracellular solutions. In the same voltage range, about 1% (defined as percentage of D) extracellular solutions. In the same voltage range, about 1% (defined as percentage of gmax) of the macroscopic Ca2+ current was available (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>E</italic> and <italic>F</italic></xref>, grey triangles).E and F, grey triangles).', 'The single Ca2+ channel open probability (Po) was voltage dependent and increased with depolarization, reaching a maximum average value of about 0.15 in active sweeps (500 ms steps: <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref>), which is consistent with that previously found in lower vertebrate hair cells (F), which is consistent with that previously found in lower vertebrate hair cells (Po= 0.24 in 5 mm Ca2+: Rodriguez-Contreras & Yamoah, 2003) and in salamander photoreceptors (Po=∼0.12; Thoreson et al. 2000). The percentage of null sweeps was very high (on average > 60%) at all membrane potentials. The possibility that rapid single-channel openings were missed was excluded since no significant difference was found in the variance and the standard deviation of the current recorded in null sweeps at −67 mV and at −17 mV, which corresponds to the voltage where Po was minimal and maximal, respectively. In contrast to our findings, a significantly higher maximal Po and smaller elementary conductance was estimated in cochlear IHCs using ensemble variance analysis (Po: ∼0.8; 0.62 pA at −60 mV: Brandt et al. 2005), which could reflect the different experimental conditions used between the two studies (e.g. extracellular Ca2+ concentration, age range and voltage protocol duration, which affect the possible contribution of Ca2+ channel inactivation).', 'Although the single-channel and whole-cell measurements were performed using the same recording conditions (5 mm Ca2+ and BayK 8644: <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3</xref>), we investigated to what extent the voltage and time dependence of the macroscopic Ca), we investigated to what extent the voltage and time dependence of the macroscopic Ca2+ current was affected when using 1.3 mm Ca2+ (the physiological perilymphatic Ca2+ concentration; Wangemann & Schacht, 1996) and without the gating modifier BayK 8644. The size and voltage of half-maximal activation of ICa were significantly increased when the extracellular Ca2+ concentration was elevated from 1.3 mm to 5 mm with or without BayK 8644 (overall: P < 0.0001, one-way ANOVA, for both panels A and B in <xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6</xref>). In particular, BayK 8644 by prolonging the single Ca). In particular, BayK 8644 by prolonging the single Ca2+ channel open time caused the amplitude of ICa to increase about 3-fold, due to an increased channel Po. However, the voltage sensitivity of the current activation, defined by the slope factor S, was similar in all conditions tested (<xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6<italic>B</italic></xref>, right panel). This indicates that the effect of BayK 8644 on CaB, right panel). This indicates that the effect of BayK 8644 on CaV1.3 channel activation was not significantly voltage dependent. The activation time constant (τ) of the macroscopic Ca2+ current (<xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6<italic>C</italic></xref>) was also found to be significantly smaller in 1.3 mC) was also found to be significantly smaller in 1.3 mm Ca2+ (overall: P < 0.0001, two-way ANOVA; <xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6<italic>D</italic></xref>).D).', 'The left axis indicates the mean number of open Ca2+ channels as a function of membrane potential obtained from N×Po, where N= 180 Ca2+ channels per ribbon synapse and Po is that estimated in 1.3 mm extracellular Ca2+ from the fit in <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref> (maximal F (maximal Po at −20 mV = 0.05: see Discussion). The right axis shows the RRP measured from ΔCm responses in early postnatal IHCs in response to 100 ms depolarization (from <xref ref-type="fig" rid="tjp0588-0187-f5">Fig. 5<italic>A</italic></xref> in A in Johnson et al. 2005), using a conversion factor of 37 aF per vesicle (Lenzi et al. 1999). The number of fused synaptic vesicles per ribbon was estimated assuming 15 active zones (see <xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>G</italic></xref>). Note that on average, due to the very low G). Note that on average, due to the very low Po of Ca2+ channels, only a few of them will be open at any instant during the 100 ms depolarization (e.g. 9 Ca2+ channels out of 180 at −10 mV). Due to stochastic channel gating, the [Ca2+]i required to release the RRP/ribbon is provided by a large number (180) of Ca2+ channels, each randomly opening for a small fraction of time over the 100 ms. The [Ca2+]i provided by a total number of 9 Ca2+ channels per ribbon would be sufficient to control the RRP, instead of 180, if Po was near to a hypothetical value of 1 (i.e. open 100% of the time).'], 'tjp0588-0187-f1': ['Immature mouse (P6 and P7) cochleae were isolated, fixed, cryosectioned and stained as described (Knipper et al. 2000; Knirsch et al. 2007). Animals were killed by exposure to a rising concentration of CO2 gas in accordance with the ethical guidelines approved by the University of Tübingen and the Tierschutzgesetz (Germany). Apical IHCs were stained using rabbit polyclonal anti-CaV1.3 (Alomone Labs, 1:50) and mouse monoclonal anti-CtBP2/RIBEYE (BD Transduction Laboratories, CA, USA; 1:50) antibodies. Primary antibodies were detected with Cy3-conjugated (Jackson ImmunoResearch Laboratories, USA) or Alexa Fluor 488-conjugated antibodies (Molecular Probes, USA). Sections were embedded with Vectashield mounting medium with DAPI (Vector Laboratories, USA). Sections were viewed using an Olympus AX70 microscope equipped with epifluorescence illumination (×100 objective, NA = 1.35) and a motorized z-axis. Images were acquired using a CCD camera and the imaging software Cell∧F (OSIS GmbH, Münster, Germany). For CaV1.3 and CtBP2/RIBEYE immunopositive spot counting, cryo-sectioned cochleae were imaged over a distance of 8 μm with the complete coverage of the IHC nucleus and beyond in an image-stack along the z-axis (z-stack). Typically z-stacks consisted of 30 layers with a z-increment of 0.276 μm, for each layer one image per fluorochrome was acquired. z-stacks were 3-dimensionally deconvoluted using Cell∧F\'s RIDE module with the Nearest Neighbour algorithm (OSIS GmbH, Münster, Germany). Panels A–C, E and F in <xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1</xref> are composite images, which represent the maximum intensity projection over all layers of the are composite images, which represent the maximum intensity projection over all layers of the z-stack. <xref ref-type="fig" rid="tjp0588-0187-f1">Figure 1<italic>D</italic></xref> is a single layer image at the nuclear level from D is a single layer image at the nuclear level from z-stack-deconvoluted pictures.', 'The distribution of CaV1.3 channels within immature IHCs was investigated using immunolabelling experiments (<xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1</xref>). Calcium channel clusters were not only found at the IHC presynaptic region, as for post-hearing IHCs (). Calcium channel clusters were not only found at the IHC presynaptic region, as for post-hearing IHCs (<xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>F</italic></xref>: see also F: see also Brandt et al. 2005; Meyer et al. 2009), but also in their neck region (<xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>A</italic>, <italic>C</italic> and <italic>D</italic></xref>). Although the total number of immunopositive CaA, C and D). Although the total number of immunopositive CaV1.3 spots measured in eight P7 mouse IHCs was 80 ± 13 (<xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>G</italic></xref>), only about 70% of them (56 spots) were associated with the plasma membrane (G), only about 70% of them (56 spots) were associated with the plasma membrane (<xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>D</italic></xref>). Some of these CaD). Some of these Ca2+ spots (15 ± 3) co-localized with synaptic ribbons found in the basal pole of immature IHCs (31 ± 4 ribbons: <xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>E</italic> and <italic>G</italic></xref>). Co-localization was evaluated using E and G). Co-localization was evaluated using z-stack images (see Methods for details). The finding that only about 50% of ribbons were co-localized with Ca2+ channels in immature IHCs was surprising since in adult cells all, or the majority of them, co-localized with Ca2+ channel spots (<xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>F</italic></xref>: see also F: see also Brandt et al. 2005). Such a difference could indicate that the synaptic machinery from early postnatal cells has yet to fully mature, as recently shown for proteins involved in ribbon synapse formation in immature photoreceptor cells (Regus-Leidig et al. 2009). Nevertheless, the above findings show that only a small proportion (∼27%: 15 out of 56 CaV1.3 spots in the membrane) of Ca2+ channels expressed in these immature cells are directly associated with presynaptic active zones, assuming a similar distribution of Ca2+ channels among the immunopositive spots. However, this is not surprising since Ca2+ channels in immature IHCs also have a purely electrical function in the generation of action potentials (APs: Marcotti et al. 2003b) and activation of the small conductance Ca2+-activated K+ current SK2, which has a crucial role in the AP repolarization (Marcotti et al. 2004; Johnson et al. 2007). The specificity of the CaV1.3 antibody was verified by performing experiments on CaV1.3 knockout mice (Platzer et al. 2000) and pre-incubating the antibody with the antigenic peptide (see Supplemental Fig. 1, available online only).', 'Single Ca2+ channel recordings were only performed on the basal pole of IHCs (from around the level of the horizontal dotted line and below in <xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>A–C</italic></xref>), which contains roughly 50% of the total CaA–C), which contains roughly 50% of the total Ca2+ channel spots associated with the membrane. At first, the success rate for observing single Ca2+ channel openings was extremely low (less than 3%), most likely because of channels being masked by afferent terminals contacting immature IHCs (Pujol et al. 1998). In order to improve the recording success rate, IHCs were very briefly and topically perfused with trypsin prior to attempting to seal. After this procedure, 43 out of 313 patches were successful in that they showed Ca2+ channel activity. For the only single Ca2+ channel activity recorded without trypsin, where a current–voltage relation for the Ca2+ current (ICa) could be measured, the channel conductance did not differ from those obtained following enzyme treatment (data not shown). This finding is consistent with previous observations showing that the biophysical properties of the single-channel and macroscopic L-type Ca2+ currents recorded from enzymatic-isolated vestibular hair cells (Prigioni et al. 1992; Rodriguez-Contreras & Yamoah, 2001) were similar to those measured from slice preparations (Russo et al. 2003; Zampini et al. 2006).', 'Due to the presence of extrasynaptic Ca2+ channels within the IHC basal pole (see the single-channel recording area below the horizontal dotted line: <xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1</xref>), it is very likely that some of the patches we recorded would have contained channels located outside the cell active zones. Nevertheless, current evidence from lower vertebrate hair cells suggests that the biophysical properties of L-type Ca), it is very likely that some of the patches we recorded would have contained channels located outside the cell active zones. Nevertheless, current evidence from lower vertebrate hair cells suggests that the biophysical properties of L-type CaV1.3 channels appear homogeneous irrespective of their location within a cell (Rodriguez-Contreras & Yamoah, 2001; Zampini et al. 2006).', 'The number and elementary properties of CaV1.3 Ca2+ channels present at the presynaptic release sites of cochlear IHCs is currently unknown. We estimated that the minimum number of Ca2+ channels present in immature IHCs, considering the low channel Po (0.15 at −20 mV) found in these cells, is likely to be in the order of 10 000 channels (range: 9600–11 000 channels: see Results). This is about six times larger than that previously reported using non-stationary fluctuation analysis in IHCs from young post-hearing mice (about 1800 channels: Brandt et al. 2005; Meyer et al. 2009). A few thousand Ca2+ channels have also been calculated using single Ca2+ channel recordings from lower vertebrate hair cells (∼4500 channels; Rodriguez-Contreras & Yamoah, 2001). Considering that only a small proportion (∼27%) of these 10 000 channels expressed in immature IHCs are likely to be associated with ribbons, each of the 15 active zones present in these cells (ribbons co-localized with plasma membrane Ca2+ channels: <xref ref-type="fig" rid="tjp0588-0187-f1">Fig. 1<italic>E</italic> and <italic>G</italic></xref>) is likely to contain ∼180 CaE and G) is likely to contain ∼180 Ca2+ channels. Therefore, we would expect an average of ∼27 Ca2+ channels to be simultaneously open at each IHC release site near the peak of an action potential (using a Po of 0.15). This value can be assumed to decrease to about one-third in the presence of 1.3 mm extracellular Ca2+ (9 Ca2+ channels simultaneously open/active zone: <xref ref-type="fig" rid="tjp0588-0187-f7">Fig. 7</xref>), which is mainly due to a decreased channel ), which is mainly due to a decreased channel Po (<xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6</xref>). These findings indicate that, because of the low open probability, a large number (∼180) of Ca). These findings indicate that, because of the low open probability, a large number (∼180) of Ca2+ channels per ribbon is required in order to provide, at any instant, sufficient Ca2+ ions to trigger vesicle fusion in the physiological voltage range (<xref ref-type="fig" rid="tjp0588-0187-f7">Fig. 7</xref>).).'], 'tjp0588-0187-f4': ['To calculate the single-channel open and closed times at each membrane potential (<xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4</xref>), data from IHCs were pooled to obtain a distribution of dwell times on a log scale (20 bins/decade) with no normalization of the number of observations for bin amplitude (), data from IHCs were pooled to obtain a distribution of dwell times on a log scale (20 bins/decade) with no normalization of the number of observations for bin amplitude (Sigworth & Sine, 1987). The plots obtained were interpolated, using the maximum-likelihood method, with the following transform of the sum of n (two or three) exponential functions (Sigworth & Sine, 1987):(3)where Pi and τi are the relative area and time constant of the ith component of the distribution. When more Ca2+ channels were present in the patch, time constants were calculated by excluding sweeps containing multiple openings.', 'Theoretical mean dwell times , used to estimate Po in <xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>E</italic></xref>, were derived from the exponential fitting functions according to the relation:\nE, were derived from the exponential fitting functions according to the relation:\n(4)where Pi has the same meaning as above. Open channel probability was also estimated from the mean open and closed times (calculated according to eqn (4)) using the following function:\n(5)', 'Fitting the dwell time distributions revealed three closed (τc1, τc2 and τc3) and two open (τo1 and τo2) time constants (<xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4</xref>). Of the three closed time constants, only τ). Of the three closed time constants, only τc2 was clearly voltage dependent, decreasing with membrane depolarization from 16.5 ms at −67 mV to 1.9 ms at −17 mV (<xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>A</italic></xref>). By contrast, open time constants did not change significantly with membrane depolarization (A). By contrast, open time constants did not change significantly with membrane depolarization (<xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>B</italic></xref>). The relative contribution of the different closed time constants (B). The relative contribution of the different closed time constants (Table 1; see also <xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>C</italic></xref>) showed a decrease in τC) showed a decrease in τc3 (from 50% to 4%) but an increase in τc1 (from 21% to 73%) with depolarization from −67 mV to −17 mV. These data, together with a moderate increase in the relative contribution of τo2 with membrane depolarization (from 5% to 21%, <xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>D</italic></xref>), indicate an overall higher rate of transition to the channel open state. This is consistent with the increased CaD), indicate an overall higher rate of transition to the channel open state. This is consistent with the increased Ca2+ channel Po upon depolarization (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref>). The fitting parameters obtained from the open- and closed-time distribution analysis (F). The fitting parameters obtained from the open- and closed-time distribution analysis (Table 1) were used to derive the Ca2+ channel opening probability (<xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>E</italic></xref>). The advantage of this alternative method, compared to that described in E). The advantage of this alternative method, compared to that described in <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref> (black circles), was that F (black circles), was that Po was only estimated using the Ca2+ channel kinetics and therefore independent of the number of channel openings in each sweep. The very similar voltage dependence and amplitude of Po between the plots in <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref> (black circles) and F (black circles) and <xref ref-type="fig" rid="tjp0588-0187-f4">Fig. 4<italic>E</italic></xref> confirmed the very low open probability of CaE confirmed the very low open probability of CaV1.3 Ca2+ channels. More importantly, the voltage dependence of the single-channel Po closely resembled that of the macroscopic Ca2+ current activation (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>F</italic></xref>, grey triangles), indicating that the estimated kinetic properties of single-channel openings determine those of the whole-cell current.F, grey triangles), indicating that the estimated kinetic properties of single-channel openings determine those of the whole-cell current.'], 'tjp0588-0187-f5': ['The first latency distribution was investigated by measuring the time interval between 63% of the capacitative transient decay (τ: 0.14 ± 0.07 ms, n= 5) and the first opening. These values were corrected for the number of channels in the patch (Colquhoun & Hawkes, 1987). The number of events used for this analysis was smaller than those used for the dwell times, since only the time to the first opening from each trace could be used. The distribution of the first latency was analysed using log–log plots (McManus et al. 1987). Lower and upper bin limits were first set according to a logarithmic scale (6.64 bins per decade). After binning, the number of events (n) was divided by the corresponding bin width (δti), and the natural logarithm of ni/δti ratio was calculated. These values were plotted as a function of x= lnt to construct log–log frequency distribution graphs (see <xref ref-type="fig" rid="tjp0588-0187-f5">Fig. 5</xref>). Exponential fitting of log–log histograms was performed by applying the following double-logarithmic transform of a sum of exponential equations (). Exponential fitting of log–log histograms was performed by applying the following double-logarithmic transform of a sum of exponential equations (McManus et al. 1987):\n(6)where xoj= lnτj, and Wj and τj are the weight coefficient and time constant, respectively, for each exponential component. The above fits were based on a minimum χ2 method.', 'The first latency (e.g. delay between the stimulus onset and first observed Ca2+ channel opening) was investigated in six patches showing two Ca2+ channels per patch. At the membrane potential of −17 mV, the distribution was best fitted by the sum of three exponentials (τ1: 0.7 ms; τ2: 10.5 ms; τ3: 170.5 ms: <xref ref-type="fig" rid="tjp0588-0187-f5">Fig. 5</xref>). Assuming identical gating properties for both Ca). Assuming identical gating properties for both Ca2+ channels present in the recordings, the mean fastest single-channel latency (τ1) is likely to be in the order of 1.4 ms. This value is significantly longer than the activation time constant measured for the ensemble average (∼0.1 ms: obtained from the same patches used for the estimation of the first latency, <xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>C</italic></xref>) and macroscopic (∼0.3 ms, C) and macroscopic (∼0.3 ms, <xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6<italic>D</italic></xref>) CaD) Ca2+ currents. This is likely to result from Ca2+ channel inactivation shortening the time at which the macroscopic Ca2+ current reaches the peak, which could in turn affect the estimation of its activation time constant. However, the capacitive artefact (see Methods) could have masked some initial single Ca2+ channel events resulting in the overestimation of the first latency, thus contributing to the above discrepancy.'], 'tjp0588-0187-f2': ['Single Ca2+ channel currents were recorded from P5–P10 IHCs. The majority of the experiments were performed at body temperature and using near-physiological Ca2+ concentrations (5 mm) and BayK 8644 (5 μm). The use of BayK 8644 was essential when working at body temperature since in its absence the majority of single-channel openings were not resolved and the apparent sub-conductive open states became very frequent. This behaviour was observed even when the signal-to-noise ratio of the recordings was enhanced by increasing the single-channel current with 70 mm Ba2+ (<xref ref-type="fig" rid="tjp0588-0187-f2">Fig. 2<italic>A</italic> and <italic>B</italic></xref>). Although BayK 8644 is known to produce longer CaA and B). Although BayK 8644 is known to produce longer Ca2+ channel openings, it does not significantly affect the single Ca2+ channel amplitude (Hess et al. 1984), as also shown in <xref ref-type="fig" rid="tjp0588-0187-f2">Fig. 2</xref>. The slope conductance of the single Ca. The slope conductance of the single Ca2+ channel current (<xref ref-type="fig" rid="tjp0588-0187-f2">Fig. 2<italic>C</italic></xref>) recorded with BayK 8644 (39.0 ± 1.8 pS, C) recorded with BayK 8644 (39.0 ± 1.8 pS, n= 7) in the pipette solution was similar to that measured in its absence (36.8 ± 3.3 pS, n= 5). In the presence of 5 mm Ca2+, unitary CaV1.3 Ca2+ currents were observed as rare openings at most hyperpolarized membrane potentials that became more frequent with depolarization (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>A</italic></xref>). Sub-conductive states were rarely observed in this condition and accounted for less than 1% of the conductive state (data not shown). The analysis of ensemble-average single CaA). Sub-conductive states were rarely observed in this condition and accounted for less than 1% of the conductive state (data not shown). The analysis of ensemble-average single Ca2+ channel currents showed a fast activation time constant and a slow time-dependent inactivation (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>C</italic></xref>: τ= 0.1 ms and τ= 771 ms, respectively), consistent with that of the macroscopic CaC: τ= 0.1 ms and τ= 771 ms, respectively), consistent with that of the macroscopic Ca2+ current (Marcotti et al. 2003b). The single-channel current–voltage (I–V) relation was linear (<xref ref-type="fig" rid="tjp0588-0187-f3">Fig. 3<italic>D</italic></xref>) with an average slope conductance of 14.4 pS (D) with an average slope conductance of 14.4 pS (n= 9).'], 'tjp0588-0187-f6': ['The majority (>90%) of the macroscopic Ca2+ current in IHCs is carried by L-type Ca2+ channels containing the CaV1.3 subunit (Platzer et al. 2000). The nature of the residual Ca2+ current in IHCs is still unclear. In agreement with whole-cell recordings, our single-channel measurements from the basal pole region of IHCs indicated the presence of a homogeneous population of Ca2+ channels both in terms of current amplitude and kinetics. These biophysical properties also resembled those described in lower vertebrate hair cells from the bullfrog sacculus (Rodriguez-Contreras & Yamoah, 2001) and the chicken semicircular canal, where P/Q and N-type Ca2+ channel blockers were used (Zampini et al. 2006). CaV1.3 Ca2+ channels in immature IHCs can activate at a membrane potential as negative as −70 mV, indicating that they would be capable of generating spontaneous Ca2+ AP activity present in immature IHCs (Marcotti et al. 2003b) without the need of external depolarizing stimuli (Tritsch et al. 2007). The Ca2+ channel fastest time constant of the first latency distribution (τ1: 1.4 ms near −20 mV) can be assumed to decrease by 20% (to ∼1.1 ms) in the presence of 1.3 mm Ca2+ (see <xref ref-type="fig" rid="tjp0588-0187-f6">Fig. 6<italic>D</italic></xref>). Although the first opening delay of these CaD). Although the first opening delay of these Ca2+ channels was found to be sufficiently rapid to support the relatively slow rising phase of IHC action potentials it is unlikely to be suitable for supporting the high-frequency signalling of adult IHCs. However, recent findings have shown that the kinetics of the macroscopic Ca2+ current become faster in adult IHCs (Johnson & Marcotti, 2008), suggesting that some variation in the channel composition and/or their modulation is likely to occur during development.']}
|
V 2+ Elementary properties of Ca1.3 Ca channels expressed in mouse cochlear inner hair cells
| null |
J Physiol
|
1262332800
|
Label-free LC-MS analysis allows determining the differential expression level of proteins in multiple samples, without the use of stable isotopes. This technique is based on the direct comparison of multiple runs, obtained by continuous detection in MS mode. Only differentially expressed peptides are selected for further fragmentation, thus avoiding the bias toward abundant peptides typical of data-dependent tandem MS. The computational framework includes detection, alignment, normalization and matching of peaks across multiple sets, and several software packages are available to address these processing steps. Yet, more care should be taken to improve the quality of the LC-MS maps entering the pipeline, as this parameter severely affects the results of all downstream analyses. In this paper we show how the inclusion of a preprocessing step of background subtraction in a common laboratory pipeline can lead to an enhanced inclusion list of peptides selected for fragmentation and consequently to better protein identification.
|
[
"Chromatography, Liquid",
"Dry Eye Syndromes",
"Eye Proteins",
"Humans",
"Mass Spectrometry",
"Peptide Fragments",
"Proteomics",
"Signal Processing, Computer-Assisted",
"Software",
"Tears"
] |
other
|
PMC2817446
| null |
15
|
[
"{'Citation': 'Iliuk A, Galan J, Tao WA. Playing tag with quantitative proteomics. Analytical and Bioanalytical Chemistry. 2009;393(2):503–513.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18843486'}}}",
"{'Citation': 'Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B. Quantitative mass spectrometry in proteomics: a critical review. Analytical and Bioanalytical Chemistry. 2007;389(4):1017–1031.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17668192'}}}",
"{'Citation': 'America AHP, Cordewener JHG. Comparative LC-MS: a landscape of peaks and valleys. Proteomics. 2008;8(4):731–749.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18297651'}}}",
"{'Citation': 'Hilario M, Kalousis A, Pellegrini C, Müller M. Processing and classification of protein mass spectra. Mass Spectrometry Reviews. 2006;25(3):409–449.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16463283'}}}",
"{'Citation': 'Listgarten J, Emili A. Statistical and computational methods for comparative proteomic profiling using liquid chromatography-tandem mass spectrometry. Molecular and Cellular Proteomics. 2005;4(4):419–434.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15741312'}}}",
"{'Citation': 'Cappadona S, Levander F, Jansson M, James P, Cerutti S, Pattini L. Wavelet-based method for noise characterization and rejection in high-performance liquid chromatography coupled to mass spectrometry. Analytical Chemistry. 2008;80(13):4960–4968.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18510348'}}}",
"{'Citation': 'Nanni P, Mezzanotte L, Roda G, et al. Differential proteomic analysis of HT29 Cl.16E and intestinal epithelial cells by LC ESI/QTOF mass spectrometry. Journal of Proteomics. 2009;72(5):865–873.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19168159'}}}",
"{'Citation': 'Nanni P, Levander F, Roda G, Caponi A, James P, Roda A. A label-free nano-liquid chromatography-mass spectrometry approach for quantitative serum peptidomics in Crohn’s disease patients. Journal of Chromatography B. 2009;877(27):3127–3136.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19683480'}}}",
"{'Citation': 'Windig W, Phalp JM, Payne AW. A noise and background reduction method for component detection in liquid chromatography/mass spectrometry. Analytical Chemistry. 1996;68(20):3602–3606.'}",
"{'Citation': 'Andreev VP, Rejtar T, Chen H-S, Moskovets EV, Ivanov AR, Karger BL. A universal denoising and peak picking algorithm for LC-MS based on matched filtration in the chromatographic time domain. Analytical Chemistry. 2003;75(22):6314–6326.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '14616016'}}}",
"{'Citation': 'Bellew M, Coram M, Fitzgibbon M, et al. A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution LC-MS. Bioinformatics. 2006;22(15):1902–1909.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16766559'}}}",
"{'Citation': 'Häkkinen J, Vincic G, Månsson O, Wårell K, Levander F. The proteios software environment: an extensible multiuser platform for management and analysis of proteomics data. Journal of Proteome Research. 2009;8(6):3037–3043.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19354269'}}}",
"{'Citation': 'Versura P, Nanni P, Bavelloni A, et al. Tear protein changes in mild evaporative dry eye. submitted.'}",
"{'Citation': 'Report of the Dry Eye Workshop (DEWS) The Ocular Surface. 2007;5(2)', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '0'}}}",
"{'Citation': 'Versura P, Frigato M, Mulé R, Malavolta N, Campos EC. A proposal of new ocular items in Sjögren’s syndrome classification criteria. Clinical and Experimental Rheumatology. 2006;24(5):567–572.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17181927'}}}"
] |
J Physiol. 2010 Jan 1; 588(Pt 1):187-199
|
NO-CC CODE
|
|
(A) NHS-ester
formation and labeling of 13. (B) Fluorescence
microscopy images of live MDA-MB-468 and MCF-7 cells treated with
100 nM labeled panitumumab and Hoechst 33342 (1 μM). (C) Flow
cytometry of MDA-MB-468 and MCF-7 cells treated with 100 nM labeled
panitumumab.
|
ol-2014-03398f_0006
|
2
|
eb68626c6824b9353db5dc1fc9e8ca273c8c2eab6a024edf314c9a4d87394219
|
ol-2014-03398f_0006.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
641,
821
] |
[{'image_id': 'ol-2014-03398f_0010', 'image_file_name': 'ol-2014-03398f_0010.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0010.jpg', 'caption': 'Oxygen-Selective\nElectrophiles', 'hash': 'bcef6ba00e4becc670fff4d0f965d5ef9ba1663360351372b40374d114d52f67'}, {'image_id': 'ol-2014-03398f_0006', 'image_file_name': 'ol-2014-03398f_0006.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0006.jpg', 'caption': '(A) NHS-ester\nformation and labeling of 13. (B) Fluorescence\nmicroscopy images of live MDA-MB-468 and MCF-7 cells treated with\n100 nM labeled panitumumab and Hoechst 33342 (1 μM). (C) Flow\ncytometry of MDA-MB-468 and MCF-7 cells treated with 100 nM labeled\npanitumumab.', 'hash': 'eb68626c6824b9353db5dc1fc9e8ca273c8c2eab6a024edf314c9a4d87394219'}, {'image_id': 'ol-2014-03398f_0008', 'image_file_name': 'ol-2014-03398f_0008.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0008.jpg', 'caption': 'Scope of N- to O-Rearrangement', 'hash': 'eb9996c999e90823e51c4b7c6128ec9fd5af0d3e9c5af26f4ced57c60f8dcae7'}, {'image_id': 'ol-2014-03398f_0011', 'image_file_name': 'ol-2014-03398f_0011.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0011.jpg', 'caption': 'Oxygen-Selective\nElectrophiles', 'hash': '0f4d5309c0542412b2d28c5a12873839cc9e956c8e5697937c42d8d3c1a9c88e'}, {'image_id': 'ol-2014-03398f_0009', 'image_file_name': 'ol-2014-03398f_0009.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0009.jpg', 'caption': 'Scope of N- to O-Rearrangement', 'hash': '00ddbbcd920f95a31dbc0ddd04e50a8d6bb068500ecaee42abe84739fdcb7666'}, {'image_id': 'ol-2014-03398f_0007', 'image_file_name': 'ol-2014-03398f_0007.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0007.jpg', 'caption': 'O- to N-Rearrangement\nof 9 to 6', 'hash': 'f278900c90d7b26a61078d74ef43791dcef33eb83f86377a19498a590a82e3ed'}, {'image_id': 'ol-2014-03398f_0003', 'image_file_name': 'ol-2014-03398f_0003.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0003.jpg', 'caption': 'General considerations.', 'hash': 'ec7738478594e35e00b7b2c4601785fc57a2688ffc24ba917dc60d6b1d9aaac2'}, {'image_id': 'ol-2014-03398f_0004', 'image_file_name': 'ol-2014-03398f_0004.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0004.jpg', 'caption': 'Relative rearrangement kinetics of 6 and 18.', 'hash': '2bf0c26130c4ea00091eb22c199ac289cc9c5b15ca7c49df5e46cf0dfb3ca4c7'}, {'image_id': 'ol-2014-03398f_0012', 'image_file_name': 'ol-2014-03398f_0012.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0012.jpg', 'caption': 'No caption found', 'hash': '35420c68340831cf965ae253ecaa306da0ade0f5bba356a072ad728b902acedf'}, {'image_id': 'ol-2014-03398f_0005', 'image_file_name': 'ol-2014-03398f_0005.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0005.jpg', 'caption': 'Stability of 21, 22, and 13 in the presence of 1 mM glutathione\n(GSH) in pH 7.4 PBS. (A) HPLC\nconversion of starting material (10 μM). These data were used\nto obtain the indicated half-lives. (B) Fluorescent signal over time\n(2 μM, λex = 740 nm, λem =\n790 nm).', 'hash': '811827d243aa0ea404f3175217e20f6ed57c491ec5d17f2476e63405b29eb4b4'}, {'image_id': 'ol-2014-03398f_0002', 'image_file_name': 'ol-2014-03398f_0002.jpg', 'image_path': '../data/media_files/PMC4301176/ol-2014-03398f_0002.jpg', 'caption': 'No caption found', 'hash': 'd95d8472d86b290d5328d6092b7aad7276e9d19e2cc2e1d215941dd355737921'}]
|
{'ol-2014-03398f_0003': ['Given the central role of fluorescent\nsmall molecules in many modern biological techniques, it is surprising\ntheir preparation still often relies on inefficient condensation reactions\nrequiring harsh reaction conditions with poor substrate scope.1,2 New synthetic methodologies are needed to enable the identification\nof optimal agents. This is particularly true for near-IR fluorophores,\nwhich find increasing use for a variety of techniques, including in\nclinical settings, due to the low autofluorescence and high tissue\npenetration of light in this range.3 Heptamethine\ncyanines represent a privileged scaffold, and various derivatives\nare the small molecule bioimaging agents of choice with emission maxima\napproaching 800 nm.4−7 Current state-of-the-art compounds are often substituted at the\nC4′ position with phenols (Figure <xref rid="ol-2014-03398f_0003" ref-type="fig">1</xref>A).A).8−11 These widely used molecules suffer from significant liabilities\nincluding poor chemical stability, likely arising from nucleophilic\nexchange reactions at the C4′ position.12−15 Cyanines modified at the C4′\nposition with an O-alkyl substituent are desirable\nbecause these are likely to be quite stable, while maintaining the\nexcellent optical and physical properties associated with C4′-O-substitution. However, such molecules have only rarely\nbeen described16 and are unknown when functionalized\nfor biomolecule conjugation.', 'Here we report a general approach to access C4′-O-alkyl heptamethine cyanines through a new variant of the\nclassical Smiles rearrangement. Traditionally, Smiles rearrangements\nemploy a combination of strong bases and elevated temperatures to\ninduce the α,δ-transposition of heteroatoms in reactions\ndriven by formation of the thermodynamically preferred isomer (Figure <xref rid="ol-2014-03398f_0003" ref-type="fig">1</xref>B).B).17,18 A distinct alternative selectively\nincorporates an electrophile on the heteroatom originally affixed\nto the unsaturated carbon providing otherwise inaccessible products.\nHere we provide the first examples of this concept, a Smiles rearrangement\nwith concurrent electrophile incorporation, to gain access to promising\nC4′-O-alkyl heptamethine cyanines (2) from easily synthesized C4′-N-methylethanolamine-substituted\nprecursors (1). This method avoids the shortcomings of\nthe more orthodox preparative approach for these compounds, intermolecular\nC4′-chloride substitution by direct addition of an alkoxide.\nSuch chloride substitution reactions, which proceed rapidly in excellent\nyield with amine, phenol, and thiol nucleophiles, generally fail in\nthis case due to the poor kinetics of alkoxides in electron transfer\nSRN1 pathways, the putative substitution mechanism.8a In one report, competitive addition of alkoxides\nto the imine-like C2 position has been suggested to intercede.19 In our own initial studies, which are consistent\nwith these previous findings, we found that 5 was not\nobserved when 3 and 4 were combined under\na variety of basic conditions (eq 1).1'], 'ol-2014-03398f_0004': ['We sought\nto provide initial insight regarding the various mechanistic\npossibilities. Divergent reaction pathways were observed between several\nmethylation and acylation conditions (Table 2). While methyl iodide yields the rearrangement product, 12, trimethyloxonium tetrafluoroborate affords methyl ether 16. Similarly, unlike the peptide coupling conditions of entry 4 in\nTable 1, a combination of acetyl chloride and\n2,6-lutidine provided only the O-acetylation product 17 in high yield. The latter result indicates that O-acylated compounds are unlikely to be reaction intermediates\nen route to N-acylated products (e.g., 11). We also have compared the rearrangement kinetics of N-methylethanolamine substituted 6, and the\none-carbon N-methylpropanolamine homologue, 18 (Figure <xref rid="ol-2014-03398f_0004" ref-type="fig">2</xref>). Derivative ). Derivative 6 and 18 were exposed to identical conditions (4 equiv\nof EDCI, 4 equiv of AcOH, 1 equiv of DIPEA), and the reaction progress\nwas monitored by HPLC. While 6 reacts relatively rapidly\n(t1/2 = 37 min) to form 11, 18 proceeds much more slowly to 19 (t1/2 = 720 min), though ultimately in good conversion\nand with satisfying isolated yield (72%). One reasonable mechanistic\nscenario involves initial nitrogen quaternerization and facile intramolecular\ndisplacement. This mechanism rationalizes the kinetic dependence on\nring size (5 faster than 6) by suggesting that acyl-ammonium formation,\nwhich is likely reversible,26 precedes\nrate determining and, presumably, irreversible tetrahedral intermediate\nformation. An alternative mechanism consisting of N-selective electrophilic trapping of a minor equilibrating C4′-O-substituted component of 6 or 7 is also conceivable. However, the lack of a ∼780 nm peak\nor peak shoulder in the absorption spectra under relevant reaction\nconditions suggests that O-linked species are, at\nmost, negligible components of 6 or 7. Nevertheless,\nfurther studies are required.'], 'ol-2014-03398f_0005': ['With access to a range of C4′-O-alkyl heptamethine\ncyanines, we explored their suitability for imaging purposes. Representative\ncompounds 8 and 13 possess similar absorption\nproperties and improved quantum yields relative to a standard heptamethine\ncyanine, indocyanine green (ICG) (Table 3).\nPrevious studies have shown that widely used C4′ phenol- and\nthiol-substituted heptamethine cyanines can be rapidly exchanged by\nthiol nucleophiles under aqueous conditions, with problematic consequences\nduring conjugation reactions of cysteine-containing peptides and macromolecules\nand during DNA sequencing applications.12,14 In addition,\nphenol-to-thiol exchange has been recently observed intracellularly\nin the context of a thiol-sensing platform.15 The thiol reactivity of 13 was compared with phenol-substituted 21 and to S-mercaptoethanol-substituted 22. Solutions of 13, 21, and 22 (10 μM in pH 7.4 PBS buffer with no intentional O2 exclusion) were exposed to 1 mM glutathione, and the reaction\ncourse was monitored by HPLC (Figure <xref rid="ol-2014-03398f_0005" ref-type="fig">3</xref>A). With A). With 21 and 22, rapid conversion to the glutathione\nadduct 23 was observed (t1/2 = 95 and 40 min, respectively). By contrast, alkyl-ether 13 showed no decomposition over the same time period and >90% was\npresent\nafter 3 days. These experiments clearly demonstrate the superior chemical\nstability of these new alkyl-ether variants over conventional C4′\nphenol- or thiol-substituted heptamethine cyanines. We also measured\nthe near-IR fluorescence signal under identical conditions. As shown\nin Figure <xref rid="ol-2014-03398f_0005" ref-type="fig">3</xref>B, the C4′-B, the C4′-O-linked compounds, 13 and 21, initially\nexhibit significantly greater signal than C4′-S-linked 22. Whereas 13 maintains the initial\nvalue, fluorescence from the mixture of 21 and forming 23 diminishes and approaches that of the mixture of 22 and 23. Thus, the loss of C4′-O-linkage is detrimental to the emissive properties of these\nmolecules.'], 'ol-2014-03398f_0006': ['We have examined the use of these\nmolecules as antibody labels\n(Figure <xref rid="ol-2014-03398f_0006" ref-type="fig">4</xref>).).27 Carboxylate 13 was converted to its NHS-ester (TSTU, DMF, 35 °C)\nand then incubated with the anti-HER1 antibody, panitumumab, to provide\nthe labeled antibody with a degree of labeling (DOL) of 2.1–2.2.\nThe conjugate was incubated with HER1+ (MDA-MB-468) and HER1–\n(MCF-7) cells. Characteristic antibody labeling was only observed\nin HER1+ cells by fluorescence microscopy using a standard Cy7 filter\nset (λex = 710 nm, λem = 775 nm,\nFigure <xref rid="ol-2014-03398f_0006" ref-type="fig">4</xref>B). The efficient cellular labeling\nof the fluorophore–antibody conjugate was also confirmed using\nFACS (Figure B). The efficient cellular labeling\nof the fluorophore–antibody conjugate was also confirmed using\nFACS (Figure <xref rid="ol-2014-03398f_0006" ref-type="fig">4</xref>C). These results suggest that C). These results suggest that 13 and other fluorophores that emerge from this new approach\nare likely to be suitable for a range of near-IR fluorescence applications.']}
|
O Electrophile-Integrating Smiles Rearrangement Provides
Previously Inaccessible C4′--Alkyl
Heptamethine Cyanine Fluorophores
| null |
Org Lett
|
1421395200
|
[{'@Label': 'BACKGROUND', '@NlmCategory': 'BACKGROUND', '#text': "It has been reported that the ATP13A2 gene is one of the most susceptible pathogenic genes of Parkinson's disease (PD). PARK9 mutations are found in early-onset PD and familial PD patients. Uygur and Han PD patients in the Xinjiang area were recruited as research subjects to study the differences in the Thr12Met and Ala1144Thr loci mutations of the ATP13A2 gene in these PD populations. This study explored the mutations at the Thr12Met and Ala1144Thr gene loci of the ATP13A2 gene in Parkinson's disease patients in the Uygur and Han populations in the Xinjiang province."}, {'@Label': 'MATERIAL/METHODS', '@NlmCategory': 'METHODS', '#text': 'The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze the Thr12Met and Ala1144Thr mutations of the ATP13A2 gene in a case-control study of 200 age- and sex- matched Uygur and Han PD patients.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'Of the 200 PD patients were studied, 2 from the Han group had a Thr12Met mutation, but Ala1144Thr mutations were not found. Among the Uygur PD patients, no Thr12Met or Ala1144Thr mutations were found.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'Thr12Met and Ala1144Thr mutations of the ATP13A2 gene are rare in the Uygur PD patients in Xinjiang. Overall, the mutation rates of Thr12Met and Ala1144Thr in the Uygur and Han PD patients in the Xinjiang region are low.'}]
|
[
"Adult",
"Aged",
"Aged, 80 and over",
"Amino Acid Substitution",
"Asian People",
"Base Sequence",
"DNA Mutational Analysis",
"Ethnicity",
"Female",
"Genetic Predisposition to Disease",
"Humans",
"Male",
"Middle Aged",
"Molecular Sequence Data",
"Mutation",
"Parkinson Disease",
"Proton-Translocating ATPases"
] |
other
|
PMC4301176
| null |
20
|
[
"{'Citation': 'Xiromerisiou G, Dardiotis E, Tsimourtou V, et al. Genetic basis of Parkinson disease. Neuroses Focus. 2010;28:E7.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '20043722'}}}",
"{'Citation': 'Lesage S, Brice A. Parkinson’s disease: from monogenic forms to genetic susceptibility factors. Hum Mol Genet. 2009;18:R48–59.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19297401'}}}",
"{'Citation': 'Bekris LM, Data IF, Elizabethan CP. The genetics of Parkinson disease. J Geriatr Psychiatry Neurol. 2010;23:228–42.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3044594'}, {'@IdType': 'pubmed', '#text': '20938043'}]}}",
"{'Citation': 'García S, López-Hernández LB, Suarez-Cuenca JA, et al. Low prevalence of most frequent pathogenic variants of six PARK genes in sporadic Parkinson’s disease. Folia Neuropathol. 2014;52(1):22–29.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '24729340'}}}",
"{'Citation': 'Dehay B, Ramirez A, Martinez-Vicente M, et al. Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads toParkinson disease neurodegeneration. Proc Natl Acad Sci USA. 2012;109(24):9611–16.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3386132'}, {'@IdType': 'pubmed', '#text': '22647602'}]}}",
"{'Citation': 'Ramirez A, Heimbach A, Gründemann J, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet. 2006;38:1184–91.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16964263'}}}",
"{'Citation': 'Yang X, Xu Y. Mutations in the ATP13A2 gene and Parkinsonism: a preliminary review. Biomed Res Int. 2014;2014:371256.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4147200'}, {'@IdType': 'pubmed', '#text': '25197640'}]}}",
"{'Citation': 'Chan AY, Baum L, Tang NL, et al. The role of the Ala746Thr variant in the ATP13A2 gene among Chinese patients with Parkinson’s disease. J Clin Neurosci. 2013;20(5):761–62.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4209840'}, {'@IdType': 'pubmed', '#text': '23522931'}]}}",
"{'Citation': 'Dan H, Jifeng G, Lei W, et al. Mutation analysis of ATP13A2 gene in Chinese patients with familial autusomal recessive early-onset Parkinsonism. Chinese Journal of Medical Genetics. 2009;26:567–70. [in Chinese]', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19806583'}}}",
"{'Citation': 'Ning YP, Kanai K, Tomiyama H, et al. PARK9-linked Parkinsonism in eastern Asia: mutation detection in ATP13A2 and clinical phenotype. Neurology. 2008;70:1491–93.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18413573'}}}",
"{'Citation': 'Schultheis PJ, Hagen TT, O’Toole KK, et al. Characterization of the P5 subfamily of P-type transport ATPases in mice. Biochem Biophys Res Commun. 2004;323:731–38.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15381061'}}}",
"{'Citation': 'Coppedè F. Genetics and epigenetics of Parkinson’s disease. Scientific World Journal. 2012;2012:489830.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3353471'}, {'@IdType': 'pubmed', '#text': '22623900'}]}}",
"{'Citation': 'Schulte C, Gasser T. Genetic basis of Parkinson’s disease: inheritance, penetrance, and expression. Appl Clin Genet. 2011;4:67–80.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3681179'}, {'@IdType': 'pubmed', '#text': '23776368'}]}}",
"{'Citation': 'Giller AD, Chesi A, Geddie ML, et al. Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity. Nat Genet. 2009;41:308–15.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC2683786'}, {'@IdType': 'pubmed', '#text': '19182805'}]}}",
"{'Citation': 'Kühlbrandt W. Biology, structure and mechanism of P-type ATPases. Nat Rev Mol Cell Biol. 2004;5:282–95.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15071553'}}}",
"{'Citation': 'Djarmati A, Hagenah J, Reetz K, et al. ATP13A2 variants in early-onset Parkinson’s disease patients and controls. Mov Disord. 2009;24:104–11.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19705361'}}}",
"{'Citation': 'Park JS, Mehta P, Cooper AA, et al. Pathogenic effects of novel mutations in the P-type ATPase ATP13A2 (PARK9) causing Kufor-Rakeb syndrome, a form of early-onset parkinsonism. Hum Mutat. 2011;32:956–64.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '21542062'}}}",
"{'Citation': 'Crosiers D, Ceulemans B, Meeus B, et al. Juvenile dystonia-parkinsonism and dementia caused by a novel ATP13A2 frameshift mutation. Parkinsonism Relat Disord. 2011;17:135–38.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '21094623'}}}",
"{'Citation': 'Masliah E, Rockenstein E, Veinbergs I, et al. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science. 2000;287:1265–69.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '10678833'}}}",
"{'Citation': 'Di Fonzo A, Chien HF, Socal M, et al. ATP13A2 missense mutations in juvenile Parkinsonism and young onset Parkinson disease. Neurology. 2007;68:1557–62.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17485642'}}}"
] |
Org Lett. 2015 Jan 16; 17(2):302-305
|
NO-CC CODE
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|
Selection for reporter activation by Gal4–sfGFP–Fis1p reveals TA mutations inhibiting mitochondrial localization. (A) Missense mutations within the Fis1p TA provide selectable marker activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 or variants expressed from plasmids b128 (V145E), b129 (L139P), b130 (L129P, V138A), or b101 (∆TA) were treated as in Figure 1C. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.
|
691fig2
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2
|
043b34757c0eaf50bc8cc420c5fc576f84b2c63a42885a6b018737c43be14ae7
|
691fig2.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
677,
524
] |
[{'image_id': '691fig2', 'image_file_name': '691fig2.jpg', 'image_path': '../data/media_files/PMC5289845/691fig2.jpg', 'caption': 'Selection for reporter activation by Gal4–sfGFP–Fis1p reveals TA mutations inhibiting mitochondrial localization. (A) Missense mutations within the Fis1p TA provide selectable marker activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 or variants expressed from plasmids b128 (V145E), b129 (L139P), b130 (L129P, V138A), or b101 (∆TA) were treated as in Figure 1C. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.', 'hash': '043b34757c0eaf50bc8cc420c5fc576f84b2c63a42885a6b018737c43be14ae7'}, {'image_id': '691fig5', 'image_file_name': '691fig5.jpg', 'image_path': '../data/media_files/PMC5289845/691fig5.jpg', 'caption': 'Proline substitution is acceptable at a discrete position within the Fis1p TA. (A) Replacement of specific amino acids within the TA of Gal4–sfGFP–Fis1p with proline can lead to Gal4p-mediated selectable marker activation. Strain MaV203, expressing Gal4–sfGFP–Fis1p variants from plasmids b100 (WT), b188 (V134P), b189 (G137P), b129 (L139P), b190 (A140P), b296 (A144P), or b101 (∆TA), was cultured in SC −Trp medium and then spotted to SC −Trp or SMM −His + 20 mM 3-AT medium for 2 days. (B) TAs with specific proline replacements can reduce mitochondrial targeting of a linked fluorescent protein. Variants of the Fis1p TA fused to mCherry were expressed in WT strain CDD961 from plasmids b109 (WT), b208 (V134P), b209 (G137P), b135 (L139P), b210 (A140P), b211 (A144P) and examined, along with mitochondria-targeted GFP, as in Figure 2B. Bar, 5 µm.', 'hash': 'f75c21afdf49a00499721d50a4584297763b767d6e216bf6bb8f386a0e6d10a7'}, {'image_id': '691fig4', 'image_file_name': '691fig4.jpg', 'image_path': '../data/media_files/PMC5289845/691fig4.jpg', 'caption': 'Identification of abundant Gal4–sfGFP–Fis1p clones that are highly enriched upon selection for Gal4–sfGFP–Fis1p nuclear translocation. (A) TA substitution mutations are plotted, with log2 enrichment values provided on the x-axis and sequence counts recovered from the starting pool (SC −Trp) provided on the y-axis. Those replacement mutations that are within the top 75th percentile of mutant abundance in the starting pool and enriched at least fourfold following selection in SMM −Trp −His medium containing 20 mM 3-AT are highlighted in a blue box. (B) Expansion of the highlighted region in A showing specific TA mutations.', 'hash': '3bf096c1823a3d72dc2027ae319727e92f1b2aa56248c6acd5af4cba8a5b8a17'}, {'image_id': '691fig3', 'image_file_name': '691fig3.jpg', 'image_path': '../data/media_files/PMC5289845/691fig3.jpg', 'caption': 'Global discovery of mutations within the TA of a Gal4–sfGFP–Fis1 fusion protein that allow Gal4p-driven transcription. The log2 of enrichment values for each amino acid were calculated for each position following selection in SMM −Trp −His medium containing 20 mM 3-AT. Enrichment values are generated for individual amino acid positions within the TA and not across positions. Black outlines denote the native amino acid at each position. Amino acid replacements not detectable under selective conditions are denoted by black, filled squares. The predicted MAD is indicated by a red line. “X” represents substitution by a stop codon.', 'hash': 'e721dd5ec92755d68638ae0f69684e138efc21ee78f771b54e1390727153f410'}, {'image_id': '691fig7', 'image_file_name': '691fig7.jpg', 'image_path': '../data/media_files/PMC5289845/691fig7.jpg', 'caption': 'Targeting of Fis1p is not dependent upon a specific TA length. (A) Deletion of up to three amino acids or insertion of up to three amino acids does not allow Gal4–sfGFP–Fis1p to activate transcription. MaV203 cells expressing Gal4–sfGFP–Fis1p variants from plasmids b229 (∇1A), b230 (∇2A), b231 (∇3A), b226 (∆G136), b227 (∆A135–G136), b228 (∆A135–G137), or b101 (∆TA) were treated as in Figure 5A. (B) mCherry fused to a Fis1p TA containing an insertion of up to three amino acids in length localizes properly to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b235 (∇1A), b236 (∇2A), or b237 (∇3A), was visualized as in Figure 2B. (C) mCherry, fused to a Fis1p TA deleted of up to three amino acids, is properly targeted to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b232 (∆G136), b233 (∆A135–G136), or b234 (∆A135–G137), was examined as in Figure 2B. Bar, 5 µm.', 'hash': 'fb381982c545c068cd9669524a5190de1c254e599b98d7d02c20105f1a353302'}, {'image_id': '691fig6', 'image_file_name': '691fig6.jpg', 'image_path': '../data/media_files/PMC5289845/691fig6.jpg', 'caption': 'The positively charged carboxyl terminus of the Fis1p TA is important for specific localization to and insertion at the mitochondrial outer membrane. (A) Deletion of the final five amino acids from the Fis1p TA permits transcriptional activation by Gal4–sfGFP–Fis1p. Strain MaV203, harboring plasmids b100 (WT), b253 (R151X), or b101 (∆TA), was treated as in Figure 5A. (B) Removal of the last five amino acids from the Fis1p TA allows mislocalization to the ER. Strain CDD961, expressing mCherry fused to the WT Fis1p TA from plasmid b109 or expressing mCherry linked to a truncated Fis1p TA (R151X) from plasmid b254, was evaluated as in Figure 2B. (C) Strain CDD961 was cured of plasmid pHS1. The resulting strain was transformed with plasmid pJK59 to label ER by expression of Sec63p–GFP, then transformed with either plasmid b109 or plasmid b254 to localize the WT and R151X TAs, and examined by fluorescence microscopy. Bar, 5 µm.', 'hash': '9e691017d93a96099a69b422911bda10e72420f91bf73e6412fe63fce5532709'}, {'image_id': '691fig1', 'image_file_name': '691fig1.jpg', 'image_path': '../data/media_files/PMC5289845/691fig1.jpg', 'caption': 'A genetic selection based on protein mislocalization allows recovery of mutations blocking Fis1p TA localization to mitochondria. (A) Scheme for selection of mutations preventing mitochondrial targeting of the Fis1p TA. Full-length Fis1p is fused to the transcription factor Gal4p. Upon failure of Fis1p to be localized to the mitochondrial OM, Gal4p may be free to translocate to the nucleus and activate the selectable markers HIS3 and URA3. (B) The Gal4–sfGFP–Fis1 fusion localizes to mitochondria. Strain CDD898 was transformed with plasmid b102, which overexpresses the Gal4–sfGFP–Fis1p construct used in this study. Mitochondria were visualized using mCherry fused to the Cox4 presequence expressed from plasmid pHS12–mCherry. Bar, 5 µm. (C) Removal of the TA from Gal4–sfGFP–Fis1p allows proliferation on medium requiring HIS3 activation or URA3 activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 (WT TA), a variant lacking the Fis1p TA from plasmid b101 (∆TA), or harboring empty vector pKS1 was cultured in SC −Trp medium, then, following serial dilution, spotted to SC −Trp, SMM −His + 20 mM 3-AT, or SC −Ura and incubated for 2 days.', 'hash': 'a38c2d99e56ea4252c237c1d7b6faab8fdfd4982461bd2dd8243c4c96b1cf363'}, {'image_id': '691fig8', 'image_file_name': '691fig8.jpg', 'image_path': '../data/media_files/PMC5289845/691fig8.jpg', 'caption': 'Fis1p TA targeting is hindered to a greater extent by inclusion of negatively charged amino acids in the MAD than by positively charged amino acids. (A) Negative charges allow higher transcriptional activity than positive charges when placed at specific positions within the Gal4–sfGFP–Fis1p TA. Strain MaV203 was transformed with plasmids pKS1 (vector), b100 (Gal4–sfGFP–Fis1p), b101 [Gal4–sfGFP–Fis1p(∆TA)], or plasmids encoding the indicated charge replacements within the Fis1p TA (plasmids b173–b187 and b295). The resulting transformants were spotted to SC −Trp medium (1 day, 30°) or SMM −His + 20 mM 3-AT medium (2 days, 30°). (B) mCherry–Fis1(TA)p localization is disrupted more severely by negatively charged amino acids within the MAD than by positively charged amino acids. Strain CDD961 was transformed with plasmids (b192–b207) expressing mCherry linked to Fis1p TAs harboring the indicated substitutions. Cells were visualized as in Figure 2B. Bar, 5 µm.', 'hash': 'da8533a3ba2e41bfa5258029ce31dd1428d94ad383cc7e11c81319c11ff60921'}]
|
{'691fig1': ['The TA of Fis1p is necessary (Mozdy et al. 2000; Beilharz 2003) and sufficient (Kemper et al. 2008; Förtsch et al. 2011) for insertion of this polypeptide into the mitochondrial OM. No cellular machinery involved in Fis1p insertion has been identified (Kemper et al. 2008; Krumpe et al. 2012). Fis1p has been suggested to reach a final topology in the OM in which the amino-terminal bulk of the protein faces the cytosol, a very short and positively charged carboxyl terminus protrudes into the mitochondrial intermembrane space, and the two are connected by a membrane-anchoring domain (MAD) passing through the OM (Mozdy et al. 2000). In developing our selection for TA mutations that diminish Fis1p targeting, we reasoned that fusion of a transcription factor to Fis1p would lead to insertion within the mitochondrial OM and a lack of nuclear function (<xref ref-type="fig" rid="691fig1">Figure 1A</xref>). Mutations within the TA of ). Mutations within the TA of Fis1p that prevent effective membrane insertion would, however, presumably allow the linked transcription factor to enter the nucleus, promote expression of its targets, and allow survival under specific selective conditions, provided that the fusion protein is not degraded, aggregated, or misdirected to another cellular location. Toward this goal, we generated a construct containing the Gal4p transcription factor at the amino terminal end of the polypeptide and full-length Fis1p at the carboxyl terminal end of the protein, since S. cerevisiae strains allowing titratable selection based upon nuclear entry and subsequent binding to Gal4p-responsive DNA elements are readily available. sfGFP (Pédelacq et al. 2005) was placed between the Gal4 and Fis1 moieties and was visible at mitochondria upon overexpression of this fusion protein (<xref ref-type="fig" rid="691fig1">Figure 1B</xref>). While ). While Fis1p has been reported to be homogenously distributed on the mitochondrial surface (Mozdy et al. 2000), puncta containing Gal4–sfGFP–Fis1p are observed, perhaps due to the formation of heteromeric complexes with nuclear import components attempting to transport Gal4–sfGFP–Fis1p to the nucleus.', 'To assess failed Gal4–sfGFP–Fis1p targeting to mitochondria, we specifically took advantage of the Gal4p-driven HIS3 and URA3 auxotrophic markers in MaV203, a strain commonly used for yeast two-hybrid assays (Vidal et al. 1996a). Similar to cells containing an empty vector, Gal4p fused to Fis1p was unable to allow proliferation on medium lacking histidine and containing 20 mM 3-AT to competitively inhibit any His3p produced independently of Gal4p activation (Durfee et al. 1993) or in medium lacking uracil (SC −Ura) (<xref ref-type="fig" rid="691fig1">Figure 1C</xref>). However, the same ). However, the same Gal4–sfGFP–Fis1 polypeptide devoid of its TA [Gal4–sfGFP–Fis1p(∆TA)] permitted ample proliferation on the same two selective media. This result indicated that our fusion protein could translocate to the nucleus upon TA disruption and that any potential lipid binding mediated by the cytosolic domain of Fis1p (Wells and Hill 2011) will not prevent genetic assessment of TA localization.', 'Selection for reporter activation by Gal4–sfGFP–Fis1p reveals TA mutations inhibiting mitochondrial localization. (A) Missense mutations within the Fis1p TA provide selectable marker activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 or variants expressed from plasmids b128 (V145E), b129 (L139P), b130 (L129P, V138A), or b101 (∆TA) were treated as in <xref ref-type="fig" rid="691fig1">Figure 1C</xref>. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.'], '691fig2': ['In the strain used for our selective scheme, a Ura+ phenotype requires greater Gal4p-dependent transcriptional activation than is required for a His+ phenotype (Vidal et al. 1996b). Therefore, we reasoned that initial selection of TA mutants based on a His+ phenotype may provide informative mutations that weaken, but do not totally inhibit membrane association. We used mutagenic PCR to generate altered TAs within the context of a Gal4–sfGFP–Fis1 fusion protein. We then isolated four colonies that proliferated upon SMM −His medium containing 20 mM 3-AT, yet exhibited diminished proliferation on SC −Ura medium when compared to cells expressing the Gal4–sfGFP–Fis1(∆TA) polypeptide. Sanger sequencing of the region encoding the TA of Gal4–sfGFP–Fis1p within these colonies revealed one clone containing a V145E mutation (amino acid numbering provided in this study will correspond to that of the unmodified, full-length Fis1 protein; the necessary and sufficient region for mitochondrial association of Fis1p begins at amino acid L129), two clones containing a L139P mutation, and one clone harboring two mutations: L129P and V138A. Serial dilution assays (<xref ref-type="fig" rid="691fig2">Figure 2A</xref>) confirmed that V145E and L139P provided a less than maximal, but still apparent Ura) confirmed that V145E and L139P provided a less than maximal, but still apparent Ura+ phenotype, with the V145E mutant allowing more rapid proliferation on medium lacking uracil than the L139P mutant. The L129P/V138A mutant provided a His+ phenotype, but could not drive uracil prototrophy, suggesting a less severe localization defect than that exhibited by the other two mutant TAs. Interestingly, the V145E mutation falls within the predicted MAD of the Fis1p TA (Figure S1A), consistent with poor accommodation of a negatively charged amino acid within this hydrophobic stretch of amino acids. Moreover, the Fis1p TA is predicted to be mostly α-helical in nature, and some evidence suggests that helicity is an important determinant of TA targeting to mitochondria (Wattenberg et al. 2007). Therefore, isolation of the potentially helix-disrupting L139P replacement by selection supports the need for TA helicity during mitochondrial targeting.', 'The location of mCherry fused to Fis1p TAs was consistent with our genetic findings. V145E and L139P mutations in the Fis1p TA led to substantial cytosolic and nuclear accumulation of mCherry (<xref ref-type="fig" rid="691fig2">Figure 2B</xref>). Moreover, the L129P/V138A TA, in agreement with its weaker activation of ). Moreover, the L129P/V138A TA, in agreement with its weaker activation of Gal4p targets in our selection system, provided still discernible mitochondrial localization of the mCherry signal, but extraorganellar levels of this mutant fusion protein appeared to be increased compared to mCherry fused to the WT TA. These results suggest that our genetic approach is likely to allow recovery of mutations affecting the ability of the Fis1p TA to moor proteins to the mitochondrial outer membrane.', 'Proline substitution is acceptable at a discrete position within the Fis1p TA. (A) Replacement of specific amino acids within the TA of Gal4–sfGFP–Fis1p with proline can lead to Gal4p-mediated selectable marker activation. Strain MaV203, expressing Gal4–sfGFP–Fis1p variants from plasmids b100 (WT), b188 (V134P), b189 (G137P), b129 (L139P), b190 (A140P), b296 (A144P), or b101 (∆TA), was cultured in SC −Trp medium and then spotted to SC −Trp or SMM −His + 20 mM 3-AT medium for 2 days. (B) TAs with specific proline replacements can reduce mitochondrial targeting of a linked fluorescent protein. Variants of the Fis1p TA fused to mCherry were expressed in WT strain CDD961 from plasmids b109 (WT), b208 (V134P), b209 (G137P), b135 (L139P), b210 (A140P), b211 (A144P) and examined, along with mitochondria-targeted GFP, as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. Bar, 5 µm.. Bar, 5 µm.', 'Fis1p TA targeting is hindered to a greater extent by inclusion of negatively charged amino acids in the MAD than by positively charged amino acids. (A) Negative charges allow higher transcriptional activity than positive charges when placed at specific positions within the Gal4–sfGFP–Fis1p TA. Strain MaV203 was transformed with plasmids pKS1 (vector), b100 (Gal4–sfGFP–Fis1p), b101 [Gal4–sfGFP–Fis1p(∆TA)], or plasmids encoding the indicated charge replacements within the Fis1p TA (plasmids b173–b187 and b295). The resulting transformants were spotted to SC −Trp medium (1 day, 30°) or SMM −His + 20 mM 3-AT medium (2 days, 30°). (B) mCherry–Fis1(TA)p localization is disrupted more severely by negatively charged amino acids within the MAD than by positively charged amino acids. Strain CDD961 was transformed with plasmids (b192–b207) expressing mCherry linked to Fis1p TAs harboring the indicated substitutions. Cells were visualized as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. Bar, 5 µm.. Bar, 5 µm.'], '691fig3': ['While all potential replacement mutations could not be detected within our starting library (Figure S2), and some biases did exist at each TA position, the vast majority of potential amino acid mutations were represented within our pool. A total of 98.9% of potential amino acid replacements were identified in the starting pool cultured in SC −Trp, and 95.9% of TAs with single mutations were represented by at least 10 counts. Quantification of counts from all samples can be found in Table S1. When comparing the mutant pool cultured in SC −Trp with selection in SMM −Trp −His without added 3-AT, there was no appreciable difference in the relative abundance of most mutant TAs, including truncation mutations expected to totally prevent mitochondrial targeting of Gal4–sfGFP–Fis1p (Figure S3A). Such a result is consistent with “leaky” expression of HIS3 independent of Gal4p-driven activation (Durfee et al. 1993). However, upon addition of 3-AT at concentrations of 5 mM (Figure S3B), 10 mM (Figure S3C), or 20 mM (<xref ref-type="fig" rid="691fig3">Figure 3</xref>) to medium lacking histidine, there were substantial shifts in the composition of the mutant pools toward specific amino acids, prompting further experiments that we describe below. The pool cultured in SC −Ura medium showed very strong selection for nonsense mutations within the TA () to medium lacking histidine, there were substantial shifts in the composition of the mutant pools toward specific amino acids, prompting further experiments that we describe below. The pool cultured in SC −Ura medium showed very strong selection for nonsense mutations within the TA (Figure S3D), but less prominent biases among amino acids. When considering our initial findings, in which recovery of uracil prototrophs by our genetic scheme led to a high recovery of frameshift and nonsense mutations, assessment of HIS3 activation seems more informative regarding determinants of Fis1p TA targeting than competition assays performed in the more strongly selective medium lacking uracil. Independently of the primary amino acid sequence, the specific codons used to direct synthesis of a protein can affect that polypeptide’s translation rate, folding, or even synthesis of its transcript (Yu et al. 2015; Zhou et al. 2016). While the data are more “noisy” due to a lack of representation of certain codons at each position, codons encoding the same amino acid generally acted in concert with one another within our selection scheme (Figure S4). Therefore, our focus remained upon the amino acid sequence of library variants rather than on codon sequence.', 'Previous analyses of various tail-anchored mitochondrial proteins similar in general structure to Fis1p suggested that no primary consensus sequence is required for TA insertion (Horie et al. 2002; Beilharz 2003; Rapaport 2003). While meaningful alignment of Fis1p TAs across species is difficult due to constraints in amino acid choice within hydrophobic domains and as a consequence of the apparently variable Fis1p TA length across orthologs, only G131 (as pertains to the S. cerevisiae\nFis1p sequence) might be considered highly conserved (Figure S1B). Our comprehensive analysis supports the idea that no consensus sequence within the Fis1p TA is necessary to achieve membrane insertion, since most amino acid replacements within the necessary and sufficient region required for Fis1p targeting, including at position G131, fail to lead to notable selectable reporter activation (<xref ref-type="fig" rid="691fig3">Figure 3</xref>). Consequently, we focused our subsequent analysis on structural characteristics of the TA that might be most important for mitochondrial OM targeting.). Consequently, we focused our subsequent analysis on structural characteristics of the TA that might be most important for mitochondrial OM targeting.', 'The recovery of the L139P mutation during preliminary selection for Fis1p TA mutations indicated that proline may not be acceptable within the hydrophobic core of the Fis1p TA. Our deep mutational scan of the Fis1p TA in SMM −Trp −His + 20 mM 3-AT (<xref ref-type="fig" rid="691fig3">Figure 3</xref>) also strongly indicated that proline insertion across many positions disrupted mitochondrial TA localization. When focusing specifically upon those mutants that were in the top 75% most commonly tallied variants in the starting pool (>126 counts) and enriched at least fourfold in SMM −Trp −His + 20 mM 3-AT, 12 of 33 missense mutations within this set caused an amino acid change to proline () also strongly indicated that proline insertion across many positions disrupted mitochondrial TA localization. When focusing specifically upon those mutants that were in the top 75% most commonly tallied variants in the starting pool (>126 counts) and enriched at least fourfold in SMM −Trp −His + 20 mM 3-AT, 12 of 33 missense mutations within this set caused an amino acid change to proline (<xref ref-type="fig" rid="691fig4">Figure 4</xref>), further indicating failure of TA targeting following placement of proline at many TA positions.), further indicating failure of TA targeting following placement of proline at many TA positions.', 'Subsequently, we carried out directed experiments to further examine poor accommodation of proline within the Fis1p TA. We further examined the L139P mutant that was initially isolated during selection for Fis1p TA targeting mutants, and we also generated four additional, individual proline replacements within Gal4–sfGFP–Fis1p and tested for Gal4p-driven reporter activation. Newly constructed V134P, A140P, and A144P substitutions, consistent with our larger scale analysis (<xref ref-type="fig" rid="691fig3">Figure 3</xref> or or <xref ref-type="fig" rid="691fig4">Figure 4</xref>), provided ample proliferation on medium selective for ), provided ample proliferation on medium selective for HIS3 activation (<xref ref-type="fig" rid="691fig5">Figure 5A</xref>). Upon visualization of mCherry fused to these ). Upon visualization of mCherry fused to these Fis1p TA mutants, V134P, L139P, A140P, and A144P replacements all clearly diminished mCherry localization to mitochondria (<xref ref-type="fig" rid="691fig5">Figure 5B</xref>). Our results suggest that the secondary structure of the ). Our results suggest that the secondary structure of the Fis1p TA is important for its function, and that disruption of helicity at many locations may make targeting to the mitochondrial OM unfavorable. We also noted, however, variability in the propensity of proline replacements to disrupt Fis1p TA targeting. Most prominently, the G137P substitution allowed apparently normal targeting to mitochondria, as assessed by Gal4p-driven reporter activation (<xref ref-type="fig" rid="691fig3">Figure 3</xref> and and <xref ref-type="fig" rid="691fig5">Figure 5A</xref>) and by microscopic analysis () and by microscopic analysis (<xref ref-type="fig" rid="691fig5">Figure 5B</xref>), potentially suggesting the existence of two separable, helical segments within the ), potentially suggesting the existence of two separable, helical segments within the Fis1p TA rather than a single, monolithic helix.', 'Analysis of the data from our deep mutational scan suggested that nonsense mutations throughout much of the TA can allow Gal4–sfGFP–Fis1p to move to the nucleus and activate transcription (<xref ref-type="fig" rid="691fig3">Figure 3</xref>, , <xref ref-type="fig" rid="691fig4">Figure 4</xref>, and , and Figure S3). Stop codons placed within the sequence encoding the highly charged RNKRR pentapeptide near the carboxyl terminus of Fis1p, however, seem to permit some membrane localization, as reported by proliferation rates in selective medium. Therefore, we examined the behavior of a R151X mutant, which lacks all charged amino acids following the predicted MAD (X represents mutation to a stop codon). Supporting partial localization to a cellular membrane, the R151X mutant of Gal4–sfGFP–Fis1p did not activate Gal4-controlled expression of HIS3 to the same extent as a Gal4–sfGFP–Fis1p construct lacking the entire TA (<xref ref-type="fig" rid="691fig6">Figure 6A</xref>). Consistent with those results, the R151X TA directed mCherry to intracellular organelles (). Consistent with those results, the R151X TA directed mCherry to intracellular organelles (<xref ref-type="fig" rid="691fig6">Figure 6B</xref>). However, along with some apparent mitochondrial association, the R151X TA was also clearly localized to the ER (). However, along with some apparent mitochondrial association, the R151X TA was also clearly localized to the ER (<xref ref-type="fig" rid="691fig6">Figure 6C</xref>). Interestingly, ER localization of mCherry fused to the R151X TA was not completely dependent upon ). Interestingly, ER localization of mCherry fused to the R151X TA was not completely dependent upon Get3p, a receptor for ER tail-anchored proteins (Schuldiner et al. 2008), suggesting the existence of an alternative mechanism for localization of the R151X TA to the ER (Figure S6, A and B), such as the newly discovered SND pathway (Aviram et al. 2016). Similarly, R151X TA could be targeted to the ER in get2∆ mutants, lacking a receptor for several ER-directed tail-anchored proteins (Figure S6C). However, puncta that might indicate association with Get3p (Schuldiner et al. 2008) were apparent in some get2∆ cells, raising the unexplored possibility that Get3p can bind at least some portion of the cytosolic pool of R151X TA.', 'Our deep mutational scan of the Fis1p TA demonstrated that Gal4–sfGFP–Fis1p was generally able to activate gene expression when aspartate or glutamate was placed within the MAD (<xref ref-type="fig" rid="691fig3">Figure 3</xref>). In fact, upon examination of those amino acid replacements found within the top three quartiles of counts in the initial library and also enriched at least fourfold upon culture in SMM −Trp −His + 20 mM 3-AT, 18 of 33 missense mutations were aspartate or glutamate substitutions (). In fact, upon examination of those amino acid replacements found within the top three quartiles of counts in the initial library and also enriched at least fourfold upon culture in SMM −Trp −His + 20 mM 3-AT, 18 of 33 missense mutations were aspartate or glutamate substitutions (<xref ref-type="fig" rid="691fig4">Figure 4</xref>). We were surprised to find that placement of positively charged arginine or lysine residues appeared to be much more acceptable within the MAD of ). We were surprised to find that placement of positively charged arginine or lysine residues appeared to be much more acceptable within the MAD of Fis1p than aspartate or glutamate; none of the amino acid substitutions within this high-count, high-enrichment set were by lysine or arginine.'], '691fig5': ['The positively charged carboxyl terminus of the Fis1p TA is important for specific localization to and insertion at the mitochondrial outer membrane. (A) Deletion of the final five amino acids from the Fis1p TA permits transcriptional activation by Gal4–sfGFP–Fis1p. Strain MaV203, harboring plasmids b100 (WT), b253 (R151X), or b101 (∆TA), was treated as in <xref ref-type="fig" rid="691fig5">Figure 5A</xref>. (B) Removal of the last five amino acids from the Fis1p TA allows mislocalization to the ER. Strain CDD961, expressing mCherry fused to the WT Fis1p TA from plasmid b109 or expressing mCherry linked to a truncated Fis1p TA (R151X) from plasmid b254, was evaluated as in . (B) Removal of the last five amino acids from the Fis1p TA allows mislocalization to the ER. Strain CDD961, expressing mCherry fused to the WT Fis1p TA from plasmid b109 or expressing mCherry linked to a truncated Fis1p TA (R151X) from plasmid b254, was evaluated as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. (C) Strain CDD961 was cured of plasmid pHS1. The resulting strain was transformed with plasmid pJK59 to label ER by expression of Sec63p–GFP, then transformed with either plasmid b109 or plasmid b254 to localize the WT and R151X TAs, and examined by fluorescence microscopy. Bar, 5 µm.. (C) Strain CDD961 was cured of plasmid pHS1. The resulting strain was transformed with plasmid pJK59 to label ER by expression of Sec63p–GFP, then transformed with either plasmid b109 or plasmid b254 to localize the WT and R151X TAs, and examined by fluorescence microscopy. Bar, 5 µm.', 'Targeting of Fis1p is not dependent upon a specific TA length. (A) Deletion of up to three amino acids or insertion of up to three amino acids does not allow Gal4–sfGFP–Fis1p to activate transcription. MaV203 cells expressing Gal4–sfGFP–Fis1p variants from plasmids b229 (∇1A), b230 (∇2A), b231 (∇3A), b226 (∆G136), b227 (∆A135–G136), b228 (∆A135–G137), or b101 (∆TA) were treated as in <xref ref-type="fig" rid="691fig5">Figure 5A</xref>. (B) mCherry fused to a Fis1p TA containing an insertion of up to three amino acids in length localizes properly to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b235 (∇1A), b236 (∇2A), or b237 (∇3A), was visualized as in . (B) mCherry fused to a Fis1p TA containing an insertion of up to three amino acids in length localizes properly to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b235 (∇1A), b236 (∇2A), or b237 (∇3A), was visualized as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. (C) mCherry, fused to a Fis1p TA deleted of up to three amino acids, is properly targeted to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b232 (∆G136), b233 (∆A135–G136), or b234 (∆A135–G137), was examined as in . (C) mCherry, fused to a Fis1p TA deleted of up to three amino acids, is properly targeted to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b232 (∆G136), b233 (∆A135–G136), or b234 (∆A135–G137), was examined as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. Bar, 5 µm.. Bar, 5 µm.'], '691fig7': ['Targeting of tail-anchored proteins to specific membranes has been suggested to depend, at least in part, upon the specific length of the MAD within the TA (Isenmann et al. 1998; Horie et al. 2002). We reasoned that the region within the MAD at which prolines do not strongly disrupt mitochondrial targeting, as defined by our analysis, may be amenable to the insertion or deletion of new amino acids, thereby allowing us to test the relationship between Fis1p TA length and mitochondrial targeting. We inserted one (∇1A), two (∇2A), or three (∇3A) additional alanines between A135 and G136 within the TA of Gal4–sfGFP–Fis1p, but none of these mutant constructs led to apparent HIS3 (<xref ref-type="fig" rid="691fig7">Figure 7A</xref>) activation. We then analyzed the location of mCherry fused to a ) activation. We then analyzed the location of mCherry fused to a Fis1p TA carrying these same insertions. All constructs were localized properly to mitochondria (<xref ref-type="fig" rid="691fig7">Figure 7B</xref>).).', 'Next, we deleted one (∆G136), two (∆A135–G136), or three (∆A135–G137) amino acids within the Fis1p MAD and performed similar assays. Like our insertion mutants, deletion mutants were apparently targeted to a membrane, as assessed by Gal4p-driven reporter transcription (<xref ref-type="fig" rid="691fig7">Figure 7A</xref>). Moreover, mCherry-). Moreover, mCherry-Fis1(TA)p remained localized to mitochondria when up to three amino acids were deleted (<xref ref-type="fig" rid="691fig7">Figure 7C</xref>).).'], '691fig8': ['To further pursue the possibility that positively charged amino acids can be accommodated within the Fis1p MAD, we mutated four amino acids within the hydrophobic stretch of the Fis1p TA to aspartate, glutamate, lysine, or arginine. Specifically, we generated amino acid replacements at positions V132, A140, A144, or F148, then retested these mutants under selection for Gal4–sfGFP–Fis1p transcriptional activity. The results from our global analysis were verified, with aspartate and glutamate mutations providing stronger reporter activation than lysine and arginine mutations (<xref ref-type="fig" rid="691fig8">Figure 8A</xref>). Only the A144D mutation provided sufficient ). Only the A144D mutation provided sufficient Gal4p activity for proliferation on medium lacking uracil (Figure S9A) after 2 days of incubation, suggesting a very severe TA localization defect caused by this substitution. We noted that these mutant Gal4–sfGFP–Fis1p constructs exhibit altered behavior at different temperatures. For example, lysine and arginine substitutions at positions A140, A144, or F148 led to reduced proliferation at 37° under conditions selective for HIS3 activation (Figure S9B) when compared to the same TA substitutions assessed at 18° (Figure S9C) or 30° (extended incubation, Figure S9D). This outcome is consistent with the idea that altered phospholipid dynamics at different temperatures may lead to consequent changes in TA insertion efficiency (de Mendoza and Cronan 1983).', 'We then tested the ability of these Fis1p TAs containing charge substitutions to promote mitochondrial localization of mCherry. At positions V132 and F148, locations within the MAD nearer to the putative water–lipid bilayer interface, mutation to positively charged amino acids allowed abundant localization to mitochondria (<xref ref-type="fig" rid="691fig8">Figure 8B</xref>). In contrast, mutation to negatively charged amino acids clearly hindered mitochondrial targeting. We noted that F148D and F148E replacements hampered mitochondrial localization more severely than V132D and V132E replacements, consistent with phenotypic testing of ). In contrast, mutation to negatively charged amino acids clearly hindered mitochondrial targeting. We noted that F148D and F148E replacements hampered mitochondrial localization more severely than V132D and V132E replacements, consistent with phenotypic testing of Gal4–sfGFP–Fis1 fusion proteins. At position A144, lying more deeply within the predicted MAD, all charge mutations inhibited mCherry targeting, yet A144K and A144R substitutions allowed some mCherry localization at mitochondria, while A144D and A144E mutants appeared undetectable at mitochondria. Finally, no mitochondrial signal was apparent for any of the charge mutants tested at position A140 of the Fis1p TA. However, A140K and A140R mutants differed from A140D and A140E mutants by localizing to other membranes within the cell, including the plasma membrane, rather than providing a diffuse cytosolic signal. Msp1p removal did not permit relocalization of tail-anchored fluorescent proteins to mitochondria (Figure S5C), supporting the idea that charge replacements within the Fis1p TA lead to a failure of association with the OM rather than enhanced removal from mitochondria. Removal of UBQLN1 ortholog Dsk2p also had no discernible effect on mCherry–Fis1(TA)p mutant localization (Figure S5D).']}
|
In Vivo Evidence for Amino Acid Snorkeling from a High-Resolution, Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane
|
[
"mitochondrial protein targeting",
"mitochondrial division",
"membrane insertion",
"amino acid snorkeling",
"deep mutational scanning"
] |
Genetics
|
1487404800
|
Over the past 5 years there have been a number of new initiatives focused on improving birth outcomes and reducing infant mortality, including a renewed focus on the complex interactions between motherhood and infancy that influence lifelong health trajectories. Beginning in 2012, the Association of Maternal & Child Health Programs (AMCHP) facilitated a series of meetings to enhance coordination across initiatives. Emerging from these conversations was a shared desire across stakeholders to reimagine the postpartum visit and improve postpartum care and wellness. AMCHP convened a Postpartum Think-Tank Meeting in 2014 to map the system of postpartum care and identify levers for its transformation. The meeting findings are presented in an infographic which frames the challenges and proposed solutions from the woman's perspective. The infographic describes maternal issues and concerns along with a concise summary of the recommended solutions. Strategies include creating integrated services and seamless care transitions from preconception through postpartum and well-baby; business, community, and government support, including paid parental leave, health insurance and spaces for new parents to meet each other; and mother-centered care, including quality visits on her schedule with complete and culturally appropriate information. These solutions catalyze a postpartum system of care that supports women, children, and families by infusing new ideas and capitalizing on existing opportunities and resources.
|
[
"Child",
"Female",
"Health Promotion",
"Health Services Needs and Demand",
"Humans",
"Infant",
"Infant Mortality",
"Maternal-Child Health Centers",
"Mothers",
"Postpartum Period",
"Pregnancy",
"Primary Health Care",
"Quality Assurance, Health Care",
"Social Support"
] |
other
|
PMC5289845
| null |
7
|
[
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"{'Citation': 'Coyle S. B. Health-related quality of life of mothers: A review of the research. Health Care for Women International. 2009;30(6):484–506. doi: 10.1080/07399330902801260.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1080/07399330902801260'}, {'@IdType': 'pubmed', '#text': '19418322'}]}}",
"{'Citation': 'Declercq, E. R., Sakala, C., Corry, M. P., Applebaum, S., & Herrlich, A. III (2013). Listening to Mothers III: Report of the third national US survey of women’s childbearing experiences. New York: Childbirth Connection; 2013.'}",
"{'Citation': 'Howell E. A. Lack of patient preparation for the postpartum period and patients’ satisfaction with their obstetric clinicians. Obstetrics & Gynecology. 2010;115:284–289. doi: 10.1097/AOG.0b013e3181c8b39b.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1097/AOG.0b013e3181c8b39b'}, {'@IdType': 'pubmed', '#text': '20093900'}]}}",
"{'Citation': 'Martin A., Horowitz C., Balbierz A., Howell E. A. Views of women and clinicians on postpartum preparation and recovery. Maternal and Child Health Journal. 2014;18(3):707–713. doi: 10.1007/s10995-013-1297-7.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1007/s10995-013-1297-7'}, {'@IdType': 'pmc', '#text': 'PMC4304667'}, {'@IdType': 'pubmed', '#text': '23775250'}]}}"
] |
Genetics. 2017 Feb 18; 205(2):691-705
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NO-CC CODE
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Proline substitution is acceptable at a discrete position within the Fis1p TA. (A) Replacement of specific amino acids within the TA of Gal4–sfGFP–Fis1p with proline can lead to Gal4p-mediated selectable marker activation. Strain MaV203, expressing Gal4–sfGFP–Fis1p variants from plasmids b100 (WT), b188 (V134P), b189 (G137P), b129 (L139P), b190 (A140P), b296 (A144P), or b101 (∆TA), was cultured in SC −Trp medium and then spotted to SC −Trp or SMM −His + 20 mM 3-AT medium for 2 days. (B) TAs with specific proline replacements can reduce mitochondrial targeting of a linked fluorescent protein. Variants of the Fis1p TA fused to mCherry were expressed in WT strain CDD961 from plasmids b109 (WT), b208 (V134P), b209 (G137P), b135 (L139P), b210 (A140P), b211 (A144P) and examined, along with mitochondria-targeted GFP, as in Figure 2B. Bar, 5 µm.
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691fig5
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f75c21afdf49a00499721d50a4584297763b767d6e216bf6bb8f386a0e6d10a7
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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757,
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[{'image_id': '691fig2', 'image_file_name': '691fig2.jpg', 'image_path': '../data/media_files/PMC5289845/691fig2.jpg', 'caption': 'Selection for reporter activation by Gal4–sfGFP–Fis1p reveals TA mutations inhibiting mitochondrial localization. (A) Missense mutations within the Fis1p TA provide selectable marker activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 or variants expressed from plasmids b128 (V145E), b129 (L139P), b130 (L129P, V138A), or b101 (∆TA) were treated as in Figure 1C. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.', 'hash': '043b34757c0eaf50bc8cc420c5fc576f84b2c63a42885a6b018737c43be14ae7'}, {'image_id': '691fig5', 'image_file_name': '691fig5.jpg', 'image_path': '../data/media_files/PMC5289845/691fig5.jpg', 'caption': 'Proline substitution is acceptable at a discrete position within the Fis1p TA. (A) Replacement of specific amino acids within the TA of Gal4–sfGFP–Fis1p with proline can lead to Gal4p-mediated selectable marker activation. Strain MaV203, expressing Gal4–sfGFP–Fis1p variants from plasmids b100 (WT), b188 (V134P), b189 (G137P), b129 (L139P), b190 (A140P), b296 (A144P), or b101 (∆TA), was cultured in SC −Trp medium and then spotted to SC −Trp or SMM −His + 20 mM 3-AT medium for 2 days. (B) TAs with specific proline replacements can reduce mitochondrial targeting of a linked fluorescent protein. Variants of the Fis1p TA fused to mCherry were expressed in WT strain CDD961 from plasmids b109 (WT), b208 (V134P), b209 (G137P), b135 (L139P), b210 (A140P), b211 (A144P) and examined, along with mitochondria-targeted GFP, as in Figure 2B. Bar, 5 µm.', 'hash': 'f75c21afdf49a00499721d50a4584297763b767d6e216bf6bb8f386a0e6d10a7'}, {'image_id': '691fig4', 'image_file_name': '691fig4.jpg', 'image_path': '../data/media_files/PMC5289845/691fig4.jpg', 'caption': 'Identification of abundant Gal4–sfGFP–Fis1p clones that are highly enriched upon selection for Gal4–sfGFP–Fis1p nuclear translocation. (A) TA substitution mutations are plotted, with log2 enrichment values provided on the x-axis and sequence counts recovered from the starting pool (SC −Trp) provided on the y-axis. Those replacement mutations that are within the top 75th percentile of mutant abundance in the starting pool and enriched at least fourfold following selection in SMM −Trp −His medium containing 20 mM 3-AT are highlighted in a blue box. (B) Expansion of the highlighted region in A showing specific TA mutations.', 'hash': '3bf096c1823a3d72dc2027ae319727e92f1b2aa56248c6acd5af4cba8a5b8a17'}, {'image_id': '691fig3', 'image_file_name': '691fig3.jpg', 'image_path': '../data/media_files/PMC5289845/691fig3.jpg', 'caption': 'Global discovery of mutations within the TA of a Gal4–sfGFP–Fis1 fusion protein that allow Gal4p-driven transcription. The log2 of enrichment values for each amino acid were calculated for each position following selection in SMM −Trp −His medium containing 20 mM 3-AT. Enrichment values are generated for individual amino acid positions within the TA and not across positions. Black outlines denote the native amino acid at each position. Amino acid replacements not detectable under selective conditions are denoted by black, filled squares. The predicted MAD is indicated by a red line. “X” represents substitution by a stop codon.', 'hash': 'e721dd5ec92755d68638ae0f69684e138efc21ee78f771b54e1390727153f410'}, {'image_id': '691fig7', 'image_file_name': '691fig7.jpg', 'image_path': '../data/media_files/PMC5289845/691fig7.jpg', 'caption': 'Targeting of Fis1p is not dependent upon a specific TA length. (A) Deletion of up to three amino acids or insertion of up to three amino acids does not allow Gal4–sfGFP–Fis1p to activate transcription. MaV203 cells expressing Gal4–sfGFP–Fis1p variants from plasmids b229 (∇1A), b230 (∇2A), b231 (∇3A), b226 (∆G136), b227 (∆A135–G136), b228 (∆A135–G137), or b101 (∆TA) were treated as in Figure 5A. (B) mCherry fused to a Fis1p TA containing an insertion of up to three amino acids in length localizes properly to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b235 (∇1A), b236 (∇2A), or b237 (∇3A), was visualized as in Figure 2B. (C) mCherry, fused to a Fis1p TA deleted of up to three amino acids, is properly targeted to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b232 (∆G136), b233 (∆A135–G136), or b234 (∆A135–G137), was examined as in Figure 2B. Bar, 5 µm.', 'hash': 'fb381982c545c068cd9669524a5190de1c254e599b98d7d02c20105f1a353302'}, {'image_id': '691fig6', 'image_file_name': '691fig6.jpg', 'image_path': '../data/media_files/PMC5289845/691fig6.jpg', 'caption': 'The positively charged carboxyl terminus of the Fis1p TA is important for specific localization to and insertion at the mitochondrial outer membrane. (A) Deletion of the final five amino acids from the Fis1p TA permits transcriptional activation by Gal4–sfGFP–Fis1p. Strain MaV203, harboring plasmids b100 (WT), b253 (R151X), or b101 (∆TA), was treated as in Figure 5A. (B) Removal of the last five amino acids from the Fis1p TA allows mislocalization to the ER. Strain CDD961, expressing mCherry fused to the WT Fis1p TA from plasmid b109 or expressing mCherry linked to a truncated Fis1p TA (R151X) from plasmid b254, was evaluated as in Figure 2B. (C) Strain CDD961 was cured of plasmid pHS1. The resulting strain was transformed with plasmid pJK59 to label ER by expression of Sec63p–GFP, then transformed with either plasmid b109 or plasmid b254 to localize the WT and R151X TAs, and examined by fluorescence microscopy. Bar, 5 µm.', 'hash': '9e691017d93a96099a69b422911bda10e72420f91bf73e6412fe63fce5532709'}, {'image_id': '691fig1', 'image_file_name': '691fig1.jpg', 'image_path': '../data/media_files/PMC5289845/691fig1.jpg', 'caption': 'A genetic selection based on protein mislocalization allows recovery of mutations blocking Fis1p TA localization to mitochondria. (A) Scheme for selection of mutations preventing mitochondrial targeting of the Fis1p TA. Full-length Fis1p is fused to the transcription factor Gal4p. Upon failure of Fis1p to be localized to the mitochondrial OM, Gal4p may be free to translocate to the nucleus and activate the selectable markers HIS3 and URA3. (B) The Gal4–sfGFP–Fis1 fusion localizes to mitochondria. Strain CDD898 was transformed with plasmid b102, which overexpresses the Gal4–sfGFP–Fis1p construct used in this study. Mitochondria were visualized using mCherry fused to the Cox4 presequence expressed from plasmid pHS12–mCherry. Bar, 5 µm. (C) Removal of the TA from Gal4–sfGFP–Fis1p allows proliferation on medium requiring HIS3 activation or URA3 activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 (WT TA), a variant lacking the Fis1p TA from plasmid b101 (∆TA), or harboring empty vector pKS1 was cultured in SC −Trp medium, then, following serial dilution, spotted to SC −Trp, SMM −His + 20 mM 3-AT, or SC −Ura and incubated for 2 days.', 'hash': 'a38c2d99e56ea4252c237c1d7b6faab8fdfd4982461bd2dd8243c4c96b1cf363'}, {'image_id': '691fig8', 'image_file_name': '691fig8.jpg', 'image_path': '../data/media_files/PMC5289845/691fig8.jpg', 'caption': 'Fis1p TA targeting is hindered to a greater extent by inclusion of negatively charged amino acids in the MAD than by positively charged amino acids. (A) Negative charges allow higher transcriptional activity than positive charges when placed at specific positions within the Gal4–sfGFP–Fis1p TA. Strain MaV203 was transformed with plasmids pKS1 (vector), b100 (Gal4–sfGFP–Fis1p), b101 [Gal4–sfGFP–Fis1p(∆TA)], or plasmids encoding the indicated charge replacements within the Fis1p TA (plasmids b173–b187 and b295). The resulting transformants were spotted to SC −Trp medium (1 day, 30°) or SMM −His + 20 mM 3-AT medium (2 days, 30°). (B) mCherry–Fis1(TA)p localization is disrupted more severely by negatively charged amino acids within the MAD than by positively charged amino acids. Strain CDD961 was transformed with plasmids (b192–b207) expressing mCherry linked to Fis1p TAs harboring the indicated substitutions. Cells were visualized as in Figure 2B. Bar, 5 µm.', 'hash': 'da8533a3ba2e41bfa5258029ce31dd1428d94ad383cc7e11c81319c11ff60921'}]
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{'691fig1': ['The TA of Fis1p is necessary (Mozdy et al. 2000; Beilharz 2003) and sufficient (Kemper et al. 2008; Förtsch et al. 2011) for insertion of this polypeptide into the mitochondrial OM. No cellular machinery involved in Fis1p insertion has been identified (Kemper et al. 2008; Krumpe et al. 2012). Fis1p has been suggested to reach a final topology in the OM in which the amino-terminal bulk of the protein faces the cytosol, a very short and positively charged carboxyl terminus protrudes into the mitochondrial intermembrane space, and the two are connected by a membrane-anchoring domain (MAD) passing through the OM (Mozdy et al. 2000). In developing our selection for TA mutations that diminish Fis1p targeting, we reasoned that fusion of a transcription factor to Fis1p would lead to insertion within the mitochondrial OM and a lack of nuclear function (<xref ref-type="fig" rid="691fig1">Figure 1A</xref>). Mutations within the TA of ). Mutations within the TA of Fis1p that prevent effective membrane insertion would, however, presumably allow the linked transcription factor to enter the nucleus, promote expression of its targets, and allow survival under specific selective conditions, provided that the fusion protein is not degraded, aggregated, or misdirected to another cellular location. Toward this goal, we generated a construct containing the Gal4p transcription factor at the amino terminal end of the polypeptide and full-length Fis1p at the carboxyl terminal end of the protein, since S. cerevisiae strains allowing titratable selection based upon nuclear entry and subsequent binding to Gal4p-responsive DNA elements are readily available. sfGFP (Pédelacq et al. 2005) was placed between the Gal4 and Fis1 moieties and was visible at mitochondria upon overexpression of this fusion protein (<xref ref-type="fig" rid="691fig1">Figure 1B</xref>). While ). While Fis1p has been reported to be homogenously distributed on the mitochondrial surface (Mozdy et al. 2000), puncta containing Gal4–sfGFP–Fis1p are observed, perhaps due to the formation of heteromeric complexes with nuclear import components attempting to transport Gal4–sfGFP–Fis1p to the nucleus.', 'To assess failed Gal4–sfGFP–Fis1p targeting to mitochondria, we specifically took advantage of the Gal4p-driven HIS3 and URA3 auxotrophic markers in MaV203, a strain commonly used for yeast two-hybrid assays (Vidal et al. 1996a). Similar to cells containing an empty vector, Gal4p fused to Fis1p was unable to allow proliferation on medium lacking histidine and containing 20 mM 3-AT to competitively inhibit any His3p produced independently of Gal4p activation (Durfee et al. 1993) or in medium lacking uracil (SC −Ura) (<xref ref-type="fig" rid="691fig1">Figure 1C</xref>). However, the same ). However, the same Gal4–sfGFP–Fis1 polypeptide devoid of its TA [Gal4–sfGFP–Fis1p(∆TA)] permitted ample proliferation on the same two selective media. This result indicated that our fusion protein could translocate to the nucleus upon TA disruption and that any potential lipid binding mediated by the cytosolic domain of Fis1p (Wells and Hill 2011) will not prevent genetic assessment of TA localization.', 'Selection for reporter activation by Gal4–sfGFP–Fis1p reveals TA mutations inhibiting mitochondrial localization. (A) Missense mutations within the Fis1p TA provide selectable marker activation. Strain MaV203 expressing Gal4–sfGFP–Fis1p from plasmid b100 or variants expressed from plasmids b128 (V145E), b129 (L139P), b130 (L129P, V138A), or b101 (∆TA) were treated as in <xref ref-type="fig" rid="691fig1">Figure 1C</xref>. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.. pKS1 (vector) is also provided as a negative control. (B) Missense mutations within the Fis1p TA allow cytosolic and nuclear accumulation of a linked mCherry protein. mCherry fused to variants of the Fis1p TA were expressed in WT strain CDD961 from plasmids b109 (WT), b134 (V145E), b135 (L139P), b136 (L129P, V138A), or b252 (∆TA) and visualized by fluorescence microscopy. Mitochondria were labeled with a mitochondria-targeted GFP expressed from plasmid pHS1. Bar, 5 µm.'], '691fig2': ['In the strain used for our selective scheme, a Ura+ phenotype requires greater Gal4p-dependent transcriptional activation than is required for a His+ phenotype (Vidal et al. 1996b). Therefore, we reasoned that initial selection of TA mutants based on a His+ phenotype may provide informative mutations that weaken, but do not totally inhibit membrane association. We used mutagenic PCR to generate altered TAs within the context of a Gal4–sfGFP–Fis1 fusion protein. We then isolated four colonies that proliferated upon SMM −His medium containing 20 mM 3-AT, yet exhibited diminished proliferation on SC −Ura medium when compared to cells expressing the Gal4–sfGFP–Fis1(∆TA) polypeptide. Sanger sequencing of the region encoding the TA of Gal4–sfGFP–Fis1p within these colonies revealed one clone containing a V145E mutation (amino acid numbering provided in this study will correspond to that of the unmodified, full-length Fis1 protein; the necessary and sufficient region for mitochondrial association of Fis1p begins at amino acid L129), two clones containing a L139P mutation, and one clone harboring two mutations: L129P and V138A. Serial dilution assays (<xref ref-type="fig" rid="691fig2">Figure 2A</xref>) confirmed that V145E and L139P provided a less than maximal, but still apparent Ura) confirmed that V145E and L139P provided a less than maximal, but still apparent Ura+ phenotype, with the V145E mutant allowing more rapid proliferation on medium lacking uracil than the L139P mutant. The L129P/V138A mutant provided a His+ phenotype, but could not drive uracil prototrophy, suggesting a less severe localization defect than that exhibited by the other two mutant TAs. Interestingly, the V145E mutation falls within the predicted MAD of the Fis1p TA (Figure S1A), consistent with poor accommodation of a negatively charged amino acid within this hydrophobic stretch of amino acids. Moreover, the Fis1p TA is predicted to be mostly α-helical in nature, and some evidence suggests that helicity is an important determinant of TA targeting to mitochondria (Wattenberg et al. 2007). Therefore, isolation of the potentially helix-disrupting L139P replacement by selection supports the need for TA helicity during mitochondrial targeting.', 'The location of mCherry fused to Fis1p TAs was consistent with our genetic findings. V145E and L139P mutations in the Fis1p TA led to substantial cytosolic and nuclear accumulation of mCherry (<xref ref-type="fig" rid="691fig2">Figure 2B</xref>). Moreover, the L129P/V138A TA, in agreement with its weaker activation of ). Moreover, the L129P/V138A TA, in agreement with its weaker activation of Gal4p targets in our selection system, provided still discernible mitochondrial localization of the mCherry signal, but extraorganellar levels of this mutant fusion protein appeared to be increased compared to mCherry fused to the WT TA. These results suggest that our genetic approach is likely to allow recovery of mutations affecting the ability of the Fis1p TA to moor proteins to the mitochondrial outer membrane.', 'Proline substitution is acceptable at a discrete position within the Fis1p TA. (A) Replacement of specific amino acids within the TA of Gal4–sfGFP–Fis1p with proline can lead to Gal4p-mediated selectable marker activation. Strain MaV203, expressing Gal4–sfGFP–Fis1p variants from plasmids b100 (WT), b188 (V134P), b189 (G137P), b129 (L139P), b190 (A140P), b296 (A144P), or b101 (∆TA), was cultured in SC −Trp medium and then spotted to SC −Trp or SMM −His + 20 mM 3-AT medium for 2 days. (B) TAs with specific proline replacements can reduce mitochondrial targeting of a linked fluorescent protein. Variants of the Fis1p TA fused to mCherry were expressed in WT strain CDD961 from plasmids b109 (WT), b208 (V134P), b209 (G137P), b135 (L139P), b210 (A140P), b211 (A144P) and examined, along with mitochondria-targeted GFP, as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. Bar, 5 µm.. Bar, 5 µm.', 'Fis1p TA targeting is hindered to a greater extent by inclusion of negatively charged amino acids in the MAD than by positively charged amino acids. (A) Negative charges allow higher transcriptional activity than positive charges when placed at specific positions within the Gal4–sfGFP–Fis1p TA. Strain MaV203 was transformed with plasmids pKS1 (vector), b100 (Gal4–sfGFP–Fis1p), b101 [Gal4–sfGFP–Fis1p(∆TA)], or plasmids encoding the indicated charge replacements within the Fis1p TA (plasmids b173–b187 and b295). The resulting transformants were spotted to SC −Trp medium (1 day, 30°) or SMM −His + 20 mM 3-AT medium (2 days, 30°). (B) mCherry–Fis1(TA)p localization is disrupted more severely by negatively charged amino acids within the MAD than by positively charged amino acids. Strain CDD961 was transformed with plasmids (b192–b207) expressing mCherry linked to Fis1p TAs harboring the indicated substitutions. Cells were visualized as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. Bar, 5 µm.. Bar, 5 µm.'], '691fig3': ['While all potential replacement mutations could not be detected within our starting library (Figure S2), and some biases did exist at each TA position, the vast majority of potential amino acid mutations were represented within our pool. A total of 98.9% of potential amino acid replacements were identified in the starting pool cultured in SC −Trp, and 95.9% of TAs with single mutations were represented by at least 10 counts. Quantification of counts from all samples can be found in Table S1. When comparing the mutant pool cultured in SC −Trp with selection in SMM −Trp −His without added 3-AT, there was no appreciable difference in the relative abundance of most mutant TAs, including truncation mutations expected to totally prevent mitochondrial targeting of Gal4–sfGFP–Fis1p (Figure S3A). Such a result is consistent with “leaky” expression of HIS3 independent of Gal4p-driven activation (Durfee et al. 1993). However, upon addition of 3-AT at concentrations of 5 mM (Figure S3B), 10 mM (Figure S3C), or 20 mM (<xref ref-type="fig" rid="691fig3">Figure 3</xref>) to medium lacking histidine, there were substantial shifts in the composition of the mutant pools toward specific amino acids, prompting further experiments that we describe below. The pool cultured in SC −Ura medium showed very strong selection for nonsense mutations within the TA () to medium lacking histidine, there were substantial shifts in the composition of the mutant pools toward specific amino acids, prompting further experiments that we describe below. The pool cultured in SC −Ura medium showed very strong selection for nonsense mutations within the TA (Figure S3D), but less prominent biases among amino acids. When considering our initial findings, in which recovery of uracil prototrophs by our genetic scheme led to a high recovery of frameshift and nonsense mutations, assessment of HIS3 activation seems more informative regarding determinants of Fis1p TA targeting than competition assays performed in the more strongly selective medium lacking uracil. Independently of the primary amino acid sequence, the specific codons used to direct synthesis of a protein can affect that polypeptide’s translation rate, folding, or even synthesis of its transcript (Yu et al. 2015; Zhou et al. 2016). While the data are more “noisy” due to a lack of representation of certain codons at each position, codons encoding the same amino acid generally acted in concert with one another within our selection scheme (Figure S4). Therefore, our focus remained upon the amino acid sequence of library variants rather than on codon sequence.', 'Previous analyses of various tail-anchored mitochondrial proteins similar in general structure to Fis1p suggested that no primary consensus sequence is required for TA insertion (Horie et al. 2002; Beilharz 2003; Rapaport 2003). While meaningful alignment of Fis1p TAs across species is difficult due to constraints in amino acid choice within hydrophobic domains and as a consequence of the apparently variable Fis1p TA length across orthologs, only G131 (as pertains to the S. cerevisiae\nFis1p sequence) might be considered highly conserved (Figure S1B). Our comprehensive analysis supports the idea that no consensus sequence within the Fis1p TA is necessary to achieve membrane insertion, since most amino acid replacements within the necessary and sufficient region required for Fis1p targeting, including at position G131, fail to lead to notable selectable reporter activation (<xref ref-type="fig" rid="691fig3">Figure 3</xref>). Consequently, we focused our subsequent analysis on structural characteristics of the TA that might be most important for mitochondrial OM targeting.). Consequently, we focused our subsequent analysis on structural characteristics of the TA that might be most important for mitochondrial OM targeting.', 'The recovery of the L139P mutation during preliminary selection for Fis1p TA mutations indicated that proline may not be acceptable within the hydrophobic core of the Fis1p TA. Our deep mutational scan of the Fis1p TA in SMM −Trp −His + 20 mM 3-AT (<xref ref-type="fig" rid="691fig3">Figure 3</xref>) also strongly indicated that proline insertion across many positions disrupted mitochondrial TA localization. When focusing specifically upon those mutants that were in the top 75% most commonly tallied variants in the starting pool (>126 counts) and enriched at least fourfold in SMM −Trp −His + 20 mM 3-AT, 12 of 33 missense mutations within this set caused an amino acid change to proline () also strongly indicated that proline insertion across many positions disrupted mitochondrial TA localization. When focusing specifically upon those mutants that were in the top 75% most commonly tallied variants in the starting pool (>126 counts) and enriched at least fourfold in SMM −Trp −His + 20 mM 3-AT, 12 of 33 missense mutations within this set caused an amino acid change to proline (<xref ref-type="fig" rid="691fig4">Figure 4</xref>), further indicating failure of TA targeting following placement of proline at many TA positions.), further indicating failure of TA targeting following placement of proline at many TA positions.', 'Subsequently, we carried out directed experiments to further examine poor accommodation of proline within the Fis1p TA. We further examined the L139P mutant that was initially isolated during selection for Fis1p TA targeting mutants, and we also generated four additional, individual proline replacements within Gal4–sfGFP–Fis1p and tested for Gal4p-driven reporter activation. Newly constructed V134P, A140P, and A144P substitutions, consistent with our larger scale analysis (<xref ref-type="fig" rid="691fig3">Figure 3</xref> or or <xref ref-type="fig" rid="691fig4">Figure 4</xref>), provided ample proliferation on medium selective for ), provided ample proliferation on medium selective for HIS3 activation (<xref ref-type="fig" rid="691fig5">Figure 5A</xref>). Upon visualization of mCherry fused to these ). Upon visualization of mCherry fused to these Fis1p TA mutants, V134P, L139P, A140P, and A144P replacements all clearly diminished mCherry localization to mitochondria (<xref ref-type="fig" rid="691fig5">Figure 5B</xref>). Our results suggest that the secondary structure of the ). Our results suggest that the secondary structure of the Fis1p TA is important for its function, and that disruption of helicity at many locations may make targeting to the mitochondrial OM unfavorable. We also noted, however, variability in the propensity of proline replacements to disrupt Fis1p TA targeting. Most prominently, the G137P substitution allowed apparently normal targeting to mitochondria, as assessed by Gal4p-driven reporter activation (<xref ref-type="fig" rid="691fig3">Figure 3</xref> and and <xref ref-type="fig" rid="691fig5">Figure 5A</xref>) and by microscopic analysis () and by microscopic analysis (<xref ref-type="fig" rid="691fig5">Figure 5B</xref>), potentially suggesting the existence of two separable, helical segments within the ), potentially suggesting the existence of two separable, helical segments within the Fis1p TA rather than a single, monolithic helix.', 'Analysis of the data from our deep mutational scan suggested that nonsense mutations throughout much of the TA can allow Gal4–sfGFP–Fis1p to move to the nucleus and activate transcription (<xref ref-type="fig" rid="691fig3">Figure 3</xref>, , <xref ref-type="fig" rid="691fig4">Figure 4</xref>, and , and Figure S3). Stop codons placed within the sequence encoding the highly charged RNKRR pentapeptide near the carboxyl terminus of Fis1p, however, seem to permit some membrane localization, as reported by proliferation rates in selective medium. Therefore, we examined the behavior of a R151X mutant, which lacks all charged amino acids following the predicted MAD (X represents mutation to a stop codon). Supporting partial localization to a cellular membrane, the R151X mutant of Gal4–sfGFP–Fis1p did not activate Gal4-controlled expression of HIS3 to the same extent as a Gal4–sfGFP–Fis1p construct lacking the entire TA (<xref ref-type="fig" rid="691fig6">Figure 6A</xref>). Consistent with those results, the R151X TA directed mCherry to intracellular organelles (). Consistent with those results, the R151X TA directed mCherry to intracellular organelles (<xref ref-type="fig" rid="691fig6">Figure 6B</xref>). However, along with some apparent mitochondrial association, the R151X TA was also clearly localized to the ER (). However, along with some apparent mitochondrial association, the R151X TA was also clearly localized to the ER (<xref ref-type="fig" rid="691fig6">Figure 6C</xref>). Interestingly, ER localization of mCherry fused to the R151X TA was not completely dependent upon ). Interestingly, ER localization of mCherry fused to the R151X TA was not completely dependent upon Get3p, a receptor for ER tail-anchored proteins (Schuldiner et al. 2008), suggesting the existence of an alternative mechanism for localization of the R151X TA to the ER (Figure S6, A and B), such as the newly discovered SND pathway (Aviram et al. 2016). Similarly, R151X TA could be targeted to the ER in get2∆ mutants, lacking a receptor for several ER-directed tail-anchored proteins (Figure S6C). However, puncta that might indicate association with Get3p (Schuldiner et al. 2008) were apparent in some get2∆ cells, raising the unexplored possibility that Get3p can bind at least some portion of the cytosolic pool of R151X TA.', 'Our deep mutational scan of the Fis1p TA demonstrated that Gal4–sfGFP–Fis1p was generally able to activate gene expression when aspartate or glutamate was placed within the MAD (<xref ref-type="fig" rid="691fig3">Figure 3</xref>). In fact, upon examination of those amino acid replacements found within the top three quartiles of counts in the initial library and also enriched at least fourfold upon culture in SMM −Trp −His + 20 mM 3-AT, 18 of 33 missense mutations were aspartate or glutamate substitutions (). In fact, upon examination of those amino acid replacements found within the top three quartiles of counts in the initial library and also enriched at least fourfold upon culture in SMM −Trp −His + 20 mM 3-AT, 18 of 33 missense mutations were aspartate or glutamate substitutions (<xref ref-type="fig" rid="691fig4">Figure 4</xref>). We were surprised to find that placement of positively charged arginine or lysine residues appeared to be much more acceptable within the MAD of ). We were surprised to find that placement of positively charged arginine or lysine residues appeared to be much more acceptable within the MAD of Fis1p than aspartate or glutamate; none of the amino acid substitutions within this high-count, high-enrichment set were by lysine or arginine.'], '691fig5': ['The positively charged carboxyl terminus of the Fis1p TA is important for specific localization to and insertion at the mitochondrial outer membrane. (A) Deletion of the final five amino acids from the Fis1p TA permits transcriptional activation by Gal4–sfGFP–Fis1p. Strain MaV203, harboring plasmids b100 (WT), b253 (R151X), or b101 (∆TA), was treated as in <xref ref-type="fig" rid="691fig5">Figure 5A</xref>. (B) Removal of the last five amino acids from the Fis1p TA allows mislocalization to the ER. Strain CDD961, expressing mCherry fused to the WT Fis1p TA from plasmid b109 or expressing mCherry linked to a truncated Fis1p TA (R151X) from plasmid b254, was evaluated as in . (B) Removal of the last five amino acids from the Fis1p TA allows mislocalization to the ER. Strain CDD961, expressing mCherry fused to the WT Fis1p TA from plasmid b109 or expressing mCherry linked to a truncated Fis1p TA (R151X) from plasmid b254, was evaluated as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. (C) Strain CDD961 was cured of plasmid pHS1. The resulting strain was transformed with plasmid pJK59 to label ER by expression of Sec63p–GFP, then transformed with either plasmid b109 or plasmid b254 to localize the WT and R151X TAs, and examined by fluorescence microscopy. Bar, 5 µm.. (C) Strain CDD961 was cured of plasmid pHS1. The resulting strain was transformed with plasmid pJK59 to label ER by expression of Sec63p–GFP, then transformed with either plasmid b109 or plasmid b254 to localize the WT and R151X TAs, and examined by fluorescence microscopy. Bar, 5 µm.', 'Targeting of Fis1p is not dependent upon a specific TA length. (A) Deletion of up to three amino acids or insertion of up to three amino acids does not allow Gal4–sfGFP–Fis1p to activate transcription. MaV203 cells expressing Gal4–sfGFP–Fis1p variants from plasmids b229 (∇1A), b230 (∇2A), b231 (∇3A), b226 (∆G136), b227 (∆A135–G136), b228 (∆A135–G137), or b101 (∆TA) were treated as in <xref ref-type="fig" rid="691fig5">Figure 5A</xref>. (B) mCherry fused to a Fis1p TA containing an insertion of up to three amino acids in length localizes properly to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b235 (∇1A), b236 (∇2A), or b237 (∇3A), was visualized as in . (B) mCherry fused to a Fis1p TA containing an insertion of up to three amino acids in length localizes properly to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b235 (∇1A), b236 (∇2A), or b237 (∇3A), was visualized as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. (C) mCherry, fused to a Fis1p TA deleted of up to three amino acids, is properly targeted to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b232 (∆G136), b233 (∆A135–G136), or b234 (∆A135–G137), was examined as in . (C) mCherry, fused to a Fis1p TA deleted of up to three amino acids, is properly targeted to mitochondria. Strain CDD961, expressing mCherry–Fis1(TA) fusion proteins from plasmids b109 (WT), b232 (∆G136), b233 (∆A135–G136), or b234 (∆A135–G137), was examined as in <xref ref-type="fig" rid="691fig2">Figure 2B</xref>. Bar, 5 µm.. Bar, 5 µm.'], '691fig7': ['Targeting of tail-anchored proteins to specific membranes has been suggested to depend, at least in part, upon the specific length of the MAD within the TA (Isenmann et al. 1998; Horie et al. 2002). We reasoned that the region within the MAD at which prolines do not strongly disrupt mitochondrial targeting, as defined by our analysis, may be amenable to the insertion or deletion of new amino acids, thereby allowing us to test the relationship between Fis1p TA length and mitochondrial targeting. We inserted one (∇1A), two (∇2A), or three (∇3A) additional alanines between A135 and G136 within the TA of Gal4–sfGFP–Fis1p, but none of these mutant constructs led to apparent HIS3 (<xref ref-type="fig" rid="691fig7">Figure 7A</xref>) activation. We then analyzed the location of mCherry fused to a ) activation. We then analyzed the location of mCherry fused to a Fis1p TA carrying these same insertions. All constructs were localized properly to mitochondria (<xref ref-type="fig" rid="691fig7">Figure 7B</xref>).).', 'Next, we deleted one (∆G136), two (∆A135–G136), or three (∆A135–G137) amino acids within the Fis1p MAD and performed similar assays. Like our insertion mutants, deletion mutants were apparently targeted to a membrane, as assessed by Gal4p-driven reporter transcription (<xref ref-type="fig" rid="691fig7">Figure 7A</xref>). Moreover, mCherry-). Moreover, mCherry-Fis1(TA)p remained localized to mitochondria when up to three amino acids were deleted (<xref ref-type="fig" rid="691fig7">Figure 7C</xref>).).'], '691fig8': ['To further pursue the possibility that positively charged amino acids can be accommodated within the Fis1p MAD, we mutated four amino acids within the hydrophobic stretch of the Fis1p TA to aspartate, glutamate, lysine, or arginine. Specifically, we generated amino acid replacements at positions V132, A140, A144, or F148, then retested these mutants under selection for Gal4–sfGFP–Fis1p transcriptional activity. The results from our global analysis were verified, with aspartate and glutamate mutations providing stronger reporter activation than lysine and arginine mutations (<xref ref-type="fig" rid="691fig8">Figure 8A</xref>). Only the A144D mutation provided sufficient ). Only the A144D mutation provided sufficient Gal4p activity for proliferation on medium lacking uracil (Figure S9A) after 2 days of incubation, suggesting a very severe TA localization defect caused by this substitution. We noted that these mutant Gal4–sfGFP–Fis1p constructs exhibit altered behavior at different temperatures. For example, lysine and arginine substitutions at positions A140, A144, or F148 led to reduced proliferation at 37° under conditions selective for HIS3 activation (Figure S9B) when compared to the same TA substitutions assessed at 18° (Figure S9C) or 30° (extended incubation, Figure S9D). This outcome is consistent with the idea that altered phospholipid dynamics at different temperatures may lead to consequent changes in TA insertion efficiency (de Mendoza and Cronan 1983).', 'We then tested the ability of these Fis1p TAs containing charge substitutions to promote mitochondrial localization of mCherry. At positions V132 and F148, locations within the MAD nearer to the putative water–lipid bilayer interface, mutation to positively charged amino acids allowed abundant localization to mitochondria (<xref ref-type="fig" rid="691fig8">Figure 8B</xref>). In contrast, mutation to negatively charged amino acids clearly hindered mitochondrial targeting. We noted that F148D and F148E replacements hampered mitochondrial localization more severely than V132D and V132E replacements, consistent with phenotypic testing of ). In contrast, mutation to negatively charged amino acids clearly hindered mitochondrial targeting. We noted that F148D and F148E replacements hampered mitochondrial localization more severely than V132D and V132E replacements, consistent with phenotypic testing of Gal4–sfGFP–Fis1 fusion proteins. At position A144, lying more deeply within the predicted MAD, all charge mutations inhibited mCherry targeting, yet A144K and A144R substitutions allowed some mCherry localization at mitochondria, while A144D and A144E mutants appeared undetectable at mitochondria. Finally, no mitochondrial signal was apparent for any of the charge mutants tested at position A140 of the Fis1p TA. However, A140K and A140R mutants differed from A140D and A140E mutants by localizing to other membranes within the cell, including the plasma membrane, rather than providing a diffuse cytosolic signal. Msp1p removal did not permit relocalization of tail-anchored fluorescent proteins to mitochondria (Figure S5C), supporting the idea that charge replacements within the Fis1p TA lead to a failure of association with the OM rather than enhanced removal from mitochondria. Removal of UBQLN1 ortholog Dsk2p also had no discernible effect on mCherry–Fis1(TA)p mutant localization (Figure S5D).']}
|
In Vivo Evidence for Amino Acid Snorkeling from a High-Resolution, Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane
|
[
"mitochondrial protein targeting",
"mitochondrial division",
"membrane insertion",
"amino acid snorkeling",
"deep mutational scanning"
] |
Genetics
|
1487404800
|
Over the past 5 years there have been a number of new initiatives focused on improving birth outcomes and reducing infant mortality, including a renewed focus on the complex interactions between motherhood and infancy that influence lifelong health trajectories. Beginning in 2012, the Association of Maternal & Child Health Programs (AMCHP) facilitated a series of meetings to enhance coordination across initiatives. Emerging from these conversations was a shared desire across stakeholders to reimagine the postpartum visit and improve postpartum care and wellness. AMCHP convened a Postpartum Think-Tank Meeting in 2014 to map the system of postpartum care and identify levers for its transformation. The meeting findings are presented in an infographic which frames the challenges and proposed solutions from the woman's perspective. The infographic describes maternal issues and concerns along with a concise summary of the recommended solutions. Strategies include creating integrated services and seamless care transitions from preconception through postpartum and well-baby; business, community, and government support, including paid parental leave, health insurance and spaces for new parents to meet each other; and mother-centered care, including quality visits on her schedule with complete and culturally appropriate information. These solutions catalyze a postpartum system of care that supports women, children, and families by infusing new ideas and capitalizing on existing opportunities and resources.
|
[
"Child",
"Female",
"Health Promotion",
"Health Services Needs and Demand",
"Humans",
"Infant",
"Infant Mortality",
"Maternal-Child Health Centers",
"Mothers",
"Postpartum Period",
"Pregnancy",
"Primary Health Care",
"Quality Assurance, Health Care",
"Social Support"
] |
other
|
PMC5289845
| null |
7
|
[
"{'Citation': 'Aber C., Weiss M., Fawcett J. Contemporary women’s adaptation to motherhood the first 3 to 6weeks postpartum. Nursing Science Quarterly. 2013;26(4):344–351. doi: 10.1177/0894318413500345.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1177/0894318413500345'}, {'@IdType': 'pubmed', '#text': '24085672'}]}}",
"{'Citation': 'Bennett W. L., Chang H. Y., Levine D. M., Wang L., Neale D., Werner E. F., Clark J. M. Utilization of primary and obstetric care after medically complicated pregnancies: An analysis of medical claims data. Journal of General Internal Medicine. 2014;29(4):636–645. doi: 10.1007/s11606-013-2744-2.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1007/s11606-013-2744-2'}, {'@IdType': 'pmc', '#text': 'PMC3965743'}, {'@IdType': 'pubmed', '#text': '24474651'}]}}",
"{'Citation': 'Bennett W. L., Ennen C. S., Carrese J. A., Hill-Briggs F., Levine D. M., Nicholson W. K., Clark J. M. Barriers to and facilitators of postpartum follow-up care in women with recent gestational diabetes mellitus: A qualitative study. Journal of Women’s Health. 2011;20(2):239–245. doi: 10.1089/jwh.2010.2233.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1089/jwh.2010.2233'}, {'@IdType': 'pmc', '#text': 'PMC3064871'}, {'@IdType': 'pubmed', '#text': '21265645'}]}}",
"{'Citation': 'Coyle S. B. Health-related quality of life of mothers: A review of the research. Health Care for Women International. 2009;30(6):484–506. doi: 10.1080/07399330902801260.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1080/07399330902801260'}, {'@IdType': 'pubmed', '#text': '19418322'}]}}",
"{'Citation': 'Declercq, E. R., Sakala, C., Corry, M. P., Applebaum, S., & Herrlich, A. III (2013). Listening to Mothers III: Report of the third national US survey of women’s childbearing experiences. New York: Childbirth Connection; 2013.'}",
"{'Citation': 'Howell E. A. Lack of patient preparation for the postpartum period and patients’ satisfaction with their obstetric clinicians. Obstetrics & Gynecology. 2010;115:284–289. doi: 10.1097/AOG.0b013e3181c8b39b.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1097/AOG.0b013e3181c8b39b'}, {'@IdType': 'pubmed', '#text': '20093900'}]}}",
"{'Citation': 'Martin A., Horowitz C., Balbierz A., Howell E. A. Views of women and clinicians on postpartum preparation and recovery. Maternal and Child Health Journal. 2014;18(3):707–713. doi: 10.1007/s10995-013-1297-7.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1007/s10995-013-1297-7'}, {'@IdType': 'pmc', '#text': 'PMC4304667'}, {'@IdType': 'pubmed', '#text': '23775250'}]}}"
] |
Genetics. 2017 Feb 18; 205(2):691-705
|
NO-CC CODE
|
|
Graphs showing: a) quantification of apoptosis within osteocytes in nonfractured and fractured samples; b) raced and non-raced samples; and c) samples from the sagittal ridge. There is a statistically significant difference in the numbers of apoptotic cells (p < 0.05) between fractured and nonfractured samples (a) and in the numbers of apoptotic cells on the sagittal ridge, with a significant increase in apoptotic cells in the contralateral limb samples (c). No difference was recorded in the numbers of osteocytes in the samples d) and e). f) Representative microscope image of a fluorometric TUNEL apoptosis analysis. Blue stain shows live cell nuclei, green stain shows apoptotic cells. Scale bar 100 μm. F, fractured bones; CL, contralateral bones, C, control bones.
|
bonejointres-07-94-g004
|
2
|
352dadf023f379b7ba2509d77e9c866b9c65f0bf7008a90268fadce93721f347
|
bonejointres-07-94-g004.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
790,
392
] |
[{'image_id': 'bonejointres-07-94-g003', 'image_file_name': 'bonejointres-07-94-g003.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g003.jpg', 'caption': 'Representative micrographs of acid fuchsin-labelled structures in metacarpal bones: a) linear microcrack extending from articular surface; b) staining around blood vessels; and c) diffuse microdamage extending from articular surface. d) Graph showing the amount of damage per surface area of section for microcracks and diffuse damage. There was no difference in the amount of diffuse damage quantified in the three groups; however, there was a statistically significant difference between the amount of microcrack damage/surface area in the lateral condyle (site D, Figure 2) compared with both contralateral and control bones. Scale bar 100\u2009μm. F, fractured bones; Cl, contralateral bones; C, control bones. *p ≤ 0.5; †p ≤ 0.005.', 'hash': '6533454296f0194cbd31dde40f3b48802b28ad084410a92e37a235e273a9feca'}, {'image_id': 'bonejointres-07-94-g004', 'image_file_name': 'bonejointres-07-94-g004.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g004.jpg', 'caption': 'Graphs showing: a) quantification of apoptosis within osteocytes in nonfractured and fractured samples; b) raced and non-raced samples; and c) samples from the sagittal ridge. There is a statistically significant difference in the numbers of apoptotic cells (p\u2009<\u20090.05) between fractured and nonfractured samples (a) and in the numbers of apoptotic cells on the sagittal ridge, with a significant increase in apoptotic cells in the contralateral limb samples (c). No difference was recorded in the numbers of osteocytes in the samples d) and e). f) Representative microscope image of a fluorometric TUNEL apoptosis analysis. Blue stain shows live cell nuclei, green stain shows apoptotic cells. Scale bar 100\u2009μm. F, fractured bones; CL, contralateral bones, C, control bones.', 'hash': '352dadf023f379b7ba2509d77e9c866b9c65f0bf7008a90268fadce93721f347'}, {'image_id': 'bonejointres-07-94-g005', 'image_file_name': 'bonejointres-07-94-g005.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g005.jpg', 'caption': 'Representative photomicrographs of immunohistochemistry in osteocytes in the subchondral bone: a) MMP-13, b) cathepsin K, and c) HTrA1. Osteocytes stained positively are seen as black cells in the fractured samples. In a), the cartilage is stained with toluidine blue; in b), the cartilage is stained with methyl green. Scale bar 100\u2009μm.', 'hash': 'e6bc99af9590dac9f106ea09e541e98c82722fa5e5ba6caa20f604c844767af8'}, {'image_id': 'bonejointres-07-94-g002', 'image_file_name': 'bonejointres-07-94-g002.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g002.jpg', 'caption': 'Dorsopalmar radiographs of a) fractured third metacarpal bone and b) control and contralateral bone. The different regions used in the analysis of staining are shown. A, medial condyle; B, medial condylar groove; C, sagittal ridge; D, lateral condylar fracture site. In c), the sampling site regions are shown, corresponding to A to D in a) and b).', 'hash': '241ce95df7640b4a905efe5fc62df8fa98797f7514fcc1aff5acaaff7931d124'}, {'image_id': 'bonejointres-07-94-g001', 'image_file_name': 'bonejointres-07-94-g001.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g001.jpg', 'caption': 'Dorsopalmar (anterior/posterior) radiographs of a) intact third metacarpal bone (Mc-III) and b) fractured bone of same horse. Photograph of articular surfaces of c) intact bone and d) fractured Mc-III. Left medial and right lateral side.', 'hash': '72498f8889f9d5fd400ed7095770b19bbd34f902a4c4c533172f244f74da39c0'}, {'image_id': 'bonejointres-07-94-g006', 'image_file_name': 'bonejointres-07-94-g006.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g006.jpg', 'caption': 'Representative photomicrographs of sclerostin immunohistochemistry: a) subchondral bone area control sample; b) subchondral area fracture sample; c) deep zone control sample; and d) deep zone fracture sample. Osteocytes – arrowheads in a) and c) – stained positively for sclerostin are seen as black cells in b) and d) (Group F) and are shown by black arrows. In a), the cartilage is stained with toluidine blue. Scale bar 100\u2009μm. c, cartilage; scb, subchondral bone. The cartilage is stained with toluidine blue and is visible in a) and b). The fracture site is to the bottom of the figures in b) and d) (black arrowhead).', 'hash': 'c9fa6ff6ac50aae572c3f183cd383530655eca37fa23d7625266f4c0261ef27f'}, {'image_id': 'bonejointres-07-94-g007', 'image_file_name': 'bonejointres-07-94-g007.jpg', 'image_path': '../data/media_files/PMC5805827/bonejointres-07-94-g007.jpg', 'caption': 'Quantification of sclerostin immunohistochemistry within osteocytes in fractured (F), contralateral (CL) and control limbs (C). There is a significant increase (*p\u2009<\u20090.05) in sclerostin immunoreactivity in the deep zone of the fractured bone. In the photograph, the approximate site of the cartilage/bone interface region (white arrow) and the deeper region is indicated (black arrow).', 'hash': 'cd56b98b508ad658494b26088884622d14c0d33bf6c0b7625e33c131aaa2669c'}]
|
{'bonejointres-07-94-g001': ['Mc-III bones were obtained from Thoroughbred racehorses that had been euthanized on racetracks in California (in one of Stockton, Arcadia, Berkeley, Inglewood, or Pomona), United States following a catastrophic fracture, and were collected as part of the California Horse Racing Board post-mortem programme (<xref ref-type="fig" rid="bonejointres-07-94-g001">Fig. 1</xref>). The study groups were: Group F, distal Mc-III lateral condylar fractures that occurred on the racetrack immediately prior to euthanasia (n\u2009=\u200910), six of which were left-leg fractures and four of which were right-leg fractures; and the contralateral (CL) Group, distal Mc-III contralateral (uninjured) legs from horses in Group F (n\u2009=\u200910). All horses raced on anticlockwise racecourses, so the increased stress was on the left leg. Horses with bilateral fractures were excluded, as were those with concurrent fracture pathology, such as pre-existing stress fractures. A control group, Group C, consisting of distal Mc-III, comprised Thoroughbred horses who had sustained fatal, non-orthopaedic injuries on the racetrack (n\u2009=\u200910). For Groups F and CL, the mean age was 4.1 years (). The study groups were: Group F, distal Mc-III lateral condylar fractures that occurred on the racetrack immediately prior to euthanasia (n\u2009=\u200910), six of which were left-leg fractures and four of which were right-leg fractures; and the contralateral (CL) Group, distal Mc-III contralateral (uninjured) legs from horses in Group F (n\u2009=\u200910). All horses raced on anticlockwise racecourses, so the increased stress was on the left leg. Horses with bilateral fractures were excluded, as were those with concurrent fracture pathology, such as pre-existing stress fractures. A control group, Group C, consisting of distal Mc-III, comprised Thoroughbred horses who had sustained fatal, non-orthopaedic injuries on the racetrack (n\u2009=\u200910). For Groups F and CL, the mean age was 4.1 years (sd 1.2); for Group C, the mean age was 3.9 years (sd 1.5). For all samples, distal thoracic limbs were transected at the level of the carpal bone and stored at -20°C after euthanasia. The time to euthanasia was ten minutes, and samples were preserved frozen for up to six hours post-mortem.'], 'bonejointres-07-94-g002': ['A frontal plane bone block of the distal Mc-III,22,23 approximately 1\u2009cm thick, was prepared using a band saw. The bone block of the joint surface was then divided into four pieces using sagittal plane cuts to create separate blocks of each of the regions of interest: lateral condylar fracture site, medial condyle, medial condylar groove, and sagittal ridge (<xref ref-type="fig" rid="bonejointres-07-94-g002">Fig. 2</xref>).).23', 'Representative micrographs of acid fuchsin-labelled structures in metacarpal bones: a) linear microcrack extending from articular surface; b) staining around blood vessels; and c) diffuse microdamage extending from articular surface. d) Graph showing the amount of damage per surface area of section for microcracks and diffuse damage. There was no difference in the amount of diffuse damage quantified in the three groups; however, there was a statistically significant difference between the amount of microcrack damage/surface area in the lateral condyle (site D, <xref ref-type="fig" rid="bonejointres-07-94-g002">Figure 2</xref>) compared with both contralateral and control bones. Scale bar 100\u2009μm. F, fractured bones; Cl, contralateral bones; C, control bones. *p ≤ 0.5; †p ≤ 0.005.) compared with both contralateral and control bones. Scale bar 100\u2009μm. F, fractured bones; Cl, contralateral bones; C, control bones. *p ≤ 0.5; †p ≤ 0.005.'], 'bonejointres-07-94-g003': ['This study identified both microcracks and diffuse damage in the samples studied (<xref ref-type="fig" rid="bonejointres-07-94-g003">Fig. 3</xref>). There was significantly increased microcrack damage/area in the lateral condylar fracture site of Group F (7.04 cracks/mm). There was significantly increased microcrack damage/area in the lateral condylar fracture site of Group F (7.04 cracks/mm2 (sd 2.91)), compared with Group CL (3.18 cracks/mm2 (sd 4.26)) and Group C (2.93 cracks/mm2 (sd 3.85)), p\u2009=\u20090.002 and p\u2009=\u20090.005, respectively. The p-value for Group F was not significant. When the total microcrack damage was compared against the other three sites, there was no significant difference. Nor was there any significant difference between and of the groups at any site in the amount of diffuse damage/area, with Group F having 3.43 (sd 1.5) discrete areas of staining, compared with 2.92 (sd 1.45) in Group CL and 2.55 (sd 1.67) in Group C.'], 'bonejointres-07-94-g004': ['The DeadEnd fluorometric apoptosis analysis detected apoptotic cells in all samples studied (<xref ref-type="fig" rid="bonejointres-07-94-g004">Fig. 4</xref>). When data from all four sites were pooled, there were significantly fewer apoptotic osteocytes in Group F compared with Group CL (p\u2009=\u20090.002), but there was no difference detected in the percentage of apoptotic osteocytes when Groups F and CL were compared with Group C. The difference was greatest on the sagittal ridge where the rate of apoptotic cells was 22.2% (). When data from all four sites were pooled, there were significantly fewer apoptotic osteocytes in Group F compared with Group CL (p\u2009=\u20090.002), but there was no difference detected in the percentage of apoptotic osteocytes when Groups F and CL were compared with Group C. The difference was greatest on the sagittal ridge where the rate of apoptotic cells was 22.2% (sd 11.0) in Group F compared with 47.0% (sd 19.6) in Group CL (p\u2009=\u20090.007).'], 'bonejointres-07-94-g005': ['Matrix metalloproteinase-13 (MMP-13), HtrA1, and cathepsin K immunoreactivity were detected in all samples studied. Positive staining was detected in the cytoplasm of the osteocytes in the bone. MMP-13, HtrA1, and cathepsin K immunoreactivity was not different among groups or anatomical sites (<xref ref-type="fig" rid="bonejointres-07-94-g005">Fig. 5</xref>).).'], 'bonejointres-07-94-g006': ['Sclerostin immunoreactivity was detected in all samples studied (<xref ref-type="fig" rid="bonejointres-07-94-g006">Fig. 6</xref>). Positive staining was detected in the cytoplasm of the osteocytes in the bone. No staining was detected in the cartilage or within the blood vessels. No differences between sites were detected, except in the lateral condylar fracture site groove. At this site, sclerostin immunohistochemistry showed that in the subchondral bone under the articular surface (‘surface zone’), staining was in the range of 3.9% (). Positive staining was detected in the cytoplasm of the osteocytes in the bone. No staining was detected in the cartilage or within the blood vessels. No differences between sites were detected, except in the lateral condylar fracture site groove. At this site, sclerostin immunohistochemistry showed that in the subchondral bone under the articular surface (‘surface zone’), staining was in the range of 3.9% (sd 2.9) of osteocytes staining positive for sclerostin in all samples studied. In the deep zone, however, sclerostin immunohistochemistry showed that there was a significant increase in positive staining in Group F compared with Group CL, with a mean of 24.4% (sd 19.4) of osteocytes staining positive for sclerostin (p\u2009=\u20090.03) (<xref ref-type="fig" rid="bonejointres-07-94-g007">Fig. 7</xref>). In Group F samples, there was a 4.5-fold increase (p\u2009=\u20090.03) in sclerostin protein-positive cells in the deep zone (24.4%) compared with the cartilage and bone interface (5.4%). In Group CL samples, there was no change in the sclerostin protein between surface and deep zones (2.3% and 3.1%, respectively) (). In Group F samples, there was a 4.5-fold increase (p\u2009=\u20090.03) in sclerostin protein-positive cells in the deep zone (24.4%) compared with the cartilage and bone interface (5.4%). In Group CL samples, there was no change in the sclerostin protein between surface and deep zones (2.3% and 3.1%, respectively) (<xref ref-type="fig" rid="bonejointres-07-94-g007">Fig. 7</xref>).).'], 'bonejointres-07-94-g007': ['In Group C, there was no change in the sclerostin protein between surface and deep zones (9.5% and 10.5%, respectively). However, in the deep zone there was a 2.3-fold increase in sclerostin protein-positive cells in Group F (24.4%) compared with Group C (10.5%). Additionally, in the deep zone there was a 7.9-fold increase (p\u2009=\u20090.03) in sclerostin protein-positive cells in Group F (24.4%) compared with Group C (3.1%) (<xref ref-type="fig" rid="bonejointres-07-94-g007">Fig. 7</xref>).).']}
|
Increased sclerostin associated with stress fracture of the third metacarpal bone in the Thoroughbred racehorse
|
[
"Sclerostin",
"Microdamage",
"Bone fracture",
"Apoptosis",
"Osteocyte"
] |
Bone Joint Res
|
1518076800
|
[{'@Label': 'OBJECTIVES', '@NlmCategory': 'OBJECTIVE', '#text': "Patient-specific (PS) implantation surgical technology has been introduced in recent years and a gradual increase in the associated number of surgical cases has been observed. PS technology uses a patient's own geometry in designing a medical device to provide minimal bone resection with improvement in the prosthetic bone coverage. However, whether PS unicompartmental knee arthroplasty (UKA) provides a better biomechanical effect than standard off-the-shelf prostheses for UKA has not yet been determined, and still remains controversial in both biomechanical and clinical fields. Therefore, the aim of this study was to compare the biomechanical effect between PS and standard off-the-shelf prostheses for UKA."}, {'@Label': 'METHODS', '@NlmCategory': 'METHODS', '#text': 'The contact stresses on the polyethylene (PE) insert, articular cartilage and lateral meniscus were evaluated in PS and standard off-the-shelf prostheses for UKA using a validated finite element model. Gait cycle loading was applied to evaluate the biomechanical effect in the PS and standard UKAs.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'The contact stresses on the PE insert were similar for both the PS and standard UKAs. Compared with the standard UKA, the PS UKA did not show any biomechanical effect on the medial PE insert. However, the contact stresses on the articular cartilage and the meniscus in the lateral compartment following the PS UKA exhibited closer values to the healthy knee joint compared with the standard UKA.'}, {'@Label': 'CONCLUSION', '@NlmCategory': 'CONCLUSIONS', 'b': 'Cite this article', 'i': 'Bone Joint Res', '#text': 'The PS UKA provided mechanics closer to those of the normal knee joint. The decreased contact stress on the opposite compartment may reduce the overall risk of progressive osteoarthritis.: K-T. Kang, J. Son, D-S. Suh, S. K. Kwon, O-R. Kwon, Y-G. Koh. Patient-specific medial unicompartmental knee arthroplasty has a greater protective effect on articular cartilage in the lateral compartment: A Finite Element Analysis. 2018;7:20-27. DOI: 10.1302/2046-3758.71.BJR-2017-0115.R2.'}]
|
[] |
other
|
PMC5805827
| null |
46
|
[
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] |
Bone Joint Res. 2018 Feb 8; 7(1):94-102
|
NO-CC CODE
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|
Phosphorylation of S327 is required for generation of ssDNA in DT40 cells(a) Cells were labelled with 20 μM BrdU and treated with 8 Gy X-rays (where indicated). ssDNA was detected over time by staining with antibody against BrdU and analysed using confocal microscopy. Staining was performed under DNA denaturing (2N HCl) or native conditions as indicated. (b) Kinetics of ssDNA generation from (a). Cells containing 10 or more foci were scored as positive. (c) HR assay, performed as in Fig. 3 with indicated cell lines. Shows that expression of “constitutively activated” CtIPPM restores HR in ctip mutant cells but not in brca1 cells.
|
ukmss-4240-f0004
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2
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b139365902fece93ff47239a98334c19467932e56546a9779affdb938c2bbfc9
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ukmss-4240-f0004.jpg
|
multiple
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multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
727,
439
] |
[{'image_id': 'ukmss-4240-f0001', 'image_file_name': 'ukmss-4240-f0001.jpg', 'image_path': '../data/media_files/PMC2857324/ukmss-4240-f0001.jpg', 'caption': 'Sensitivity of ctip mutant cells to DNA damaging agentsClonogenic survival assays with asynchronous cell populations after exposure to X-rays (upper panel), cisplatin (CDDP, middle panel) and ultraviolet light (UV, lower panel). In this and the following figures, the data presented with error bars are the mean of three independent experiments, and the error bar indicates one standard deviation.', 'hash': '3db6321befd428faf8e672d3d09d8d9fb4fcdc1e395822f3f93dd0f17ce29aa8'}, {'image_id': 'ukmss-4240-f0002', 'image_file_name': 'ukmss-4240-f0002.jpg', 'image_path': '../data/media_files/PMC2857324/ukmss-4240-f0002.jpg', 'caption': 'ctip mutant cells are sensitive to X-rays in both G1 and S/G2 phases of the cell cycle(a) Clonogenic colony survival assay after exposure of cell lines to X-rays, synchronised by elutriation in either S/G2 (upper panel) or G1 (lower panel) stages of the cell cycle. Data represent three independent experiments. (b) Western-blot showing the presence of CtIP in G1 and S/G2. Phosphorylated CtIP is indicated with an arrow. Only unphosphorylated CtIP (*) is seen in G1. Whole cell extracts were prepared from elutriated cell poulations. Where indicated, S/G2 cell extract were treated with λ phosphatase for 2 h at 30°C. Western blot with antibody against actin was used as protein loading control.', 'hash': '487dfcf56b6873be76f2c9ad96f1ded4fbc0a22ab4654f94286951caaf30891a'}, {'image_id': 'ukmss-4240-f0003', 'image_file_name': 'ukmss-4240-f0003.jpg', 'image_path': '../data/media_files/PMC2857324/ukmss-4240-f0003.jpg', 'caption': 'ctip mutant cells are defective for homologous recombination and microhomology-mediated end joining (MMEJ)Repair is indicated by percentage of cells expressing GFP as described in Supplementary Fig. 5. Repair by (a) homologous recombination (HR); (b) single-strand annealing (SSA); (c) accurate non-homologous end-joining (accurate EJ); (d) microhomology-mediated end joining (MMEJ); (e) analysis of DNA sequences at repaired break sites in wild-type and ctip mutant cells. Individual sequences (shown in Supplementary Fig. 4) were classified according to the nature of their joints into “accurate EJ”, “inaccurate EJ” and ”MMEJ”.', 'hash': '192673726a370bb56b90622fcaf3e73ca8c8047312617f14e44b667273e3a6e2'}, {'image_id': 'ukmss-4240-f0004', 'image_file_name': 'ukmss-4240-f0004.jpg', 'image_path': '../data/media_files/PMC2857324/ukmss-4240-f0004.jpg', 'caption': 'Phosphorylation of S327 is required for generation of ssDNA in DT40 cells(a) Cells were labelled with 20 μM BrdU and treated with 8 Gy X-rays (where indicated). ssDNA was detected over time by staining with antibody against BrdU and analysed using confocal microscopy. Staining was performed under DNA denaturing (2N HCl) or native conditions as indicated. (b) Kinetics of ssDNA generation from (a). Cells containing 10 or more foci were scored as positive. (c) HR assay, performed as in Fig. 3 with indicated cell lines. Shows that expression of “constitutively activated” CtIPPM restores HR in ctip mutant cells but not in brca1 cells.', 'hash': 'b139365902fece93ff47239a98334c19467932e56546a9779affdb938c2bbfc9'}]
|
{'ukmss-4240-f0001': ['In common with other DNA repair-defective mutants9,10\u200b, ctip cells exhibit reduced proliferation rate, compared with wild-type cells (Supplementary Fig. 3a). Moreover, in clonogenic survival assays they are highly sensitive to X-rays, which cause DSBs (<xref ref-type="fig" rid="ukmss-4240-f0001">Fig. 1a</xref>). They are also sensitive to cisplatin (CDDP), which generates interstrand DNA crosslinks that may also lead to the generation of DSB during replication (). They are also sensitive to cisplatin (CDDP), which generates interstrand DNA crosslinks that may also lead to the generation of DSB during replication (<xref ref-type="fig" rid="ukmss-4240-f0001">Fig. 1a</xref>). In contrast, ). In contrast, ctip cells are not very sensitive to UV light, which causes pyrimidine dimers and 6-4 photoproducts11 (<xref ref-type="fig" rid="ukmss-4240-f0001">Fig. 1a</xref>). Importantly, expression of human CtIP (). Importantly, expression of human CtIP (HsCtIP) in ctip mutant cells fully restored the resistance of these cells to X-rays, confirming that the human and avian CtIP proteins are functionally conserved (<xref ref-type="fig" rid="ukmss-4240-f0001">Fig. 1a</xref>, , Supplementary Fig. 2d).', 'Previously Yu and Chen demonstrated that CtIP is phosphorylated on serine residue 327 as cells enter S phase, which mediates its interaction with the tumour suppressor BRCA1 that is required for the transient G2/M checkpoint12,17. Therefore, we next considered whether phosphorylation of serine 327 might also regulate the function of CtIP in DSB repair. To investigate this we expressed a mutant form of HsCtIP, in which serine 327 was substituted by alanine (HsCtIPS327A), in ctip cells and examined its sensitivity to X-rays. While expression of HsCtIPS327A improved the survival of ctip cells to X-rays, complementation was only partial (<xref ref-type="fig" rid="ukmss-4240-f0001">Fig.1a</xref>), suggesting that mutation of serine 327 results in loss of some but not all the repair functions of CtIP.), suggesting that mutation of serine 327 results in loss of some but not all the repair functions of CtIP.'], 'ukmss-4240-f0002': ['It was reported that CtIP promotes resistance to DSB-inducing agents exclusively during S and G2 phases5,12. Accordingly, ctip cells isolated by elutriation in S/G2 phase (Supplementary Fig. 4), are 5- to 6-fold more sensitive to X-ray damage (LD10 = 2.5 Gy) than are wild-type cells and approximately 2-fold more sensitive than NHEJ-defective ku70 mutant cells (<xref ref-type="fig" rid="ukmss-4240-f0002">Fig. 2a</xref>, upper panel). Surprisingly, , upper panel). Surprisingly, ctip cells isolated in G1 phase are also sensitive to X-ray-induced DNA damage (LD10 = 2.2 Gy) (<xref ref-type="fig" rid="ukmss-4240-f0002">Fig. 2a</xref>, lower panel), suggesting that CtIP function is not limited to S/G2 phase, as previously proposed, but contributes to the repair of DSB throughout the cell cycle. And, since HR does not function in G1 phase, CtIP must be involved in a second pathway for repairing DSBs., lower panel), suggesting that CtIP function is not limited to S/G2 phase, as previously proposed, but contributes to the repair of DSB throughout the cell cycle. And, since HR does not function in G1 phase, CtIP must be involved in a second pathway for repairing DSBs.', 'A role for CtIP in DSB repair during G1 was unexpected. Previous studies suggested that CtIP is present at very low levels outside of the S and G2 phases12,16. Nevertheless, CtIP was present in extracts from DT40 cells in G1 albeit at reduced levels compared to cells in S/G2 (<xref ref-type="fig" rid="ukmss-4240-f0002">Fig. 2b</xref>). Furthermore, whereas in G1 phase CtIP is largely unmodified, the majority of CtIP in S/G2 is phosphorylated (). Furthermore, whereas in G1 phase CtIP is largely unmodified, the majority of CtIP in S/G2 is phosphorylated (<xref ref-type="fig" rid="ukmss-4240-f0002">Fig. 2b</xref>).).'], 'ukmss-4240-f0003': ['For HR we measured the repair of a defined I-SceI-induced genomic DSB in a defective GFP reporter gene (Sce-GFP13) (Supplementary Fig. 5). In line with previous studies7 we observed a 10-fold defect in HR for ctip mutant cells compared to wild-type cells (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3a</xref>). A second form of homology directed DNA repair is single-strand annealing (SSA). This occurs when a DSB is generated between two directly repeated sequences and is achieved by resection of DNA ends to produce homologous ssDNA tails that can anneal to promote joining). A second form of homology directed DNA repair is single-strand annealing (SSA). This occurs when a DSB is generated between two directly repeated sequences and is achieved by resection of DNA ends to produce homologous ssDNA tails that can anneal to promote joining14. Again we found that ctip mutant cells are 10-fold defective in SSA compared with wild-type cells (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3b</xref>). We conclude that CtIP plays an important role in DSB repair by HR and this accounts for the sensitivity of ). We conclude that CtIP plays an important role in DSB repair by HR and this accounts for the sensitivity of ctip cells to X-ray damage in S and G2 phases.', 'DNA end-joining is achieved either through NHEJ, whereby broken DNA ends are directly rejoined, or by MMEJ in which DNA ends are resected locally to reveal short regions of complementary DNA (4 to 6 nucleotides) which stabilise broken ends for ligation1. We tested ctip cells for both types of end-joining and found no defect in accurate NHEJ (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3c</xref>). In fact we observed a slight increase in NHEJ activity in the ). In fact we observed a slight increase in NHEJ activity in the ctip mutant compared with wild-type cells. On the other hand, for MMEJ we observed a 4- to 5-fold defect in the ctip mutant compared with wild-type cells (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3d</xref>).).', 'In a second assay we transfected cells with linearised plasmid, recovered the repaired plasmids after 24 hours and examined the DNA sequences surrounding the joints (Supplementary Fig. 6). Both wild-type and ctip mutant cells repaired the majority of breaks through a combination of accurate and inaccurate NHEJ (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig 3e</xref>). Where NHEJ was inaccurate, the spectrum of deletions at the break site was similar in ). Where NHEJ was inaccurate, the spectrum of deletions at the break site was similar in ctip and wild-type cells. Nevertheless, in the ctip mutant cells we again detected a reduction in MMEJ (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3e</xref>). Together, with observations in 293 cells that siRNA-mediated knockdown of CtIP alters the balance of DSB repair). Together, with observations in 293 cells that siRNA-mediated knockdown of CtIP alters the balance of DSB repair15, these data establish a role for CtIP in MMEJ and provide an explanation for the defects in DSB repair observed in ctip cells during G1 phase.', 'The picture became clearer when we looked at the survival to X-ray damage in different phases of the cell cycle. We found that expression of either HsCtIP or HsCtIPS327A in ctip cells fully restored resistance to X-rays in G1 phase and restored MMEJ to wild-type levels in a plasmid assay (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig 3d</xref>), suggesting that phosphorylation on serine 327 is not required for the repair of DSB by MMEJ (), suggesting that phosphorylation on serine 327 is not required for the repair of DSB by MMEJ (<xref ref-type="fig" rid="ukmss-4240-f0002">Fig 2a</xref>). However, expression of ). However, expression of HsCtIPS327A in ctip cells did not restore HR, suggesting that phosphorylation of serine 327 is important for this function (<xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3a,b</xref>). Accordingly, the sensitivity ). Accordingly, the sensitivity ctip cells to X-rays in S/G2, was only partially restored by HsCtIPS327A (<xref ref-type="fig" rid="ukmss-4240-f0002">Fig. 2a</xref>), which can be accounted for by restoration of MMEJ, but not HR, in these cells.), which can be accounted for by restoration of MMEJ, but not HR, in these cells.', '(a) Cells were labelled with 20 μM BrdU and treated with 8 Gy X-rays (where indicated). ssDNA was detected over time by staining with antibody against BrdU and analysed using confocal microscopy. Staining was performed under DNA denaturing (2N HCl) or native conditions as indicated. (b) Kinetics of ssDNA generation from (a). Cells containing 10 or more foci were scored as positive. (c) HR assay, performed as in <xref ref-type="fig" rid="ukmss-4240-f0003">Fig. 3</xref> with indicated cell lines. Shows that expression of “constitutively activated” CtIPPM restores HR in ctip mutant cells but not in brca1 cells. with indicated cell lines. Shows that expression of “constitutively activated” CtIPPM restores HR in ctip mutant cells but not in brca1 cells.'], 'ukmss-4240-f0004': ['To test this we took advantage of the fact that BrdU incorporated into the genome is detectable by anti-BrdU antibody only in regions of ssDNA18. We cultured cells in BrdU, treated them with X-rays and stained for BrdU at intervals up to 2 hours (<xref ref-type="fig" rid="ukmss-4240-f0004">Fig. 4a</xref>). Approximately 25% of unirradiated cells exhibit 10 or more BrdU foci. After exposure to X-rays (8 Gy) we observed a time-dependent increase in BrdU staining in wild-type cells until, after 2 hours, approximately 50% contained 10 or more BrdU foci. Over the same time period only 30% of ). Approximately 25% of unirradiated cells exhibit 10 or more BrdU foci. After exposure to X-rays (8 Gy) we observed a time-dependent increase in BrdU staining in wild-type cells until, after 2 hours, approximately 50% contained 10 or more BrdU foci. Over the same time period only 30% of ctip mutant cells stained with BrdU, suggesting that while these mutant cells are not completely defective in the generation of ssDNA after exposure to X-rays, it occurs more slowly. Moreover, expression of HsCtIP in the ctip fully rescued this delay. On the other hand, expression of HsCtIPS327A cells did not complement this defect in the ctip mutant, suggesting that DNA damage-dependent increase in ssDNA is linked to the phosphorylation of serine 327. The delayed generation of ssDNA is not linked to reduced growth rate as cells expressing HsCtIPS327A exhibit reduced BrdU foci but proliferate normally (Supplementary Fig. 7).', 'Our data place CtIP at the ‘crossroads’ between DNA end-joining and HR pathways for the repair of DSB, with phosphorylation of serine 327 acting as a cell-cycle dependent switch that regulates CtIP-dependent DNA end resection. Phosphorylation of serine 327 is known to control the interaction of CtIP with BRCA1 in DT4012 (Supplementary Fig. 8a). Moreover, like serine 327 of CtIP, BRCA1 is required for repair of DSB by HR but not MMEJ (<xref ref-type="fig" rid="ukmss-4240-f0004">Fig. 4c</xref> and and Supplementary Fig. 8b), suggesting that the recruitment of BRCA1 to CtIP may be a determining factor in this switch.', 'While in yeast the switch to accurate DSB repair in S phase is controlled by phosphorylation of a single CDK site in Sae2, our data demonstrate that phosphorylation of CtIP at two independent CDK sites (S327 and T847) is required in vertebrates. The particular importance of S327 and the requirement of BRCA1 for this switch were established in two ways. Firstly, while expression of CtIPPM restores HR to ctip mutant cells without the requirement for phosphorylation at residue 847, we found that the CtIPS327A,PM mutant, in which phosphorylation at S327 is not possible, is defective in HR (Supplementary Fig. 10e). Secondly, we show that a CtIPPM mutant does not restore HR to brca1 mutant cells, confirming that recruitment of BRCA1 by CtIP is required for efficient HR function independently from the activation at T847 (<xref ref-type="fig" rid="ukmss-4240-f0004">Fig 4c</xref>).).']}
|
CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle
| null |
Nature
|
1242889200
|
Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ATR kinases during checkpoint activation; however, how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell-cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345 and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/M and S/M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, as the mutants are either partially (317A and 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated by a de-repression mechanism.
|
[
"Animals",
"Aphidicolin",
"Ataxia Telangiectasia Mutated Proteins",
"Binding Sites",
"Blotting, Western",
"Catalysis",
"Cell Cycle",
"Cell Cycle Proteins",
"Cell Line, Tumor",
"Checkpoint Kinase 1",
"Chickens",
"DNA Damage",
"DNA-Binding Proteins",
"Enzyme Activation",
"Enzyme Inhibitors",
"G2 Phase",
"Immunoprecipitation",
"Mutation",
"Phosphorylation",
"Protein Binding",
"Protein Kinases",
"Protein Serine-Threonine Kinases",
"Radiation, Ionizing",
"S Phase",
"Serine",
"Tumor Suppressor Proteins"
] |
other
|
PMC2857324
| null |
36
|
[
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"{'Citation': 'Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J, Jackson SP. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat Cell Biol. 2006;8:37–45.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16327781'}}}",
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"{'Citation': 'Kaneko YS, Watanabe N, Morisaki H, Akita H, Fujimoto A, Tominaga K, Terasawa M, Tachibana A, Ikeda K, Nakanishi M. Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene. 1999;18:3673–3681.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '10391675'}}}",
"{'Citation': 'Katsuragi Y, Sagata N. Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation. Mol Biol Cell. 2004;15:1680–1689.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC379266'}, {'@IdType': 'pubmed', '#text': '14767054'}]}}",
"{'Citation': 'Kramer A, Mailand N, Lukas C, Syljuasen RG, Wilkinson CJ, Nigg EA, Bartek J, Lukas J. Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase. Nat Cell Biol. 2004;6:884–891.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15311285'}}}",
"{'Citation': 'Lee JH, Paull TT. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science. 2005;308:551–554.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15790808'}}}",
"{'Citation': 'Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev. 2000;14:1448–1459.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC316686'}, {'@IdType': 'pubmed', '#text': '10859164'}]}}",
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"{'Citation': 'Niida H, Katsuno Y, Banerjee B, Hande MP, Nakanishi M. Specific role of Chk1 phosphorylations in cell survival and checkpoint activation. Mol Cell Biol. 2007;27:2572–2581.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC1899884'}, {'@IdType': 'pubmed', '#text': '17242188'}]}}",
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"{'Citation': 'Puc J, Keniry M, Li HS, Pandita TK, Choudhury AD, Memeo L, Mansukhani M, Murty VV, Gaciong Z, Meek SE, Piwnica-Worms H, Hibshoosh H, Parsons R. Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell. 2005;7:193–204.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15710331'}}}",
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"{'Citation': 'Smith GC, Cary RB, Lakin ND, Hann BC, Teo SH, Chen DJ, Jackson SP. Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM. Proc Natl Acad Sci USA. 1999;96:11134–11139.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC17999'}, {'@IdType': 'pubmed', '#text': '10500142'}]}}",
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"{'Citation': 'Wilsker D, Petermann E, Helleday T, Bunz F. Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control. Proc Natl Acad Sci USA. 2008;105:20752–20757.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC2634938'}, {'@IdType': 'pubmed', '#text': '19091954'}]}}",
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"{'Citation': 'Zhang YW, Otterness DM, Chiang GG, Xie W, Liu YC, Mercurio F, Abraham RT. Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway. Mol Cell. 2005;19:607–618.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16137618'}}}",
"{'Citation': 'Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol. 2001;21:4129–4139.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC87074'}, {'@IdType': 'pubmed', '#text': '11390642'}]}}",
"{'Citation': 'Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 2003;300:1542–1548.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '12791985'}}}"
] |
Nature. 2009 May 21; 459(7245):460-463
|
NO-CC CODE
|
|
The conserved ULK phosphorylation site in C. elegans Bec-1 is required for autophagyUnless otherwise stated all experiments were repeated three times and data shown is representative. (a) Bec-1 C. elegans were reconstituted with either wild-type or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti-PGL-1 antibody. Arrow indicates normal PGL-1 staining in germline cells. Scale bars represent 10μm. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents standard deviation between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mut) levels in Bec -/- stable worms were compared by Western blot. Mean value presented. (c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos displayed either high levels of diffuse PGL-1 staining (middle-left panel, 1/3 of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel. 2/3 of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy deficient embryos. (d) Embryos from the lines described in Fig.6a were labeled with anti-LGG-1, along with wild-type and unc-51 worms. Representative embryos at ~100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labeling in panel d. Error bars generated as in panel b. Mean value presented.
|
nihms495811f7
|
2
|
c08cc65e6288754444f842b28425fa70802c7b00e14698395e86d7fd8647c976
|
nihms495811f7.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
741,
938
] |
[{'image_id': 'nihms495811f8', 'image_file_name': 'nihms495811f8.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f8.jpg', 'caption': 'A working model of VPS34 complex regulation by ULK upon amino acid withdrawalAmino acid starvation inactivates TORC1, de-repressing ULK1. ULK1 is recruited to VPS34-Beclin-1 complexes via binding to ATG14L, phosphorylating Beclin-1, activating the VPS34 kinase and PI3P production at the nascent autophagosome. Additionally, UVRAG-bound Beclin-1 is phosphorylated by ULK1, which may promote autophagosome maturation.', 'hash': 'f534f26dc0209999f1cb2e72d3081d468492169b5cf4fd5dae2af2b5126bb1d9'}, {'image_id': 'nihms495811f6', 'image_file_name': 'nihms495811f6.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f6.jpg', 'caption': 'Beclin-1 S14 phosphorylation plays a critical role in autophagy induction by amino acid starvationUnless otherwise stated all experiments were repeated three times and data shown is representative. (a) 293 cells were transfected with the indicated plasmids. Cells grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies. (b) 293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Error bars represent the SD of three unique experiments. (c) shBeclin-1 reconstituted lines (wild-type or mutant) and controls (shScramble or shBeclin-1) were grown with or without amino acids were assessed for autophagy (left panel) and Beclin-1 levels (right panel). (d) Autophagosome (denoted by arrow heads) generation upon amino acid withdrawal was analyzed by electron microscopy. Cell lines and conditions from panel c were used and representative images from the indicated amino acid starved lines are shown; scale bar in bottom right represents 0.4μm. (e) Quantification of panel d. Fold induction was determined by arbitrarily making the nutrient rich condition 1 (solid bars) for each line. Error bars represent the standard deviation on the mean value over an average of 20 fields of view within a representative experiment. (f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200 -/- MEF grown under nutrient rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars represent 20μm. (g) Quantification of LC3B puncta from confocal displayed in panel f. In the HA-Beclin-1 or HA-Beclin-1-S14D transfected samples, only the HA staining positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value displayed, p-value determined by Student’s T-Test using 10 unique fields of view from panel f.', 'hash': '0a2f3acbf5e30228a0af2c988736ad9e2e59db7f6e0a4e73ba894d9d2bdc38b0'}, {'image_id': 'nihms495811f1', 'image_file_name': 'nihms495811f1.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f1.jpg', 'caption': 'ULK is essential for activation of the ATG14L-associated VPS34 upon amino acid starvationUnless otherwise stated experiments were repeated three times, data shown is representative. (a) Different VPS34 complexes were immunoprecipitated (IP) from MEF in the presence (N) or absence (-A) of amino acids using indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel, error bars denote S.D). (b) IP of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient rich or starvation conditions. VPS34 binding partners were analyzed by Western blot. (c) ATG14L-containing VPS34 complexes were immunopurified from wild-type, ULK1-/- 2KD (ULK def), and FIP200 -/- MEFs, and measured for lipid kinase activity similar to panel a. (d) VPS34 was immunopurified from wild-type, ULK def, and FIP200 -/- MEFs. Kinase assay was performed as in panel a. (e) IP of Beclin-1-containing VPS34 complexes from wild-type, ULK-def, and FIP200 -/- MEFs. Kinase assay was performed as in panel a. (f) LC3B puncta and PI3P levels were analyzed with anti-LC3B and Biotin-2XFYVE domain probe. Representative immunofluorescent images of LC3B and 2XFYVE domain binding were shown (scale bars,10μm). (g) Quantification of LC3B puncta from panel f. Details of quantification are provided in the methods section, data shown is mean -/+ S.D. Error bars in g-i are the standard deviation from a minimum of 6 unique fields of view from a representative experiment (see statistical source data). (h) Total PI3P was quantified from the experiment in panel f. Error bars was calculated as in g. (i) Quantification of PI3P that colocalizes with LC3B upon amino acid withdrawal from the experiment described in panel f. (j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown. * in panels a,g,h,i denote a p-value <0.05 as determined by Student’s T-Test (see statistical source data and methods section). Quantification of PI3P/VPS34 provided under the PI3P panel in c-e,j nutrient rich conditions were normalized to 1.', 'hash': '7738dfbaa53793744d26a663dde166f6758dfe48a1b3999e86beac4cab9f1223'}, {'image_id': 'nihms495811f7', 'image_file_name': 'nihms495811f7.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f7.jpg', 'caption': 'The conserved ULK phosphorylation site in C. elegans Bec-1 is required for autophagyUnless otherwise stated all experiments were repeated three times and data shown is representative. (a) Bec-1 C. elegans were reconstituted with either wild-type or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti-PGL-1 antibody. Arrow indicates normal PGL-1 staining in germline cells. Scale bars represent 10μm. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents standard deviation between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mut) levels in Bec -/- stable worms were compared by Western blot. Mean value presented. (c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos displayed either high levels of diffuse PGL-1 staining (middle-left panel, 1/3 of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel. 2/3 of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy deficient embryos. (d) Embryos from the lines described in Fig.6a were labeled with anti-LGG-1, along with wild-type and unc-51 worms. Representative embryos at ~100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labeling in panel d. Error bars generated as in panel b. Mean value presented.', 'hash': 'c08cc65e6288754444f842b28425fa70802c7b00e14698395e86d7fd8647c976'}, {'image_id': 'nihms495811f4', 'image_file_name': 'nihms495811f4.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f4.jpg', 'caption': 'ATG14L stimulates Beclin-1 S14 phosphorylation by promoting association with ULK1Unless otherwise stated all experiments were repeated three times and data shown is representative. (a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in 293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L IP (lanes 5-8). IP samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed. (b) Beclin-1 alone or bound to ATG14L was purified as described in panel a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blot was performed with the indicated antibodies. (c) An ATG14L-FLAG-6His inducible U2OS cell line was induced for 16 hours in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig.3a. ATG14L input levels are detected by immunoblotting. Two unique experiments were performed. (d) 293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1 were immunoprecipitated as indicated and blotted with the indicated antibodies. (e) 293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L-WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies. (f) 293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L-WT, or one of two mutants (ΔBATS, ΔN) that are defective in phagophore localization. Samples were handled as in panel e.', 'hash': '0bfe3b62ccc93bae4657dfc708363e48ba5e873d9b5cad5d74b6dfc164e14416'}, {'image_id': 'nihms495811f3', 'image_file_name': 'nihms495811f3.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f3.jpg', 'caption': 'Beclin-1 is a physiological target of ULK kinase in response to amino acid withdrawal and mTOR inhibitionUnless otherwise stated all experiments were repeated three times and data shown is representative. (a) Wild-type MEF were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blot was performed with the indicated antibodies. Beclin-1 S14 phosphorylation was quantified (shown under top panel) normalized to total Beclin-1. (b) Wild-type MEF were starved for the indicated time points. Beclin-1 was immunopurified by ATG14L IP and immunoblotted as indicated (top panels). Whole cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed. (c) Wild-type or FIP200 -/- MEF were incubated under nutrient rich, amino acid deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig.3b. (d) Wild-type or ULK def MEF were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in Fig.3a. Two unique experiments were performed.', 'hash': 'd816a2c72399ae5b5bab41d62379f393f6c5a11332b21a28c5b97e2c69379c9d'}, {'image_id': 'nihms495811f2', 'image_file_name': 'nihms495811f2.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f2.jpg', 'caption': 'Beclin-1 S14 is phosphorylated by ULK1 and required for VPS34 activation in response to amino acid withdrawalUnless otherwise stated all experiments were repeated three times and data shown is representative. (a) HEK293 cells were transfected with ATG14L, VPS34, and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of 32-P-ATP. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blot was performed (right panels). Results are representative of two unique experiments. (b) Full length murine GST-Beclin-1 and various truncations (as labeled) were subjected to an in vitro HA-ULK1 kinase assay. GST-Beclin-1 6(S-T) A has serine-threonine residues 4,7,10,14,29,42 mutated to alanine. ULK1 inputs were determined by Western, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by AR. (c) GST-Beclin-1 (1-85) was subjected to an in vitro ULK1 kinase reaction and analyzed by mass spectrometry. S14, S15in humans, (boxed) is the major phosphorylation site identified (for mass spectrometry data, see Fig.S2b,c). Mass spectrometry was performed on a single experiment. (d) Beclin-1 S14 is the major in vitro ULK1phosphorylation site. Beclin-1 WT, S4A, and S14A mutant were subjected to an in vitro ULK1 kinase assay. The reaction developed by autoradiography and stained for Beclin-1 input levels by Coomassie stain. Results are representative of two unique experiments. (e) 293 cells were transfected with the indicated plasmids under nutrient rich conditions. Beclin-1 was IP’d and immunoblotted with pBeclin-1 (S14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below IP samples. (f) Purified GST-Beclin-1 (1-85) was subjected to in vitro phosphorylation by GST-ULK1 (left panel) and GST-ULK2 (right panel). Reactions were immunoblotted with the indicated antibodies. (g) 293 cells were transfected with ATG14L, VPS34, and Beclin-1 and grown under nutrient rich conditions. ATG14L-containing VPS34 complexes were IP’d and lipid kinase activity was assayed as described in Fig.1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments. (h) Stable lines containing Beclin1 (WT or S14A) were used for Beclin-1 IP. Binding partners were determined by SDS-PAGE analysis and Western blot using the indicated antibodies.', 'hash': '1668e65e8343b2a94a28d8a042da1f9fec911039615468842e908ab36ce2925b'}, {'image_id': 'nihms495811f5', 'image_file_name': 'nihms495811f5.jpg', 'image_path': '../data/media_files/PMC3885611/nihms495811f5.jpg', 'caption': 'UVRAG promotes Beclin-1 S14 phosphorylation and association with ULK1(a) 293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown. (b) UVRAG bridges the interaction between Beclin-1 and ULK1. 293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti- HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown.', 'hash': '25ad52cc849da21421a5419a2362616f661765ddf09827a43d1c9424ad7e2f3d'}]
|
{'nihms495811f1': ['In order to study the regulation of VPS34 kinase activity we performed PI3P lipid kinase assays on complexes immunoprecipitated using antibodies against VPS34, Beclin-1 or ATG14L from cells grown in nutrient-rich or amino acid-starved conditions. The activity of VPS34 complexes immunoprecipitated by VPS34 or Beclin-1 was reduced upon starvation, while the activity of VPS34 complexes immunoprecipitated by ATG14L was significantly increased upon amino acid withdrawal (<xref rid="nihms495811f1" ref-type="fig">Fig.1a</xref>). This is consistent with previous reports of decreased VPS34 kinase activity under amino acid starvation using VPS34 immunoprecipitation). This is consistent with previous reports of decreased VPS34 kinase activity under amino acid starvation using VPS34 immunoprecipitation25-27. ATG14L levels were low in Beclin-1 and VPS34 immunoprecipitates explaining the starvation-induced decrease in VPS34 activity in these complexes despite their ability to bind ATG14L (<xref rid="nihms495811f1" ref-type="fig">Fig.1a,b</xref>). These data indicate that a differential regulation of VPS34 complexes exists in response to amino acid starvation. It has been reported that the autophagy specific function of VPS34 can be regulated by the disruption of the VPS34 kinase complex, under extended starvation. Starvation with Hanks’ balanced salt solution for several hours results in a phosphorylation of the nonstructured loop of Bcl-2 and dissociation from Beclin-1). These data indicate that a differential regulation of VPS34 complexes exists in response to amino acid starvation. It has been reported that the autophagy specific function of VPS34 can be regulated by the disruption of the VPS34 kinase complex, under extended starvation. Starvation with Hanks’ balanced salt solution for several hours results in a phosphorylation of the nonstructured loop of Bcl-2 and dissociation from Beclin-128. Under the starvation conditions used in this study we observed no significant change in Beclin-1-VPS34 interaction, consistent with previous observations25. The activation of ATG14L-containing VPS34 complexes provides a description of a unique mode of VPS34 regulation that does not necessitate the destabilization of the VPS34-Beclin-1 interaction.', 'In order to determine the requirement for ULK kinase in this activation we performed lipid kinase assays in wild-type MEF or two MEF lines deficient for ULK activity. Both ULK1 knockout MEFs with ULK2 stable knockdown (ULK-deficient MEF, described previously10) and FIP200 knockout MEF are defective for ULK activity. FIP200 is an essential cofactor for both ULK1&2, which perform redundant roles in autophagy3, 29. Interestingly, ATG14L-containing VPS34 complexes were not activated upon starvation in both ULK-deficient cell lines (<xref rid="nihms495811f1" ref-type="fig">Fig.1c</xref>). However, the starvation-induced repression of VPS34 complexes purified by VPS34 (). However, the starvation-induced repression of VPS34 complexes purified by VPS34 (<xref rid="nihms495811f1" ref-type="fig">Fig.1d</xref>) or Beclin-1 () or Beclin-1 (<xref rid="nihms495811f1" ref-type="fig">Fig.1e</xref>) antibody was retained in FIP200 null and ULK deficient MEF. Therefore, ULK kinase is only required for the activation of the pro-autophagic ATG14L-containing VPS34 complex and not the repression of non-autophagic VPS34 complexes. The inability of ATG14L-containing VPS34 complexes to be activated was accompanied by a reduction in autophagy induction in ULK and FIP200-deficient cells () antibody was retained in FIP200 null and ULK deficient MEF. Therefore, ULK kinase is only required for the activation of the pro-autophagic ATG14L-containing VPS34 complex and not the repression of non-autophagic VPS34 complexes. The inability of ATG14L-containing VPS34 complexes to be activated was accompanied by a reduction in autophagy induction in ULK and FIP200-deficient cells (Fig.S1a) as well as a reduction of endogenous LC3B puncta (Fig.S1b,c).', 'In order to measure PI3P at the autophagosome we performed co-staining for endogenous LC3B and PI3P using an anti-LC3B antibody and a biotinylated 2XFYVE domain probe, respectively. Biotin-2XFYVE specifically binds PI3P through its two FYVE domains and has been used previously to monitor PI3P on endosomes when costained with an endosomal marker30. Under starvation the total number of PI3P puncta decreased in wild-type cells (<xref rid="nihms495811f1" ref-type="fig">Fig.1f,h</xref>). Conversely, the number of PI3P puncta co-localizing to autophagosomes, as marked by the LC3B staining, increased (). Conversely, the number of PI3P puncta co-localizing to autophagosomes, as marked by the LC3B staining, increased (<xref rid="nihms495811f1" ref-type="fig">Fig.1f,i</xref>). Similarly to wild-type MEF, ULK deficient cells showed a decrease in total PI3P puncta under starvation conditions (). Similarly to wild-type MEF, ULK deficient cells showed a decrease in total PI3P puncta under starvation conditions (<xref rid="nihms495811f1" ref-type="fig">Fig.1f,h</xref>). In contrast ULK deficient cells showed a clear defect in the induction of PI3P-positive autophagosomes under starvation conditions (). In contrast ULK deficient cells showed a clear defect in the induction of PI3P-positive autophagosomes under starvation conditions (<xref rid="nihms495811f1" ref-type="fig">Fig.1f,i</xref>). The defect of ULK-deficient MEFs in LC3B puncta accumulation and PI3P-LC3B double positive puncta upon starvation were similarly observed in FIP200-/- MEF (). The defect of ULK-deficient MEFs in LC3B puncta accumulation and PI3P-LC3B double positive puncta upon starvation were similarly observed in FIP200-/- MEF (Fig.S1b,c,e), which retained the ability to repress total PI3P (Fig.S1b,d). Importantly, pharmacological inhibition of VPS34 completely abolished both 2XFYVE probe labeling and amino acid starvation-induced LC3B puncta, showing the specificity of these staining (Fig.S1f). Together, these data support a critical role of ULK in regulating the autophagy specific ATG14L-containing VPS34 complex activity.', 'To determine if ULK1 is sufficient to activate ATG14L-containing VPS34 complexes, ATG14L was immunoprecipitated from HEK293 cells co-transfected with myc-Beclin-1, Flag-ATG14, HA-VPS34 and empty vector or HA-ULK1 under nutrient-rich conditions. Activity of the ATG14L-containing VPS34 complexes was dramatically increased upon ectopic expression of ULK1 indicating that ULK1 activates the ATG14L-containing VPS34 complexes (<xref rid="nihms495811f1" ref-type="fig">Fig.1j</xref>). These results demonstrate that distinct VPS34 kinase complexes are differentially regulated in response to nutrient starvation and ULK activity is critical for activation of ATG14L-containing VPS34 complexes in response to amino acid starvation.). These results demonstrate that distinct VPS34 kinase complexes are differentially regulated in response to nutrient starvation and ULK activity is critical for activation of ATG14L-containing VPS34 complexes in response to amino acid starvation.', 'The regulation of VPS34 lipid kinase activity by ULK1 is limited to the ATG14L-containing complex (<xref rid="nihms495811f1" ref-type="fig">Fig.1c</xref>); therefore, we asked whether ULK1 would exhibit a preference for phosphorylation of the ATG14L-bound Beclin-1. Beclin-1 alone or ATG14L-bound Beclin-1 was immunoprecipitated from transfected cells. Endogenous ATG14L co-precipitated from cells overexpressing Beclin-1 alone was negligible compared to the levels from complexes obtained from cells transfected with both Beclin-1 and ATG14L (); therefore, we asked whether ULK1 would exhibit a preference for phosphorylation of the ATG14L-bound Beclin-1. Beclin-1 alone or ATG14L-bound Beclin-1 was immunoprecipitated from transfected cells. Endogenous ATG14L co-precipitated from cells overexpressing Beclin-1 alone was negligible compared to the levels from complexes obtained from cells transfected with both Beclin-1 and ATG14L (<xref rid="nihms495811f4" ref-type="fig">Fig.4b</xref>). Both complexes were used as substrates for ULK1 ). Both complexes were used as substrates for ULK1 in vitro kinase assays. We found that ATG14L-containing Beclin-1 was efficiently phosphorylated by ULK1 while Beclin-1 alone was a comparatively poor substrate (<xref rid="nihms495811f4" ref-type="fig">Fig.4a, b</xref>), indicating that ATG14L makes Beclin-1 a better substrate for ULK1 (), indicating that ATG14L makes Beclin-1 a better substrate for ULK1 (<xref rid="nihms495811f4" ref-type="fig">Fig.4b</xref>). In order to determine if ATG14L plays a role in promotion of Beclin-1 phosphorylation ). In order to determine if ATG14L plays a role in promotion of Beclin-1 phosphorylation in vivo we utilized an ATG14L-inducible cell line. Induction of ATG14L overexpression promoted Beclin-1 phosphorylation (<xref rid="nihms495811f4" ref-type="fig">Fig.4c</xref>), further supporting that ATG14L-bound Beclin-1 is the preferred target of ULK1 ), further supporting that ATG14L-bound Beclin-1 is the preferred target of ULK1 in vivo.', 'Unless otherwise stated all experiments were repeated three times and data shown is representative. (a) HEK293 cells were transfected with ATG14L, VPS34, and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of 32-P-ATP. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blot was performed (right panels). Results are representative of two unique experiments. (b) Full length murine GST-Beclin-1 and various truncations (as labeled) were subjected to an in vitro HA-ULK1 kinase assay. GST-Beclin-1 6(S-T) A has serine-threonine residues 4,7,10,14,29,42 mutated to alanine. ULK1 inputs were determined by Western, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by AR. (c) GST-Beclin-1 (1-85) was subjected to an in vitro ULK1 kinase reaction and analyzed by mass spectrometry. S14, S15in humans, (boxed) is the major phosphorylation site identified (for mass spectrometry data, see Fig.S2b,c). Mass spectrometry was performed on a single experiment. (d) Beclin-1 S14 is the major in vitro ULK1phosphorylation site. Beclin-1 WT, S4A, and S14A mutant were subjected to an in vitro ULK1 kinase assay. The reaction developed by autoradiography and stained for Beclin-1 input levels by Coomassie stain. Results are representative of two unique experiments. (e) 293 cells were transfected with the indicated plasmids under nutrient rich conditions. Beclin-1 was IP’d and immunoblotted with pBeclin-1 (S14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below IP samples. (f) Purified GST-Beclin-1 (1-85) was subjected to in vitro phosphorylation by GST-ULK1 (left panel) and GST-ULK2 (right panel). Reactions were immunoblotted with the indicated antibodies. (g) 293 cells were transfected with ATG14L, VPS34, and Beclin-1 and grown under nutrient rich conditions. ATG14L-containing VPS34 complexes were IP’d and lipid kinase activity was assayed as described in <xref rid="nihms495811f1" ref-type="fig">Fig.1j</xref>. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments. (h) Stable lines containing Beclin1 (WT or S14A) were used for Beclin-1 IP. Binding partners were determined by SDS-PAGE analysis and Western blot using the indicated antibodies.. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments. (h) Stable lines containing Beclin1 (WT or S14A) were used for Beclin-1 IP. Binding partners were determined by SDS-PAGE analysis and Western blot using the indicated antibodies.'], 'nihms495811f2': ['We sought to determine if ULK1 could directly phosphorylate any member of the VPS34 complex. ATG14L-containing VPS34 complexes were immunopurified from transfected cells and subjected to an in vitro ULK1 kinase assay using [γ32P]ATP. Autoradiography (AR) showed a single predominant band of approximately 60kDa (<xref rid="nihms495811f2" ref-type="fig">Fig.2a</xref>, left panel). Western blot confirmed co-migration of the AR band with Beclin-1 but not ATG14L (, left panel). Western blot confirmed co-migration of the AR band with Beclin-1 but not ATG14L (<xref rid="nihms495811f2" ref-type="fig">Fig.2a</xref>). To map the phosphorylation site on Beclin-1 we performed ULK1 ). To map the phosphorylation site on Beclin-1 we performed ULK1 in vitro kinase assays with [γ32P]ATP on various Beclin-1 deletions. ULK1 was capable of phosphorylating all truncations that shared the N-terminal 85 amino acids (Fig., S2a).', 'We next sought to identify putative ULK1 phosphorylation sites in the N-terminus of Beclin-1 by mutagenesis and truncations. Deletion of the N-terminal 40 amino acids largely abolished ULK1-mediated phosphorylation (<xref rid="nihms495811f2" ref-type="fig">Fig.2b</xref>). Conserved serine and threonine residues in the N-terminus of mouse Beclin-1 were mutated to alanine (S-T(4,7,10,14,29,42)A). The Beclin-1 S-T(4,7,10,14,29,42) A mutant was not phosphorylated by ULK1 (). Conserved serine and threonine residues in the N-terminus of mouse Beclin-1 were mutated to alanine (S-T(4,7,10,14,29,42)A). The Beclin-1 S-T(4,7,10,14,29,42) A mutant was not phosphorylated by ULK1 (<xref rid="nihms495811f2" ref-type="fig">Fig.2b</xref>, lane 2), indicating that one or more of the six residues are ULK1 phosphorylation sites. In conjunction we performed mass spectrometry analysis on an N-terminal fragment of Beclin-1 after performing an , lane 2), indicating that one or more of the six residues are ULK1 phosphorylation sites. In conjunction we performed mass spectrometry analysis on an N-terminal fragment of Beclin-1 after performing an in vitro ULK1 kinase reaction. Two phosphorylation sites were detected (<xref rid="nihms495811f2" ref-type="fig">Fig.2c</xref> and and S2b,c), one with low confidence, serine 4, and one with high confidence, serine 14, which is conserved to C. elegans (<xref rid="nihms495811f2" ref-type="fig">Fig.2c</xref> bottom). bottom).', 'The peptide encompassing conserved serine 63 was not detected by mass spectrometry so the GST-Beclin-1 1-85 S-T(4,7,10,14,29,42,63) A mutant was made. In this background alanine 4 and 14 were singly mutated back to serine. Recovery of serine 14 restored ULK-mediated phosphorylation, while recovery of serine 4 had no effect (Fig.S2d). In order to confirm the major phosphorylation site for ULK1, serine 4 and 14 were singly mutated to alanine in mouse Beclin-1. Mutation of serine 14 abolished ULK1-mediated phosphorylation while mutation of serine 4 had no effect, indicating that serine 14 (corresponding to S15 in human) is the primary ULK1 phosphorylation site in Beclin-1 (<xref rid="nihms495811f2" ref-type="fig">Fig.2c,d</xref>).).', 'To determine if ULK1 phosphorylates Beclin-1 S14 in vivo we generated a phospho-specific antibody. To test the specificity of the antibody cells were transfected with Beclin-1 (wild-type or S14A) with or without ULK1 (wild-type or kinase inactive). Co-expression of the wild-type ULK1, but not a catalytically inactive mutant, induced Beclin-1 S14 phosphorylation (<xref rid="nihms495811f2" ref-type="fig">Fig.2e</xref>))31. As expected no phosphorylation was observed in Beclin-1 S14A (<xref rid="nihms495811f2" ref-type="fig">Fig.2e</xref>, lane 5). These data indicate that ULK1 can phosphorylate Beclin-1 in cells and validate the specificity of the phospho-antibody. To exclude the possibility that an ULK-associated kinase was responsible for Beclin-1 phosphorylation, we used ULK1 purified from insect cells for an , lane 5). These data indicate that ULK1 can phosphorylate Beclin-1 in cells and validate the specificity of the phospho-antibody. To exclude the possibility that an ULK-associated kinase was responsible for Beclin-1 phosphorylation, we used ULK1 purified from insect cells for an in vitro kinase assay using recombinant Beclin-1 from E. coli. Immunoblot of the resulting kinase reaction showed robust Beclin-1 phosphorylation indicating that Beclin-1 is a direct target of ULK1 (<xref rid="nihms495811f2" ref-type="fig">Fig.2f</xref> and and S2e). We asked if ULK2 is similarly capable of phosphorylating Beclin-1. ULK2 kinase purified from insect cells also phosphorylated Beclin-1 at S14, indicating a redundancy for the ULK kinases in promoting Beclin-1 phosphorylation (<xref rid="nihms495811f2" ref-type="fig">Fig.2f</xref>, right panel)., right panel).', 'We next sought to determine if Beclin-1 (S14) phosphorylation was required for ULK1-mediated activation of the ATG14L-associated VPS34 lipid kinase. ATG14L was immunoprecipitated from transfected cells and an in vitro PI3P-lipid kinase assay was performed. As previously shown ULK1 cotransfection enhanced VPS34 kinase activity (<xref rid="nihms495811f2" ref-type="fig">Fig.2g</xref>, compare lanes 2&3 with 6&7); however, ATG14L VPS34 complexes containing mutant Beclin-1 did not respond to ULK1 co-transfection (, compare lanes 2&3 with 6&7); however, ATG14L VPS34 complexes containing mutant Beclin-1 did not respond to ULK1 co-transfection (<xref rid="nihms495811f2" ref-type="fig">Fig.2g</xref>, compare lanes 4&5 with 8&9). Importantly, we found that abrogation of the ULK1 phosphorylation site in Beclin-1 had no discernible effect on its ability to bind VPS34, ATG14L, p150, dynein, and Bcl2 (, compare lanes 4&5 with 8&9). Importantly, we found that abrogation of the ULK1 phosphorylation site in Beclin-1 had no discernible effect on its ability to bind VPS34, ATG14L, p150, dynein, and Bcl2 (<xref rid="nihms495811f2" ref-type="fig">Fig.2h</xref>). These data indicate that direct phosphorylation of Beclin-1 on S14 by ULK1 is required for activation of the autophagy specific VPS34 kinase complex.). These data indicate that direct phosphorylation of Beclin-1 on S14 by ULK1 is required for activation of the autophagy specific VPS34 kinase complex.', 'We previously showed that ATG14L-associated VPS34 complexes containing Beclin-1 S14A could not be activated by ULK1 (<xref rid="nihms495811f2" ref-type="fig">Fig.2g</xref>). We tested if this lack of activation results in an overall deficiency in autophagy. Cotransfection of ULK1 with wild-type Beclin-1 and ATG14L strongly increased autophagic flux as indicated by the increase of LC3B II accumulation (). We tested if this lack of activation results in an overall deficiency in autophagy. Cotransfection of ULK1 with wild-type Beclin-1 and ATG14L strongly increased autophagic flux as indicated by the increase of LC3B II accumulation (<xref rid="nihms495811f6" ref-type="fig">Fig.6a</xref>). Comparatively, cotransfection of phospho-defective Beclin-1-S14A attenuated ULK-mediated activation of autophagic flux (). Comparatively, cotransfection of phospho-defective Beclin-1-S14A attenuated ULK-mediated activation of autophagic flux (<xref rid="nihms495811f6" ref-type="fig">Fig.6b</xref>), indicating that activation of the ATG14L-containing VPS34 complex may be required for autophagy induction.), indicating that activation of the ATG14L-containing VPS34 complex may be required for autophagy induction.', 'In order to extend our observations at the organismal level and across species we used a C. elegans model system. During worm embryogenesis, the germline P granules (PGL granules) are selectively degraded by the autophagic machinery in somatic cells33. In autophagy mutant embryos, PGL granules remain in somatic cells during early embryonic divisions due to failed removal by autophagy34. To ascertain if the conserved serine 6 of BEC-1 (Beclin-1 worm homologue, <xref rid="nihms495811f2" ref-type="fig">Fig.2c</xref>) was also required for proper autophagic induction, we generated transgenic lines with p) was also required for proper autophagic induction, we generated transgenic lines with pbec-1∷bec-1(WT or S6A)∷gfp in the bec-1(ok700) background. In wild type worms, PGL-1, a key component of PGL granules, was exclusively expressed in the two germline precursor cells (<xref rid="nihms495811f7" ref-type="fig">Fig.7a</xref>, arrows mark germline cells). In contrast, , arrows mark germline cells). In contrast, bec-1 and unc-51 (ULK/ATG1 homologue) worms displayed somatic cell PGL-1 staining, indicating a defect in autophagic clearance (<xref rid="nihms495811f7" ref-type="fig">Fig.7a</xref>). Importantly, ). Importantly, bec-1 (WT) transgene expression fully rescued the defective degradation of the PGL-1 granules in the bec-1 background (<xref rid="nihms495811f7" ref-type="fig">Fig.7a</xref>). In contrast, ). In contrast, bec-1 (S6A) transgenic embryos contained PGL-1 body staining in the somatic cells (<xref rid="nihms495811f7" ref-type="fig">Fig.7a,b,c</xref>). Comparable expression levels of the ). Comparable expression levels of the bec-1 transgene was validated by Western blot (<xref rid="nihms495811f7" ref-type="fig">Fig.7b</xref>). The PGL-1 staining phenotype in ). The PGL-1 staining phenotype in bec-1 S6A lines varied between embryos with punctuate staining in 2/3rd of embryos and diffuse staining in 1/3rd of embryos; <xref rid="nihms495811f7" ref-type="fig">Fig.7c</xref>). We also noted that the increased PGL-1 staining is less pronounced in ). We also noted that the increased PGL-1 staining is less pronounced in bec-1 (S6A) worms than in the bec-1 worm, indicating a partial rescue of PGL body clearance by BEC-1(S6A). These data collectively demonstrate a role for both unc-51 and the conserved serine 6 of bec-1 in the autophagic clearance of protein aggregates in C. elegans embryogenesis.', 'ULK1 proteins were immunoprecipitated and extensively washed with MLB (once) and RIPA buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.05% SDS, 1% Triton X-100 and 0.5% deoxycholate) once, followed by washing with MLB buffer once followed by equilibration with ULK1 assay buffer [KBB supplemented with 0.05 mM DTT 10 μM cold ATP and 2 μCi 32PATP per reaction. Reaction was quenched by direct addition of 4X Laemmli buffer followed by boiling for 5min and resolution by SDS-PAGE. The analysis of some kinase reactions necessitated the separation of the kinase and substrate. In these cases one of the components (either kinase or substrate) was left on beads and the eluant and washed beads were loaded on separate gels. Substrate and kinase were separated in kinase assays displayed in <xref rid="nihms495811f2" ref-type="fig">Fig.2a,b,d</xref>. Fractions of the total kinase reaction are shown in . Fractions of the total kinase reaction are shown in Fig. S2a,d. All in vitro kinase reactions that were analyzed by Western with the phospho-Beclin-1 antibody were fractions from the total reaction.'], 'nihms495811f3': ['In order to determine if Beclin-1 is a physiological target of ULK1, ATG14L-associated Beclin-1 was immunopurified from wild-type MEF. Western blot analysis showed that endogenous Beclin-1 is phosphorylated upon amino acid starvation, while phosphatase treatment completely abolished Beclin-1 phospho-S14 signal (<xref rid="nihms495811f3" ref-type="fig">Fig.3a</xref>). ULK1 activity is potently repressed by TORC1 phosphorylation. To test if there is a correlation between Beclin-1(S14) phosphorylation and TORC1 signaling to ULK1, an amino acid withdrawal time course was performed. As expected, phosphorylation of ULK1 (S757, the TORC1 target site) decreased upon amino acid withdrawal, although more slowly than the dephosphorylation of S6K (another mTORC1 substrate) (). ULK1 activity is potently repressed by TORC1 phosphorylation. To test if there is a correlation between Beclin-1(S14) phosphorylation and TORC1 signaling to ULK1, an amino acid withdrawal time course was performed. As expected, phosphorylation of ULK1 (S757, the TORC1 target site) decreased upon amino acid withdrawal, although more slowly than the dephosphorylation of S6K (another mTORC1 substrate) (<xref rid="nihms495811f3" ref-type="fig">Fig.3b</xref>). Interestingly, ULK1-mediated Beclin-1 phosphorylation inversely correlated with the inhibitory phosphorylation on ULK1 (). Interestingly, ULK1-mediated Beclin-1 phosphorylation inversely correlated with the inhibitory phosphorylation on ULK1 (<xref rid="nihms495811f3" ref-type="fig">Fig.3b</xref>). In order to determine if inhibition of TORC1 was sufficient to activate ULK1-mediated Beclin-1 phosphorylation, wild-type or FIP200-/- and ULK deficient MEF were treated with mTOR catalytic inhibitor, Torin-1, under nutrient-rich conditions or with amino acid withdrawal. Inhibition of mTOR resulted in a clear induction of Beclin-1 S14 phosphorylation only in the wild-type MEF, indicating that relief of mTOR-mediated inhibition of ULK1 stimulates downstream target phosphorylation (). In order to determine if inhibition of TORC1 was sufficient to activate ULK1-mediated Beclin-1 phosphorylation, wild-type or FIP200-/- and ULK deficient MEF were treated with mTOR catalytic inhibitor, Torin-1, under nutrient-rich conditions or with amino acid withdrawal. Inhibition of mTOR resulted in a clear induction of Beclin-1 S14 phosphorylation only in the wild-type MEF, indicating that relief of mTOR-mediated inhibition of ULK1 stimulates downstream target phosphorylation (<xref rid="nihms495811f3" ref-type="fig">Fig.3c,d</xref>).).', 'Unless otherwise stated all experiments were repeated three times and data shown is representative. (a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in 293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L IP (lanes 5-8). IP samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed. (b) Beclin-1 alone or bound to ATG14L was purified as described in panel a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blot was performed with the indicated antibodies. (c) An ATG14L-FLAG-6His inducible U2OS cell line was induced for 16 hours in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in <xref rid="nihms495811f3" ref-type="fig">Fig.3a</xref>. ATG14L input levels are detected by immunoblotting. Two unique experiments were performed. (d) 293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1 were immunoprecipitated as indicated and blotted with the indicated antibodies. (e) 293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L-WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies. (f) 293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L-WT, or one of two mutants (ΔBATS, ΔN) that are defective in phagophore localization. Samples were handled as in panel e.. ATG14L input levels are detected by immunoblotting. Two unique experiments were performed. (d) 293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1 were immunoprecipitated as indicated and blotted with the indicated antibodies. (e) 293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L-WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies. (f) 293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L-WT, or one of two mutants (ΔBATS, ΔN) that are defective in phagophore localization. Samples were handled as in panel e.'], 'nihms495811f4': ['To understand the mechanism underlying the ATG14L-mediated promotion of Beclin-1 phosphorylation we performed immunoprecipitation assays to determine the interaction of ATG14L and Beclin-1 to ULK1 when expressed alone or together. Interestingly, we found that immunoprecipitation of Beclin-1 pulled down ULK1 only when cotransfected with ATG14L (<xref rid="nihms495811f4" ref-type="fig">Fig.4d</xref>, compare lanes 3 and 4). Conversely, immunoprecipitation of ATG14L could pull down ULK1 in the absence of Beclin-1 suggesting that ATG14L may recruit Beclin-1 to ULK1 for phosphorylation (, compare lanes 3 and 4). Conversely, immunoprecipitation of ATG14L could pull down ULK1 in the absence of Beclin-1 suggesting that ATG14L may recruit Beclin-1 to ULK1 for phosphorylation (<xref rid="nihms495811f4" ref-type="fig">Fig.4d</xref>, lane 2). To confirm that ATG14L stimulates Beclin-1 phosphorylation by promoting an ULK1-ATG14L-Beclin-1 complex, we cotransfected ATG14L WT or ATG14LΔCCD (a Beclin-1-binding deficient mutant) with Beclin-1 and ULK1, lane 2). To confirm that ATG14L stimulates Beclin-1 phosphorylation by promoting an ULK1-ATG14L-Beclin-1 complex, we cotransfected ATG14L WT or ATG14LΔCCD (a Beclin-1-binding deficient mutant) with Beclin-1 and ULK114, 15. We found that the ability of ATG14 to stimulate Beclin-1 phosphorylation was completely lost in ATG14LΔCCD (<xref rid="nihms495811f4" ref-type="fig">Fig.4e</xref>). The mechanistic model where ATG14L acts as an adaptor to recruit ULK1 to Beclin-1 is further supported by the fact that promotion of Beclin-1 phosphorylation is preserved in the ). The mechanistic model where ATG14L acts as an adaptor to recruit ULK1 to Beclin-1 is further supported by the fact that promotion of Beclin-1 phosphorylation is preserved in the in vitro kinase reaction (<xref rid="nihms495811f4" ref-type="fig">Fig.4b</xref>).).', 'Recruitment of ATG14L-containing VPS34 complexes to the phagophore requires ULK1. Therefore, we asked whether localization of ATG14L to the phagophore took place upstream or downstream of Beclin-1 S14 phosphorylation. We found that removal of the BATS (Barkor/Atg14L autophagosome targeting sequence) domain or extreme N-terminus of ATG14L, both of which are necessary for the localization of ATG14L to the phagophore 23, 32, but not binding to Beclin-1, severely compromised Beclin-1 phosphorylation when compared to WT control (<xref rid="nihms495811f4" ref-type="fig">Fig.4f</xref>). Previously, phosphorylation of AMBRA-1 by ULK1 was shown to be required for localization of Beclin-1 to the phagophore). Previously, phosphorylation of AMBRA-1 by ULK1 was shown to be required for localization of Beclin-1 to the phagophore18, making the activation of the ATG14L-containing VPS34 kinase a downstream event. While the reported role for ULK1 in the regulation of ATG9 cycling from the trans-golgi network to endosomes can be blocked by inhibition of VPS34, putting ATG9 regulation downstream of ULK-mediated activation of ATG14L-VPS34 complexes2.'], 'nihms495811f5': ['Beclin-1 also binds UV radiation resistance-associated gene protein (UVRAG) to promote the maturation of the autophagosome and this complex is known to be free of ATG14L. Therefore, we asked whether UVRAG might play a similar role in promoting Beclin-1 phosphorylation as ATG14L. Indeed UVRAG bound Beclin-1 preferentially associated with ULK1 and overexpression of UVRAG promoted Beclin-1 phosphorylation (<xref rid="nihms495811f5" ref-type="fig">Fig.5a,b</xref>). UVRAG binds only a small minority of the total Beclin-1 and represents a minor fraction of total Beclin-1-associated VPS34 activity). UVRAG binds only a small minority of the total Beclin-1 and represents a minor fraction of total Beclin-1-associated VPS34 activity24. Therefore, in addition to regulating autophagy initiation through ATG14L-complexes it is possible that Beclin-1 phosphorylation may also play a role in the autophagosome maturation through regulation of the UVRAG containing VPS34 complex.'], 'nihms495811f6': ['In order to further elucidate the role of ULK1-Beclin-1-S14 phosphorylation in the promotion of autophagy we generated knockdown-reconstitution stable cell lines (see material and methods). Cell lines expressing a scrambled non-targeting shRNA or Beclin-1 shRNA without reconstitution served as controls. Reconstituted Beclin-1 levels were comparable to that of endogenous Beclin-1 (<xref rid="nihms495811f6" ref-type="fig">Fig.6c</xref>). The wild-type and mutant Beclin-1 showed similar localization (). The wild-type and mutant Beclin-1 showed similar localization (Fig.S3c). Starvation of cells containing Beclin-1 knockdown or with mutant Beclin-1 reconstitution showed a reduction in LC3B II accumulation compared to shRNA-scramble or wild-type Beclin-1 reconstituted control (<xref rid="nihms495811f6" ref-type="fig">Fig.6c</xref>). In order to validate these findings we performed a similar experiment and analyzed the cells by electron microscopy. Consistent with previous observations we found that Beclin-S14A and shBeclin-1 lines did not produce a significant increase in autophagosomes upon amino acid starvation (). In order to validate these findings we performed a similar experiment and analyzed the cells by electron microscopy. Consistent with previous observations we found that Beclin-S14A and shBeclin-1 lines did not produce a significant increase in autophagosomes upon amino acid starvation (<xref rid="nihms495811f6" ref-type="fig">Fig.6e, 6d</xref>). Conversely, shScramble and WT-Beclin-1 reconstituted lines showed a significant accumulation of autophagosomes when starved for amino acids (). Conversely, shScramble and WT-Beclin-1 reconstituted lines showed a significant accumulation of autophagosomes when starved for amino acids (<xref rid="nihms495811f6" ref-type="fig">Fig.6d, 6e</xref>). The lack of autophagosome induction in mutant Beclin-1 reconstituted HeLa cells was also determined quantitatively by scoring of LC3B puncta accumulation (). The lack of autophagosome induction in mutant Beclin-1 reconstituted HeLa cells was also determined quantitatively by scoring of LC3B puncta accumulation (Fig.S3a,b). These data indicate that phosphorylation of Beclin-1 by ULK kinase is required for an appropriate autophagic response upon amino acid withdrawal.', 'We next sought to determine if autophagy could be driven by introduction of a phospho-mimetic residue at S14 of Beclin-1. HA-Beclin-1 (WT and S14D) were transiently expressed in the FIP200 -/- background. Induction of autophagy was determined by quantification of LC3B puncta in Beclin-1 expressing cells versus their non-expressing neighbors. Overexpression of wild-type Beclin-1 had no significant effect on inducing LC3B puncta (<xref rid="nihms495811f6" ref-type="fig">Fig.6f</xref>, top panels). However, the majority of cells expressing Beclin-1 –S14D had significantly more LC3B puncta than the FIP200 -/- background (, top panels). However, the majority of cells expressing Beclin-1 –S14D had significantly more LC3B puncta than the FIP200 -/- background (<xref rid="nihms495811f6" ref-type="fig">Fig.6f</xref>, bottom panels). The induction of autophagy by the Beclin-1 S14D indicates that at least some of the ATG14L-containing complexes were capable of getting to the phagophore, possibly as a result of transient overexpression, to induce autophagy., bottom panels). The induction of autophagy by the Beclin-1 S14D indicates that at least some of the ATG14L-containing complexes were capable of getting to the phagophore, possibly as a result of transient overexpression, to induce autophagy.', 'Unless otherwise stated all experiments were repeated three times and data shown is representative. (a) Bec-1 C. elegans were reconstituted with either wild-type or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti-PGL-1 antibody. Arrow indicates normal PGL-1 staining in germline cells. Scale bars represent 10μm. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents standard deviation between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mut) levels in Bec -/- stable worms were compared by Western blot. Mean value presented. (c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos displayed either high levels of diffuse PGL-1 staining (middle-left panel, 1/3 of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel. 2/3 of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy deficient embryos. (d) Embryos from the lines described in <xref rid="nihms495811f6" ref-type="fig">Fig.6a</xref> were labeled with anti-LGG-1, along with wild-type and were labeled with anti-LGG-1, along with wild-type and unc-51 worms. Representative embryos at ~100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labeling in panel d. Error bars generated as in panel b. Mean value presented.'], 'nihms495811f7': ['We next investigated autophagosome biogenesis in bec-1 (WT) and bec-1(S6A) rescued transgenic lines. The use of anti-LGG-1 (LC3B/ATG8 homologue) antibody in worms to distinguish levels of autophagy has been established in previous studies35. In bec-1 worms, the number of LGG-1 punctate structures was dramatically decreased, whereas the size and intensity were increased compared to the wild-type (<xref rid="nihms495811f7" ref-type="fig">Fig.7d,e</xref>). This phenotype was rescued by a transgenic line containing ). This phenotype was rescued by a transgenic line containing bec-1(WT) but not bec-1(S6A) (<xref rid="nihms495811f7" ref-type="fig">Fig.7d,e</xref>). Failure to rescue the autophagic defect in ). Failure to rescue the autophagic defect in bec-1(S6A) transgenic worms is consistent with phosphorylation of S6 being crucial for the initiation of autophagosome formation. Together, our data indicate that the bec-1 (S6A) displays an autophagic defect largely overlapping that of unc-51 worms, indicating that this residue is required for robust unc-51-dependent induction of autophagy.'], 'nihms495811f8': ['Our study supports a model that TORC1 inhibition by amino acid starvation leads to de-repression of ULK kinase activity. The active ULK directly phosphorylates Beclin-1 S14 and activates the pro-autophagy VPS34 complexes to promote autophagy induction and maturation (<xref rid="nihms495811f8" ref-type="fig">Fig.8</xref>). This model reveals a continuous signaling pathway amino acids-TORC1-ULK1-VPS34-Beclin-1. The identified ULK1 phospho-site on Beclin-1 has no obvious conservation in yeast ATG6. It will be interesting to see if the link between ATG1 and ATG6 is functionally maintained in yeast, one could look at the functional conservation of TOR-ATG1 as an example. Given the complex nature of autophagy biology, the regulation of VPS34 is likely to be complex and future studies are required to have a comprehensive understanding of VPS34 regulation in response to the wide range of autophagy inducing signals.). This model reveals a continuous signaling pathway amino acids-TORC1-ULK1-VPS34-Beclin-1. The identified ULK1 phospho-site on Beclin-1 has no obvious conservation in yeast ATG6. It will be interesting to see if the link between ATG1 and ATG6 is functionally maintained in yeast, one could look at the functional conservation of TOR-ATG1 as an example. Given the complex nature of autophagy biology, the regulation of VPS34 is likely to be complex and future studies are required to have a comprehensive understanding of VPS34 regulation in response to the wide range of autophagy inducing signals.']}
|
ULK1 induces autophagy by phosphorylating Beclin-1 and activating Vps34 lipid kinase
| null |
Nat Cell Biol
|
1374217200
|
Fetal skin has the intrinsic capacity for wound healing, which is not correlated with the intrauterine environment. This intrinsic ability requires biochemical signals, which start at the cellular level and lead to secretion of transforming factors and expression of receptors, and specific markers that promote wound healing without scar formation. The mechanisms and molecular pathways of wound healing still need to be elucidated to achieve a complete understanding of this remodeling system. The aim of this paper is to discuss the main biomarkers involved in fetal skin wound healing as well as their respective mechanisms of action.
|
[
"Animals",
"Biomarkers",
"Cytokines",
"Extracellular Matrix Proteins",
"Fetus",
"Humans",
"Skin",
"Wound Healing"
] |
other
|
PMC3885611
| null |
43
|
[
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] |
Nat Cell Biol. 2013 Jul 19; 15(7):741-750
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Sub cellular localization of the cysteines essential for Smo signalling(a) CRD mutants with compromised signalling activity have altered sub cellular localization. Cl8 cells expressing wild type or the indicated Myc-Smo mutant protein, in the presence of Hh (+) or empty vector control, were examined by indirect immunofluorescence. Wild type Smo translocates to the plasma membrane in response to Hh, whereas Smo CRD cysteine to alanine mutant C90A that was required for maximal Hh reporter gene induction was largely retained in the ER. Smo was detected using anti-Myc (red), Calreticulin-GFP-KDEL marks the ER (green) and DAPI (blue) marks the nucleus. Scale bar: 10uM (b) Sequence alignment of the Drosophila (D) Smo ECLD and ECL1 with the human (H) Smo ECLD and ECL1. The cysteines engaged in disulphide bond formation in human Smo are conserved in Drosophila. The red lines indicate the disulphide bond pattern.
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nihms-543083-f0004
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d12fd7eafa5a49a92be4b993e7dd68b16207fab0575f20b42a63e4a571622040
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
791,
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[{'image_id': 'nihms-543083-f0004', 'image_file_name': 'nihms-543083-f0004.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0004.jpg', 'caption': 'Sub cellular localization of the cysteines essential for Smo signalling(a) CRD mutants with compromised signalling activity have altered sub cellular localization. Cl8 cells expressing wild type or the indicated Myc-Smo mutant protein, in the presence of Hh (+) or empty vector control, were examined by indirect immunofluorescence. Wild type Smo translocates to the plasma membrane in response to Hh, whereas Smo CRD cysteine to alanine mutant C90A that was required for maximal Hh reporter gene induction was largely retained in the ER. Smo was detected using anti-Myc (red), Calreticulin-GFP-KDEL marks the ER (green) and DAPI (blue) marks the nucleus. Scale bar: 10uM (b) Sequence alignment of the Drosophila (D) Smo ECLD and ECL1 with the human (H) Smo ECLD and ECL1. The cysteines engaged in disulphide bond formation in human Smo are conserved in Drosophila. The red lines indicate the disulphide bond pattern.', 'hash': 'd12fd7eafa5a49a92be4b993e7dd68b16207fab0575f20b42a63e4a571622040'}, {'image_id': 'nihms-543083-f0003', 'image_file_name': 'nihms-543083-f0003.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0003.jpg', 'caption': "Solution structure of the Smo CRD(a) Stereo view of the backbone atoms (N, Cα, C') of the 20 superimposed structures of Smo CRD with the lowest energy. The disulphides are indicated in yellow and are labelled for clarity. (b) Ribbon diagram of the Smo CRD showing the secondary structure elements. The structure with the lowest energy is used to describe the secondary structure elements. The colour scheme is as follows: Cyan: Alpha helices; Red: β strands; Yellow: 310 helices Grey: random coil. (c) Superimposition of the Smo CRD with FzD CRD. The Smo CRD is represented in cyan and the FzD8 CRD is represented in red. The helices in both proteins are shown as cylinders and the beta-strands as arrowheads. All figures were generated using PyMol.", 'hash': 'c067e0a73bc51216429f1b55b511b01207449d18955f8512eff2bfba141f469c'}, {'image_id': 'nihms-543083-f0002', 'image_file_name': 'nihms-543083-f0002.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0002.jpg', 'caption': 'Identification of cysteines essential for Smo signallingCRD residues C90, C139, C155, C179 and the Smo ECLD residues C218, C238 and C242 are required for proper Hh reporter gene induction. Mutation of C84, 100,148,172,194,203 to alanine did not compromise the ability of Smo protein to induce the Hh reporter gene activity, and behaved similarly to the wild type protein. Percent activity for each of the experimental assays is shown relative to control. The control level of Hh-induced, ptcΔ136-luciferase activity for control dsRNA was set to 100%. For all conditions, luciferase activity is normalized to pAc-renilla control. Experiments were performed a minimum of two times in duplicate or triplicate and all data pooled. Error bars indicate standard error of the mean (s.e.m.).', 'hash': '68b7b65d8928c137999b2a48969a0cf0b3c9bbcdb6cd9aa9766ff73f02742a01'}, {'image_id': 'nihms-543083-f0005', 'image_file_name': 'nihms-543083-f0005.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0005.jpg', 'caption': 'Drosophila Smo CRD binds to the glucocorticoid Bud(a) CSPs of Smo CRD upon addition of Bud are plotted versus residue numbers. The red line indicates CSP greater than 0.01 ppm. The residues labelled in black form the Bud-binding pocket on Smo CRD as analysed from the HADDOCK docking experiments. The mouse FzD8-Wnt interacting “site 1” and “site 2” residues are shown in orange and green respectively. The corresponding secondary structure elements of the Drosophila Smo CRD are represented below the plot. (b) “Ribbon” representation of the Smo CRD. The backbone thickness of the ribbon is directly proportional to the weighted sum (in ppm) of the 1H and 15N chemical shifts upon binding to the ligand Bud. (c and d) Results of the HADDOCK docking of Bud on Smo CRD. (c) The aromatic side chains of the Bud contacting Smo CRD residues are shown. (d) Surface representation of the residues that interact with Bud are shown in yellow and the positively charged H135 is shown in blue.', 'hash': 'd72124133f7fe15b7e692b22fbfc35bed65a1f6cd8599e20ae82ed454dea0d86'}, {'image_id': 'nihms-543083-f0006', 'image_file_name': 'nihms-543083-f0006.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0006.jpg', 'caption': 'Analysis of the binding of Bud to human Smo CRD(a) CSPs of human Smo CRD upon addition of Bud are plotted versus residue numbers. Dotted red line indicates CSP greater than 0.01 ppm. L108, W109, G111, L112 and R161 are homologous to the mouse Smo residues that interact with 20-OHC. G162 is homologous to Drosophila F187 and W163 is homologous to Drosophila Smo F188 that interacts with Bud. All these residues map to the “site 1” of mouse FzD8-Wnt interaction. “Site 1” and “site 2” residues of mouse FzD8-Wnt interaction are shown in orange and green respectively. The secondary structure elements as in Drosophila Smo CRD structure are shown below the plot. (b) BLI binding assays show that Bud binds to Drosophila Smo CRD. The super streptavidin sensors with biotinylated Drosophila Smo CRD were exposed to three different concentrations of Bud (62, 41, and 31 μM). The processed data were fitted locally with the integrated fitting function by a 1:1 binding model (orange line). The respective Kd values obtained by curve fitting were 89 μM (62 μM Bud, black line), 74 μM (41 μM Bud, green line), 59 μM (41 μM Bud, red line), 318 μM (41 μM Bud, magenta line), 93 μM (31 μM Bud, cyan line), and 85 μM (31 μM Bud, blue line). The average Kd value of Drosophila Smo CRD for Bud was 120 ± 98 μM. (c) BLI binding assays show that Bud binds to human Smo CRD. The experimental and data analysis procedure were same as described above. The respective Kd values obtained by curve fitting were 74 μM (62 μM Bud, black line), 37 μM (41 μM Bud, green line), 44 μM (41 μM Bud, red line) and 54 μM (31 μM Bud, blue line). The average Kd value of human Smo CRD for Bud was 52 ± 16 μM.', 'hash': 'de93d33da5ff619e6bf6850ae7f29ddd802a9314b7342e56163e1a897c263efd'}, {'image_id': 'nihms-543083-f0001', 'image_file_name': 'nihms-543083-f0001.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0001.jpg', 'caption': 'Sequence alignment of the Smo CRDPrimary sequence alignment of the Drosophila (D), human (H), mouse (M) and chicken (C) Smo CRD with that of mouse FzD8 CRD and mouse secreted Frizzled Related Protein 3 (sFRP3). The residues conserved in Smo and FzD CRD are shown in red, whereas the residues conserved only in the Smo CRD are shown in blue. The cysteine in FzD not conserved in Smo is underlined and in green. The disulphide bond pattern for the Smo CRD is shown in thick purple lines. The secondary structure elements are shown above and below the primary sequence. The residues highlighted in orange indicate the “site 1” residues of mouse FzD8 that interact with the palmitate modification on the ligand Wnt. The residues highlighted in green indicate the “site 2” on mouse FzD8 that interact with the amino acid sidechains on the opposite side of Wnt. The sequence alignment was generated using ClustalW2.', 'hash': '45be55176517903401f88b3eb181988ae64f02402ebd49d5820877e2bf128fad'}, {'image_id': 'nihms-543083-f0007', 'image_file_name': 'nihms-543083-f0007.jpg', 'image_path': '../data/media_files/PMC3890372/nihms-543083-f0007.jpg', 'caption': 'A novel model for Smo allosteric regulation(a) Smo CRD may be flexible. The disulphide bonds stabilizing Smo extracellular linker and extracellular loop are shown in red lines. (b) Smo has more than one binding site. We propose that molecules like Bud bind to the Smo CRD (left, red rectangle) to alter its conformation and attenuate its signalling activity. Cyclopamine and vismodegib are known to bind near the orthosteric binding site located in the cavity of the Smo 7TM domains (right, pink inverted triangle). We speculate that there is the possibility of a class of molecules (middle, yellow star) which would bind to the CRD and cause a conformational change of the Smo extracellular structures to bring it closer to the 7TM domains. This in turn might change the conformation of the trans-membrane domains to regulate signalling.', 'hash': '35b160fdda398c162f6a9074eea06646d547dd634d663f347e60ad6d9d0c4ffd'}]
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{'nihms-543083-f0001': ['The Smo CRD is essential for regulating its subcellular localization and signalling19–21. The cysteines in this domain are conserved from Drosophila through vertebrates (<xref ref-type="fig" rid="nihms-543083-f0001">Fig.1</xref>). Despite this essential role, the functional contribution of the CRD to Smo regulation has remained unclear. To gain functional insight into the role of the CRD, we generated a mutant lacking this domain (ΔCRD; V85-K202), and tested its ability to rescue Hh-dependent reporter gene induction following knockdown of endogenous ). Despite this essential role, the functional contribution of the CRD to Smo regulation has remained unclear. To gain functional insight into the role of the CRD, we generated a mutant lacking this domain (ΔCRD; V85-K202), and tested its ability to rescue Hh-dependent reporter gene induction following knockdown of endogenous smo in cultured Clone 8 (Cl8) cells27. Consistent with previous in vivo studies, we found that deletion of this domain ablates Smo signalling capacity in vitro (<xref ref-type="fig" rid="nihms-543083-f0002">Fig 2</xref>). In order to better dissect this essential functional domain, we studied the structure of the Smo CRD using NMR (Nuclear Magnetic Resonance) methods.). In order to better dissect this essential functional domain, we studied the structure of the Smo CRD using NMR (Nuclear Magnetic Resonance) methods.'], 'nihms-543083-f0003': ['A recombinant Drosophila Smo CRD (V85-K202) was expressed and purified using the E. coli expression system. For the purpose of NMR studies, 15N and 13C labelled protein was prepared in 10 mM deuterated acetic acid buffer and 10% D2O (volume/volume) at pH 5. Resonance assignments were performed using the standard triple resonance strategy for 13C, 15N labelled proteins28. In the 2D 1H-15N HSQC spectra, all the amide resonances of the 118 residues of the CRD could be observed except D116. D116 is in the loop region preceding the first helix and hence could be undergoing motions in the intermediate time scale resulting in peak broadening for the backbone amide; nevertheless, all the side chain resonances of this residue were observed. Using the structural information obtained from the NMR studies, one hundred structures were calculated and the 20 structures with the lowest energy are represented as an ensemble (<xref ref-type="fig" rid="nihms-543083-f0003">Fig. 3a</xref>). The statistical details for the NMR structure calculation are given in ). The statistical details for the NMR structure calculation are given in Table 1. The structure has no distance violation > 0.03 Å and no angle violations > 1°. The structure with the lowest energy is used to describe the secondary structure elements (<xref ref-type="fig" rid="nihms-543083-f0003">Fig. 3b</xref>). The tertiary fold of the Smo CRD is very similar to that of the FzD CRD). The tertiary fold of the Smo CRD is very similar to that of the FzD CRD16,18 (<xref ref-type="fig" rid="nihms-543083-f0003">Fig.3c</xref>, cyan compared to red)., cyan compared to red).', 'Structure analysis shows that the eight conserved cysteines in Smo CRD form four disulphide bonds. The chemical shifts observed for the cysteine C-alpha and C-beta carbons are suggestive that all the cysteines are oxidized29. The disulphide bond pattern was determined based on the intermolecular NOEs observed between the β-protons of the cysteine residues and is as follows: C90-C155, C100-C148, C139-C179 and C172-C194 (<xref ref-type="fig" rid="nihms-543083-f0003">Fig. 3a</xref>). In order to study the structural and biological relevance of these disulphide bonds, we mutated the cysteines individually to alanine, and checked the ability of the cysteine to alanine Myc-Smo mutants to rescue Hh induced reporter gene in a ). In order to study the structural and biological relevance of these disulphide bonds, we mutated the cysteines individually to alanine, and checked the ability of the cysteine to alanine Myc-Smo mutants to rescue Hh induced reporter gene in a smo knockdown background. C100A, C148A, C172A and C194A were able to rescue Hh-induced reporter gene induction to wild-type levels (<xref ref-type="fig" rid="nihms-543083-f0002">Fig. 2</xref>) indicating that the C100-C148 and C172-C194 disulphide bonds are not essential for a functional CRD. Conversely, C90A, C139A, C155A and C179A showed attenuated rescue of the Hh-induced reporter gene induction () indicating that the C100-C148 and C172-C194 disulphide bonds are not essential for a functional CRD. Conversely, C90A, C139A, C155A and C179A showed attenuated rescue of the Hh-induced reporter gene induction (<xref ref-type="fig" rid="nihms-543083-f0002">Fig. 2</xref>) suggesting that the C90-C155 and C139-C179 disulphide play critical roles in maintaining the integrity of the CRD structure. Indeed, additional indirect immunofluorescence revealed that Smo cysteine to alanine mutants that were compromised in their ability to signal were retained in the endoplasmic reticulum (ER), overlapping with the ER marker protein Calreticulin-GFP-KDEL () suggesting that the C90-C155 and C139-C179 disulphide play critical roles in maintaining the integrity of the CRD structure. Indeed, additional indirect immunofluorescence revealed that Smo cysteine to alanine mutants that were compromised in their ability to signal were retained in the endoplasmic reticulum (ER), overlapping with the ER marker protein Calreticulin-GFP-KDEL (<xref ref-type="fig" rid="nihms-543083-f0004">Fig. 4a</xref> and and Supplementary Fig. S1a). ER retention is likely due to altered protein folding resulting from loss of essential disulphide bridges. However, such misfolding did not trigger obvious degradation of Smo protein, as the cysteine to alanine mutants were present at protein levels similar to and greater than that of wild type Smo (Supplementary Fig. S1b lane 2 compared to 3–6). Though it is difficult to understand the cause of higher protein levels for cysteine to alanine mutants, we speculate that it may be due to the misfolded Smo mutants failing to exit the ER and cycle to the plasma membrane to be phosphorylated and desensitized like the wild type protein30,31.', 'In addition to the cysteines of the CRD (V85-K202), the Drosophila Smo ECD has five other conserved cysteines, at positions 84, 203, 218, 238 and 242. In the 3D structure of the Drosophila Smo CRD the N- and the C-termini are in close proximity (<xref ref-type="fig" rid="nihms-543083-f0003">Fig. 3b</xref>); implying that C84 and C203 may form a disulphide bond. Nevertheless, mutation of C84 and C203 to alanine does not alter the ability of Smo to rescue reporter gene induction in the ); implying that C84 and C203 may form a disulphide bond. Nevertheless, mutation of C84 and C203 to alanine does not alter the ability of Smo to rescue reporter gene induction in the smo knockdown background (<xref ref-type="fig" rid="nihms-543083-f0002">Fig.2</xref>), suggesting that they are either not engaged in a disulphide bond or the disulphide bond is not crucial in defining the 3D fold of the CRD. However, when C218, C238 and C242 of the ECLD were mutated individually to alanine, they expressed at near-normal levels (), suggesting that they are either not engaged in a disulphide bond or the disulphide bond is not crucial in defining the 3D fold of the CRD. However, when C218, C238 and C242 of the ECLD were mutated individually to alanine, they expressed at near-normal levels (Supplementary Fig. S1c), but failed to rescue Hh-induced reporter gene induction following endogenous smo knockdown (<xref ref-type="fig" rid="nihms-543083-f0002">Fig.2</xref>). C218, C238 and C242 of ). C218, C238 and C242 of Drosophila Smo are homologous to C193, C213 and C217 of human Smo (<xref ref-type="fig" rid="nihms-543083-f0004">Fig. 4b</xref>). The crystal structure of the human Smo shows that the ECLD is stabilized towards the ECL by disulphide bonds between C193 and C213 of the ECLD and C217 of the ECLD and C295 ECL1 loop). The crystal structure of the human Smo shows that the ECLD is stabilized towards the ECL by disulphide bonds between C193 and C213 of the ECLD and C217 of the ECLD and C295 ECL1 loop14 (<xref ref-type="fig" rid="nihms-543083-f0004">Fig. 4b</xref>). Hence, C218 and C238 in the ECLD of ). Hence, C218 and C238 in the ECLD of Drosophila Smo may form a disulphide bond and C242 in the ECLD is likely to form a disulphide bond with C320 in ECL1 of Drosophila Smo. Thus, cysteines both in the Smo CRD and ECD linker play an important role in maintaining the conformation of Smo essential for downstream signalling.'], 'nihms-543083-f0005': ['Smo activity can be modulated by small molecule inhibitors such as cyclopamine, which binds in the orthosteric binding site located within a cavity in the 7TM bundle14,24. The FzD CRD binds to its endogenous ligand Wnt underscoring the ligand binding capability of the CRDs16,18. Therefore we decided to test whether the Drosophila CRD might bind to 20-OHC or Bud. Due to a solubility issue with 20-OHC, we carried out all experiments with Bud. We used NMR chemical-shift perturbations (CSPs) to identify the residues that are involved in binding32 since this method allows us to determine precisely the amino acid residues involved in the interaction33. Despite recent reports stating the inability of 20-OHC to bind to the Drosophila Smo protein we tested if Bud bound to Drosophila Smo CRD25. In the 2D 1H-15N HSQC spectra of Smo CRD in the absence and presence of an increasing concentration of the ligand Bud, CSPs induced by the binding of Bud are clearly observed indicating Bud bound to the CRD (Supplementary Figs. S2a and S3). The normalized CSP for each residue of Smo in the presence of Bud is shown in <xref ref-type="fig" rid="nihms-543083-f0005">Fig. 5a</xref>. The residues that have significant CSP are further shown in the back-bone “ribbon” representation of the Smo CRD, wherein the thickness of the ribbon is proportional to CSP values observed on binding of Bud to Smo CRD (. The residues that have significant CSP are further shown in the back-bone “ribbon” representation of the Smo CRD, wherein the thickness of the ribbon is proportional to CSP values observed on binding of Bud to Smo CRD (<xref ref-type="fig" rid="nihms-543083-f0005">Fig. 5b</xref>). The results show that the residues of Smo CRD that have the largest CSPs induced by the bound Bud are located between the first helix and the C-terminus 3). The results show that the residues of Smo CRD that have the largest CSPs induced by the bound Bud are located between the first helix and the C-terminus 310 helical domain as it folds back between the first and second helix. As a control we tested the ability of cyclopamine to bind and induce CSPs within the Smo CRD and Smo CRD did not bind to cyclopamine at a ratio of 1:2 of protein to ligand24 (Supplementary Fig. S2b).', 'Based on the NMR studies, we generated the docked structure of Bud bound to Smo CRD using the program HADDOCK34. The CSP data on the Smo CRD and the bound Bud were used as ambiguous restraints (Supplementary Table S1) for docking and generating one thousand binding poses. The structure with the lowest binding energy was selected and this agrees with the CSP data on the Smo CRD (<xref ref-type="fig" rid="nihms-543083-f0005">Fig. 5b</xref>). The modelled structure showed that Bud is buried into a hydrophobic surface formed by the residues A132, H135, F187, F188 and F191 on the Smo CRD (). The modelled structure showed that Bud is buried into a hydrophobic surface formed by the residues A132, H135, F187, F188 and F191 on the Smo CRD (<xref ref-type="fig" rid="nihms-543083-f0005">Fig. 5c and 5d</xref>). F191 is conserved in the vertebrate Smo family whereas F188 is a tryptophan in vertebrate Smo (). F191 is conserved in the vertebrate Smo family whereas F188 is a tryptophan in vertebrate Smo (<xref ref-type="fig" rid="nihms-543083-f0001">Fig.1</xref>). The conservation of these residues in the vertebrate family, and the recent demonstration that the domain engages 20-OHC). The conservation of these residues in the vertebrate family, and the recent demonstration that the domain engages 20-OHC25,26, suggests that this pocket may have an important role in binding an as yet unidentified allosteric regulator of Drosophila Smo. To determine whether Bud would attenuate signalling by the Drosophila Smo protein, we treated Cl8 cells with increasing concentrations of Bud, and observed a ~20–40% reduction in Hh-induced reporter gene activity (Supplementary Fig. S4). Thereby we speculate that Bud acts as a weak, synthetic mimic of an endogenous ligand for the Drosophila Smo CRD, displacing it from this binding pocket.'], 'nihms-543083-f0006': ['Recent reports indicate that 20-OHC binds the vertebrate Smo CRD25,26. We therefore tested the binding of Bud to human Smo CRD. The recombinant human Smo CRD (G65-G177) was expressed and purified using the E. coli expression system. Similar to the studies of Drosophila Smo CRD, 15N and 13C labelled protein was prepared in 10 mM deuterated acetic acid buffer and 10% D2O (volume/volume) at pH 5. In the 2D 1H-15N HSQC spectra of human Smo CRD, all the amide resonances of the 113 residues were assigned except two residues, L73 and R74. NMR titration experiments were performed to determine the specific residues involved in ligand binding. The 2D 1H-15N HSQC spectra of human Smo CRD in the absence and presence of an increasing concentration of Bud showed that Bud bound to human Smo CRD as well (Supplementary Figs. S5 and S6). The normalized CSP for each residue of human Smo CRD in the presence of Bud is shown in <xref ref-type="fig" rid="nihms-543083-f0006">Fig. 6a</xref>. Comparing with . Comparing with Drosophila Smo CRD (<xref ref-type="fig" rid="nihms-543083-f0005">Fig. 5a</xref>), Bud binds to human CRD in a similar fashion. However, there are some clear differences. For example, a smaller number of residues in human Smo CRD had CSPs induced by the binding of Bud, and the absolute values of CSPs observed in human Smo CRD are smaller. Moreover, the binding affinities measured from different residues in human Smo CRD are clustered around 45 μM whereas those obtained from residues in ), Bud binds to human CRD in a similar fashion. However, there are some clear differences. For example, a smaller number of residues in human Smo CRD had CSPs induced by the binding of Bud, and the absolute values of CSPs observed in human Smo CRD are smaller. Moreover, the binding affinities measured from different residues in human Smo CRD are clustered around 45 μM whereas those obtained from residues in Drosophila Smo CRD range from 200–1000 μM indicating that Bud binding induced backbone conformational stabilization in Drosophila CRD35. Nevertheless, judged by the NMR titration data, it seemed that Bud binds to human Smo CRD with higher affinity (Supplementary Figs. S3 and S6). Although NMR is a useful method for determining specific protein ligand interactions in solution, it is not the ideal method for calculating dissociation constants36. The associated systemic error in calculating Kd values from NMR is likely caused by the averaging effect during NMR experiments37. Kd values calculated by this method can only be treated as the upper limit for interaction. Therefore we validated the binding affinity measurements using additional experiments. Indeed, using Bio-Layer Interferometry (BLI) we found that Bud bound to Drosophila Smo CRD with a Kd value of 120 ± 98 μM and bound to human Smo CRD with a Kd value of 52 ± 16 μM (<xref ref-type="fig" rid="nihms-543083-f0006">Fig. 6b and c</xref>). Both NMR and BLI data suggest that Bud binds to human Smo CRD better than ). Both NMR and BLI data suggest that Bud binds to human Smo CRD better than Drosophila Smo CRD.'], 'nihms-543083-f0007': ['The cache of identified Smo modulators dock on distinct sites on Smo to regulate signalling14,24,45,46. The extracellular region of Smo may be flexible (<xref ref-type="fig" rid="nihms-543083-f0007">Fig.7a</xref>). Small molecules may bind in the cavity of the 7TM domains (). Small molecules may bind in the cavity of the 7TM domains (<xref ref-type="fig" rid="nihms-543083-f0007">Fig. 7b</xref> right) and modulate signalling irrespective of CRD binding right) and modulate signalling irrespective of CRD binding14,24. In the present study, we provide structural evidence identifying the specific residues in the Smo CRD that interact with the small molecule modulator Bud. We speculate that these same residues bind the endogenous allosteric activator, and that activator binding to the CRD may cause a conformational shift that bridges the extracellular domains and the 7TM domains (<xref ref-type="fig" rid="nihms-543083-f0007">Fig. 7b</xref> middle), similar to what is observed for class B GPCRs middle), similar to what is observed for class B GPCRs40,41,47. These shifts may enhance communication between the CRD and the 7TM domains to trigger structural alterations in the core and the cytoplasmic domains to regulate downstream signalling (<xref ref-type="fig" rid="nihms-543083-f0007">Fig. 7</xref>).).']}
|
Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling
| null |
Nat Commun
|
1357027200
|
[{'@Label': 'BACKGROUND', '@NlmCategory': 'BACKGROUND', '#text': 'Pain catastrophizing, appraisals of pain control, styles of coping, and social support have been suggested to affect functioning in patients with low back pain. We investigated the relation of chronic pain coping strategies to psychological variables and clinical data, in patients treated surgically due to lumbar disc herniation and coexisting spondylotic changes.'}, {'@Label': 'MATERIAL AND METHODS', '@NlmCategory': 'METHODS', '#text': 'The average age of study participants (n=90) was 43.47 years (SD 10.21). Patients completed the Polish versions of the Chronic Pain Coping Inventory-42 (PL-CPCI-42), Beck Depression Inventory (BDI-PL), Coping Strategies Questionnaire (CSQ-PL), Beliefs about Pain Control Questionnaire (BPCQ-PL), and Roland-Morris Disability Questionnaire (RMQ-PL).'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'In the PL-CPCI-42 results, resting, guarding and coping self-statements were frequently used as coping strategies (3.96 SD 1.97; 3.72 SD 1.72; 3.47 SD 2.02, respectively). In the CSQ-PL domains, catastrophizing and praying/hoping were frequently used as coping strategies (3.62 SD 1.19). The mean score obtained from the BDI-PL was 11.86 SD 7.23, and 12.70 SD 5.49 from the RMDQ-PL. BPCQ-PL results indicate that the highest score was in the subscale measuring beliefs that powerful others can control pain (4.36 SD 0.97). Exercise correlated significantly with beliefs about internal control of pain (rs=0.22). We identified associations between radiating pain and guarding (p=0.038) and between sports recreation and guarding (p=0.013) and task persistence (p=0.041).'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'Back pain characteristics, depressive mood, disability, and beliefs about personal control of pain are related to chronic LBP coping styles. Most of the variables related to advancement of degenerative changes were not associated with coping efforts.'}]
|
[
"Adaptation, Psychological",
"Adult",
"Attitude to Health",
"Chronic Pain",
"Humans",
"Intervertebral Disc Degeneration",
"Intervertebral Disc Displacement",
"Middle Aged",
"Mood Disorders",
"Pain Management",
"Poland",
"Spondylosis",
"Statistics, Nonparametric",
"Surveys and Questionnaires"
] |
other
|
PMC3890372
| null |
32
|
[
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] |
Nat Commun. 2013; 4:2965
|
NO-CC CODE
|
|
Analysis of PHB levels within cells. (a) Immunofluorescent staining for PHB foci within the nuclei of in situ fractionated LNCaP/Luc expressing either PHB-cDNA or PHB– RNAi, with anti-PHB antibody detected with TRITC-labelled secondary and stained with DAPI for DNA. Bar = 20μm. Corresponding comparison of PHB protein levels shown Western blot alongside. (b) Western blot analysis of PHB and H3 from cellular fractionation of LNCaP cells. Where indicated, samples were incubated for 1 min at 37°C ± 0.2 U of micrococcal nuclease. (c) Immunofluorescent staining of LNCaP cells for PHB (TRITC detection), HP1 and HDAC1 (FITC detection), also DNA (DAPI). (d) Western blot analysis of PHB, AR and Histone H3 in cell fractions from LNCaP cells, grown either in full serum (F) or charcoal-stripped serum (St). (d) Western blot analysis of PHB and Histone H3 in purified chromatin fraction from HeLa cells, grown in full serum or serum starved. Densitometry data for each blot are given underneath.
|
ukmss-38018-f0001
|
2
|
e58ba92a4b8872a86ec238385db794c9507035287ebfe2ffb9bb57cbe9636acb
|
ukmss-38018-f0001.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
554,
679
] |
[{'image_id': 'ukmss-38018-f0001', 'image_file_name': 'ukmss-38018-f0001.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0001.jpg', 'caption': 'Analysis of PHB levels within cells. (a) Immunofluorescent staining for PHB foci within the nuclei of in situ fractionated LNCaP/Luc expressing either PHB-cDNA or PHB– RNAi, with anti-PHB antibody detected with TRITC-labelled secondary and stained with DAPI for DNA. Bar = 20μm. Corresponding comparison of PHB protein levels shown Western blot alongside. (b) Western blot analysis of PHB and H3 from cellular fractionation of LNCaP cells. Where indicated, samples were incubated for 1 min at 37°C ± 0.2 U of micrococcal nuclease. (c) Immunofluorescent staining of LNCaP cells for PHB (TRITC detection), HP1 and HDAC1 (FITC detection), also DNA (DAPI). (d) Western blot analysis of PHB, AR and Histone H3 in cell fractions from LNCaP cells, grown either in full serum (F) or charcoal-stripped serum (St). (d) Western blot analysis of PHB and Histone H3 in purified chromatin fraction from HeLa cells, grown in full serum or serum starved. Densitometry data for each blot are given underneath.', 'hash': 'e58ba92a4b8872a86ec238385db794c9507035287ebfe2ffb9bb57cbe9636acb'}, {'image_id': 'ukmss-38018-f0006', 'image_file_name': 'ukmss-38018-f0006.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0006.jpg', 'caption': 'PHB knockdown increases global histone H3 acetylation and Histone H3 K9-acetylation of the PSA promoter. (a) Western blot analysis of chromatin fraction from a representative clone of LNCaP/Luc/PHB-RNAi cells (± doxycycline) treated with DHT for 0 – 240 minutes. Ac = acetyl, P = Phospho. Densitometry data are given alongside, normalised to Histone H3. (b) Western blot analysis of PHB, H3 and pan-acetyl H3 in LNCaP/Luc/PHB-cDNA cells treated with increasing doxycycline for 24hours. Densitometry data are given alongside, normalised to Histone H3. (c) ChIP analysis of Histone H3-Ac(K9) binding to the PSA promoter in a representative clone of LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). * = P<0.05 (t-test analysis). (d) ChIP analysis of PHB and Histone H3-Ac(K9) binding to the promoters of β-actin, TAP1 and Cyclin-D in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). * = P<0.05 (t-test analysis).', 'hash': '288b3defe38965f9fceb250a24afa3417499b4683db239ea52280a16b130cad5'}, {'image_id': 'ukmss-38018-f0007', 'image_file_name': 'ukmss-38018-f0007.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0007.jpg', 'caption': 'Effects of manipulating prohibitin levels within LNCaP/Luc/PHB-RNAi cells. (a) Cells were grown in starvation medium for 72hrs, either with or without doxycycline and then treated with 10nM DHT, Adione or DHEA. Cell growth was determined at 96hr post treatment by SRB assay. (b) FACS analysis of LNCaP cells which were hormonally starved for 72hr with or withour dox and then hormone treated (DHT or Adione at 10nM) for 48hours before. The results for the gated S-phase population is shown. ** = P<0.01, * = P<0.05 (t-test analysis) (c) Relative tumour volume measurements of LNCaP/Luc/PHB-RNAi xenografts grown in castrated nude male mice, treated with or without doxycycline and with either vehicle, or androstenedione daily supplementation (d) Boxplots showing relative tumour volumes of LNCaP/Luc/PHB-RNAi tumours at day 20 (n=8), treated with either vehicle (V), or Adione (A). ** = P<0.01, * = P<0.05 (Mann Whitney analysis). Previous data of similarly treated LNCaP xenografts grown with testosterone (T) supplementation is given for comparison (in grey).', 'hash': 'd1d0bc69a59ca9ea48cb14332c17a0c7f57a69c66e7902be318c713c4b3b7c2d'}, {'image_id': 'ukmss-38018-f0003', 'image_file_name': 'ukmss-38018-f0003.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0003.jpg', 'caption': 'ChIP analysis of AR and PHB on the PSA regulatory region. (a) Diagramatic representation of the PSA promoter indicating locations of ARE I, II, III and the intervening regions (negative and up/down-stream). Labelling boxes indicate amplification regions of primer pairs used for PCR. DNA immunoprecipitated with either IgG control, AR or PHB antibody was amplified by PCR and the results for each region are shown underneath, compared to their respective input DNA control. Densitometry data for each band for PHB are given underneath. (b) ChIP analysis of AR and PHB binding to the PSA promoter of LNCaP cells either grown in full serum, or hormone-starved with ±10nM DHT and for 2hr. Data represents Taqman quantification of immunoprecipitated DNA, from three replicate experiments, normalised to their input DNA controls. (c) ChIP analysis of AR binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). (d) ChIP analysis of PHB binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). Data are from a representative LNCaP/Luc/PHB-RNAi clone. ** = P<0.01, * = P<0.05 (t-test analysis).', 'hash': '3a6bce36ff44edc6897da35290fd8935d734fcf8388489364518a1fb53e63f5c'}, {'image_id': 'ukmss-38018-f0004', 'image_file_name': 'ukmss-38018-f0004.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0004.jpg', 'caption': 'ChIP analysis of AR and PHB on the promoter and enhancer regions of the PSA promoter. (a) ChIP analysis of AR binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). (b) ChIP analysis of AR binding to PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with androstenedione for 0-4hrs (± doxycycline). (c) ChIP analysis of PHB binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). Data are mean ± SD of 2 independent experiments performed in triplicate on a representative LNCaP/Luc/PHB-RNAi clone. ** = P<0.01, * = P<0.05 (t-test analysis).', 'hash': '397d62edd7e969ea485891928e691434befa809fde7d3220227a472af4f52687'}, {'image_id': 'ukmss-38018-f0005', 'image_file_name': 'ukmss-38018-f0005.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0005.jpg', 'caption': 'Analysis of gene expression in LNCaP cell lines with altered PHB levels. (a) Taqman RT-PCR analysis of PSA transcript levels collected at time intervals (0 – 8hr) from starved LNCaP/Luc/PHB-RNAi cells treated with 10nM DHT (left hand side) or 10nM androstenedione (right hand side) with or without doxycycline. (b) Taqman RT-PCR analysis of PSA transcript levels from starved LNCaP/Luc/PHB-cDNA treated with increasing concentrations of DHT or androstenedione. (c) Taqman RT-PCR analysis of PSA transcript levels from starved LNCaP/Luc/PHB-RNAi cells treated with increasing concentrations of DHT or androstenedione (± doxycycline). (d) Luciferase activity from LNCaP/Luc/PHB-RNAi cells treated with DHT (0-100nM) or Adione (0-100nM) with or without doxycycline. Data are mean ± SD of 3 independent experiments performed in triplicate on a representative LNCaP/Luc/PHB-RNAi clone.', 'hash': '150643cdb3649a9c8925b6d40c44feb968ec06d7b64ebb739d8406eaa761b2a8'}, {'image_id': 'ukmss-38018-f0002', 'image_file_name': 'ukmss-38018-f0002.jpg', 'image_path': '../data/media_files/PMC3427022/ukmss-38018-f0002.jpg', 'caption': 'Effects of PHB modulation on AR recruitment to chromatin. (a) Western blot analysis for PHB, AR and Histone H3 on chromatin fractions (or whole cell extract, bottom panel) of starved LNCaP/Luc/PHB-RNAi cells ± doxycycline treated with DHT for 0 – 240 minutes. (b) Western blot analysis for AR, Histone H3, and PHB on chromatin fractions or whole cell extract taken from LNCaP/Luc/PHB-cDNA cells (± doxycycline). Densitometry data for each blot are given underneath. (c) Densitometry analysis of AR band density from western blots of chromatin fractions taken from LNCaP/Luc/PHB-RNAi cells (± doxycycline) and the scrambled control line treated with hormone for 0 – 360 minutes. Bars represent the mean from three western blots normalised to histone H3 levels and then plotted normalized to time = 0 for the appropriate dataset. ** = P<0.01, * = P<0.05 (t-test analysis). Left hand side shows response to DHT, right hand side shows response to androstenedione. Data are from a representative LNCaP/Luc/PHB-RNAi clone.', 'hash': 'b174b54c637ac66688c96453063e58902bc233005555809fc4ea4880a3c25ea1'}]
|
{'ukmss-38018-f0001': ['A pair of doxycycline-inducible LNCaP prostate cancer cell lines was established, one ectopically expressing PHB cDNA (LNCaP/Luc/PHB-cDNA) and one expressing PHB-siRNA (LNCaP/Luc/PHB-RNAi) (27), each with its respective empty vector or scrambled control line. Upon removal of soluble nuclear proteins, PHB was evident in nuclear foci (<xref ref-type="fig" rid="ukmss-38018-f0001">Figure 1a</xref>), number and intensity of which altered with PHB modulation. Fractionation demonstrated that PHB is present in both the cytoplasmic fraction and also the nucleus – both the soluble nuclear fraction but to a greater extent the chromatin-associated fraction (), number and intensity of which altered with PHB modulation. Fractionation demonstrated that PHB is present in both the cytoplasmic fraction and also the nucleus – both the soluble nuclear fraction but to a greater extent the chromatin-associated fraction (<xref ref-type="fig" rid="ukmss-38018-f0001">Figure 1b</xref>). Micrococcal nuclease digestion of chromatin released some of the associated PHB into the soluble nuclear fraction, supporting the PHB-chromatin association. This nuclear pattern is reminiscent of known PHB-interacting proteins HP1 and HDAC1, which colocalise closely with PHB (). Micrococcal nuclease digestion of chromatin released some of the associated PHB into the soluble nuclear fraction, supporting the PHB-chromatin association. This nuclear pattern is reminiscent of known PHB-interacting proteins HP1 and HDAC1, which colocalise closely with PHB (<xref ref-type="fig" rid="ukmss-38018-f0001">Figure 1c</xref>). Chromatin association of PHB was reduced in cells grown in full serum compared to hormonally-starved cells (). Chromatin association of PHB was reduced in cells grown in full serum compared to hormonally-starved cells (<xref ref-type="fig" rid="ukmss-38018-f0001">Figure 1d</xref>), with a concomitant increase in soluble nuclear PHB, while total and cytoplasmic PHB levels were unchanged. Increased chromatin association of PHB was also seen in serum-starved HeLa cells (), with a concomitant increase in soluble nuclear PHB, while total and cytoplasmic PHB levels were unchanged. Increased chromatin association of PHB was also seen in serum-starved HeLa cells (<xref ref-type="fig" rid="ukmss-38018-f0001">Figure 1e</xref>), which neither express AR nor are steroid responsive. Returning these cells to full serum reduced the levels of PHB co-purifying with chromatin within 24hr.), which neither express AR nor are steroid responsive. Returning these cells to full serum reduced the levels of PHB co-purifying with chromatin within 24hr.', 'Chromatin purification experiments (<xref ref-type="fig" rid="ukmss-38018-f0001">Figures 1</xref> & & <xref ref-type="fig" rid="ukmss-38018-f0002">2</xref>) showed global chromatin binding patterns of PHB and AR to be in dynamic opposition. Liganded AR binds to AREs in the promoter and enhancer regions of the well-characterised androgen target gene ) showed global chromatin binding patterns of PHB and AR to be in dynamic opposition. Liganded AR binds to AREs in the promoter and enhancer regions of the well-characterised androgen target gene PSA, and PHB can bind strongly to the PSA promoter in the presence of anti-androgens (24), but little data is available on the effects of co-repressors on AR binding. To address this, chromatin immunoprecipitation assays (ChIP) were performed.'], 'ukmss-38018-f0002': ['Treating hormone-starved LNCaP cells with androgen revealed increased AR chromatin association over time with a concomitant reduction in PHB co-purification (<xref ref-type="fig" rid="ukmss-38018-f0002">Figure 2a</xref>, left hand side). Doxycycline-induced PHB-RNAi reduced the amount of PHB co-purifying with the chromatin as expected, but accelerated AR binding, resulting in increased binding at shorter time-points of androgen treatment (, left hand side). Doxycycline-induced PHB-RNAi reduced the amount of PHB co-purifying with the chromatin as expected, but accelerated AR binding, resulting in increased binding at shorter time-points of androgen treatment (<xref ref-type="fig" rid="ukmss-38018-f0002">Figure 2a</xref> right hand side). Conversely, ectopic expression of PHB cDNA resulted in increased PHB-chromatin association, with a dose-dependent reduction in AR co-purification, even in full serum ( right hand side). Conversely, ectopic expression of PHB cDNA resulted in increased PHB-chromatin association, with a dose-dependent reduction in AR co-purification, even in full serum (<xref ref-type="fig" rid="ukmss-38018-f0002">Figure 2b</xref>). Total levels of AR remained unchanged in each case.). Total levels of AR remained unchanged in each case.', 'To quantify the effects of PHB loss on the chromatin-binding rate of AR, hormone-starved LNCaP cells were treated with either the potent DHT or the weak androstenedione (both at 10nM). Without doxycycline, DHT treatment resulted in maximal AR chromatin co-purification at 120-240 minutes, whilst doxycycline-induced PHB-RNAi accelerated this, with maximal AR chromatin co-purification at 30-60 minutes. Similarly with androstenedione treatment: AR chromatin binding was lower in the absence of doxycycline, whilst PHB-RNAi resulted in a statistically significant increase in AR binding at 120-240 minutes (<xref ref-type="fig" rid="ukmss-38018-f0002">Figure 2c</xref>). No change in rate of AR binding to chromatin was evident in the scrambled RNAi control line.). No change in rate of AR binding to chromatin was evident in the scrambled RNAi control line.'], 'ukmss-38018-f0003': ['In hormone-starved LNCaP cells, DHT treatment (2 hours) resulted in AR binding to the promoter and enhancer regions as expected, with minimal binding to non-ARE containing regions (<xref ref-type="fig" rid="ukmss-38018-f0003">Figure 3a</xref>). In the absence of hormone, PHB binds across the 8kb ). In the absence of hormone, PHB binds across the 8kb PSA promoter region with no apparent regional specificity. DHT treatment led to reduced PHB across all PSA-promoter regions (<xref ref-type="fig" rid="ukmss-38018-f0003">Figure 3a</xref>), i.e. this was not limited to ARE-containing regions. Also PHB was recruited to both ARE-containing and ARE-negative regions under hormonally starved conditions as compared to full serum, and was displaced from all three regions by androgen treatment (), i.e. this was not limited to ARE-containing regions. Also PHB was recruited to both ARE-containing and ARE-negative regions under hormonally starved conditions as compared to full serum, and was displaced from all three regions by androgen treatment (<xref ref-type="fig" rid="ukmss-38018-f0003">Figure 3b</xref>), coincident with increased AR binding in the case of the enhancer and promoter regions.), coincident with increased AR binding in the case of the enhancer and promoter regions.', 'Since PHB knockdown increased global AR chromatin-binding, the effects upon AR binding to AREs in the PSA promoter were studied. DHT increased AR binding to the enhancer and promoter regions as expected, however PHB-RNAi increased AR binding still further (<xref ref-type="fig" rid="ukmss-38018-f0003">Figure 3c</xref>). DHT treatment reduced PHB binding across all regions, confirming previous results, and PHB-RNAi knockdown reduced this further (). DHT treatment reduced PHB binding across all regions, confirming previous results, and PHB-RNAi knockdown reduced this further (<xref ref-type="fig" rid="ukmss-38018-f0003">Figure 3d</xref>). No change was seen in total AR levels in these cells within this timeframe (see ). No change was seen in total AR levels in these cells within this timeframe (see <xref ref-type="fig" rid="ukmss-38018-f0002">Figure 2a</xref>) and no change in AR or PHB recruitment was seen in the scrambled siRNA control cell line () and no change in AR or PHB recruitment was seen in the scrambled siRNA control cell line (Supplemental Figure 1a). Similar data was obtained for the similarly organised androgen-responsive gene KLK2 (Supplemental Figure 1b).'], 'ukmss-38018-f0004': ['We then analysed AR binding to the PSA promoter and enhancer in more detail. LNCaP/Luc/PHB-RNAi cells were grown without steroids (± doxycycline) for 72 hours and then treated with either DHT or androstenedione. DHT treatment promoted AR binding to both regions within 30 minutes, with binding reduced at 1 hour then increasing again at 2 hours. PHB-RNAi changed the pattern of AR binding to a more linear increase in binding with no reduction at the 1-hour timepoint (<xref ref-type="fig" rid="ukmss-38018-f0004">Figure 4a</xref>). Since the kinetics of androstenedione activity were less rapid we extended the study to 4 hours. Similar results were seen: AR recruitment at 1 hour, dropping at 2 hours then peaking again at 4 hours (). Since the kinetics of androstenedione activity were less rapid we extended the study to 4 hours. Similar results were seen: AR recruitment at 1 hour, dropping at 2 hours then peaking again at 4 hours (<xref ref-type="fig" rid="ukmss-38018-f0004">Figure 4b</xref>). Doxycycline-induced PHB knockdown caused increased binding of‘ AR to the promoter and enhancer regions with a peak at 1 hour and no cyclical pattern evident within this timeframe. Confirming previous results, hormone treatment resulted in a rapid reduction (within 15 minutes) of PHB binding at the enhancer and at the promoter, with a slight increase at 30 minutes at the enhancer then a continued reduced level. Unsurprisingly, PHB-RNAi resulted in very low levels of PHB at the enhancer or promoter throughout (). Doxycycline-induced PHB knockdown caused increased binding of‘ AR to the promoter and enhancer regions with a peak at 1 hour and no cyclical pattern evident within this timeframe. Confirming previous results, hormone treatment resulted in a rapid reduction (within 15 minutes) of PHB binding at the enhancer and at the promoter, with a slight increase at 30 minutes at the enhancer then a continued reduced level. Unsurprisingly, PHB-RNAi resulted in very low levels of PHB at the enhancer or promoter throughout (<xref ref-type="fig" rid="ukmss-38018-f0004">Figure 4c</xref>). IgG controls showed no changes with either treatment and a similar pattern was observed for recruitment to the ). IgG controls showed no changes with either treatment and a similar pattern was observed for recruitment to the KLK2 enhancer/promoter (Supplemental Figure 2).'], 'ukmss-38018-f0005': ['Both testicular and adrenal androgens activate androgen-regulated gene expression, but the less efficient adrenal androgens are required at higher concentrations for similar effects. The ability of these to activate the AR in our cell lines was assayed using the expression of both endogenous androgen regulated genes (e.g. PSA) and an integrated androgen-responsive luciferase reporter (27). Kinetics of PSA transcript production were studied after treatment with DHT or androstenedione (10nM) ± doxycycline. DHT-induced PSA transcript levels were increased at 6 hours post treatment and further thereafter (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5a</xref>, left panel), while PHB knockdown resulted in transcript levels peaking earlier (by 2-4 hours) and remaining high thereafter. , left panel), while PHB knockdown resulted in transcript levels peaking earlier (by 2-4 hours) and remaining high thereafter. KLK2 and TMPRS2 showed similar trends (Supplemental Figure 3a). PHB knockdown also increased the rate of androstenedione-induced PSA transcript production, which was significantly higher from 4 hours hormone treatment (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5a</xref>, right panel)., right panel).', 'Similarly we investigated whether the increased AR binding rate caused by PHB-RNAi could increase the efficacy of various androgens. After 16h treatment with 100nM hormone, PSA expression increased 6-fold with DHT (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5b-c</xref> left panels), around 3-fold for androstenedione ( left panels), around 3-fold for androstenedione (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5b-c</xref>, right panels), while DHEA had no significant effect (data not shown). Overexpression of PHB-cDNA caused strong inhibition of androgen-induced , right panels), while DHEA had no significant effect (data not shown). Overexpression of PHB-cDNA caused strong inhibition of androgen-induced PSA expression, whether by DHT or androstenedione (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5b</xref>). Conversely, doxycycline-induced PHB-RNAi knock down increased ). Conversely, doxycycline-induced PHB-RNAi knock down increased PSA expression in response to both testicular and adrenal androgens (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5c</xref>), significant at doses above 1nM.), significant at doses above 1nM.', 'Using the integrated androgen receptor luciferase reporter, we found increasing PHB reduced luciferase activity as previously reported (27). However, PHB-RNAi resulted in increased luciferase activity at lower doses of DHT, changing the maximal activity concentration (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5d</xref>, left panel). Similarly 1nM androstenedione in the presence of PHB-RNAi had equal activation potency to 10nM without (, left panel). Similarly 1nM androstenedione in the presence of PHB-RNAi had equal activation potency to 10nM without (<xref ref-type="fig" rid="ukmss-38018-f0005">Figure 5d</xref>, right panel). To confirm that the observed stimulatory effects of PHB knockdown can be overcome by reintroducing PHB, we transfected LNCaP/Luc/PHB-RNAi cells with a form of PHB not sensitive to silencing. This showed that the increased androgen-dependent (both DHT and androstenedione) luciferase activity seen in the presence of doxycycline-induced PHB knockdown was abolished by increasing PHB levels (, right panel). To confirm that the observed stimulatory effects of PHB knockdown can be overcome by reintroducing PHB, we transfected LNCaP/Luc/PHB-RNAi cells with a form of PHB not sensitive to silencing. This showed that the increased androgen-dependent (both DHT and androstenedione) luciferase activity seen in the presence of doxycycline-induced PHB knockdown was abolished by increasing PHB levels (Supplemental Figure 3b).'], 'ukmss-38018-f0006': ['PHB is known to recruit HDACs and chromatin remodelling complexes including HP-1 (19, 20, 25). We saw that PHB reduction resulted in a decrease in HP-1 and HDAC1 association with chromatin (<xref ref-type="fig" rid="ukmss-38018-f0006">Figure 6a</xref>), and analysed the chromatin for changes in histone acetylation in response to PHB knockdown. This resulted in an increase in overall acetylation of Histone H3, with increased levels of histone H3-Ac(K9) and Ac(K9) P(S10), even before androgen treatment (), and analysed the chromatin for changes in histone acetylation in response to PHB knockdown. This resulted in an increase in overall acetylation of Histone H3, with increased levels of histone H3-Ac(K9) and Ac(K9) P(S10), even before androgen treatment (<xref ref-type="fig" rid="ukmss-38018-f0006">Figure 6a</xref>). Additionally, androgen induction of these histone H3 modifications was also higher in the doxycycline-treated samples. Histone H3-Ac(). Additionally, androgen induction of these histone H3 modifications was also higher in the doxycycline-treated samples. Histone H3-Ac(18) showed no significant changes (data not shown). Conversely, increasing PHB levels by exogenous PHB-cDNA expression resulted in a dose-dependent reduction in global histone H3 acetylation (<xref ref-type="fig" rid="ukmss-38018-f0006">Figure 6b</xref>).).', 'ChIP analysis of the PSA regulatory region showed androgen-induced enrichment of H3-Ac(K9) across most of the promoter region as expected, however when PHB was reduced a further enrichment was observed, most notably at the enhancer and negative regions both before and after DHT treatment (<xref ref-type="fig" rid="ukmss-38018-f0006">Figure 6c</xref>). Interestingly, the RNAi-mediated reduction in PHB also increased levels of acetylated histone H3 at the promoters of the inducible ). Interestingly, the RNAi-mediated reduction in PHB also increased levels of acetylated histone H3 at the promoters of the inducible TAP1 and Cyclin D1 genes, but had little effect at the constitutively active ß-actin gene (<xref ref-type="fig" rid="ukmss-38018-f0006">Figure 6d</xref>).).'], 'ukmss-38018-f0007': ['Having previously shown that PHB-RNAi increased prostate cell and tumour growth in response to testosterone (27), we tested whether it could similarly increase the response to adrenal androgens and translate into a tumour growth effect. Interestingly, in the absence of doxycycline we observed no cell growth in response to DHEA at doses up to and including 10nM for 96h (<xref ref-type="fig" rid="ukmss-38018-f0007">Figure 7a</xref> and data not shown). In culture, PHB-RNAi increased cell growth in response to all androgens: DHT, androstenedione and DHEA (10nM) ( and data not shown). In culture, PHB-RNAi increased cell growth in response to all androgens: DHT, androstenedione and DHEA (10nM) (<xref ref-type="fig" rid="ukmss-38018-f0007">Figure 7a</xref>). PHB-RNAi also increased the percentage of cells in S-phase, under all conditions tested (). PHB-RNAi also increased the percentage of cells in S-phase, under all conditions tested (<xref ref-type="fig" rid="ukmss-38018-f0007">Figure 7b</xref>).).', 'In testosterone-treated castrated mice, LNCaP tumours grow with a mean doubling time of 20 days, while in vehicle-treated mice tumours stop growing and shrink within 48hr (27). Tumours in androstenedione-treated castrated mice did not show rapid tumour growth as seen for testosterone, but unlike vehicle controls, tumours were maintained with no regression (<xref ref-type="fig" rid="ukmss-38018-f0007">Figure 7c</xref>, dashed line). Under the same conditions plus PHB-RNAi, tumours showed a statistically significant increase in RTV as compared to control (, dashed line). Under the same conditions plus PHB-RNAi, tumours showed a statistically significant increase in RTV as compared to control (<xref ref-type="fig" rid="ukmss-38018-f0007">Figure 7c</xref>), i.e. PHB reduction promoted androstenedione-induced tumour growth ), i.e. PHB reduction promoted androstenedione-induced tumour growth in vivo. <xref ref-type="fig" rid="ukmss-38018-f0007">Figure 7d</xref> summarises RTVs for vehicle, androstenedione and testosterone (± doxycycline) at the end of the experiment. summarises RTVs for vehicle, androstenedione and testosterone (± doxycycline) at the end of the experiment.']}
|
Reducing prohibitin increases histone acetylation, and promotes androgen independence in prostate tumours by increasing androgen receptor activation by adrenal androgens
|
[
"prohibitin",
"androgen receptor",
"prostate",
"androgens",
"castrate-resistant prostate cancer",
"corepressor"
] |
Oncogene
|
1351148400
|
[{'@Label': 'STUDY DESIGN', '@NlmCategory': 'METHODS', '#text': 'Systematic reviewObjective:\u2003To compare the safety and effectiveness of fusion versus denervation for chronic sacroiliac joint pain after failed conservative management.'}, {'@Label': 'SUMMARY OF BACKGROUND', '@NlmCategory': 'BACKGROUND', '#text': 'METHODS of confirming the sacroiliac joint as a pain source have been extensively studied and reported in the literature. After confirmation of the origin of the pain by positive local anesthetic blocks, chronic sacroiliac joint pain is usually managed with a combination of medication, physical therapies, and injections. We have chosen to compare two alternative treatments for sacroiliac pain that was refractory to conservative therapies.'}, {'@Label': 'METHODS', '@NlmCategory': 'METHODS', '#text': 'A systematic review of the English-language literature was undertaken for articles published between 1970 and June 2010. Electronic databases and reference lists of key articles were searched to identify studies evaluating fusion or denervation for chronic sacroiliac joint pain after failed conservative management. Studies involving only conservative treatment or traumatic onset of injury were excluded. Two independent reviewers assessed the level of evidence quality using the grading of recommendations assessment, development and evaluation (GRADE) system, and disagreements were resolved by consensus.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'We identified eleven articles (six fusion, five denervation) meeting our inclusion criteria. The majority of patients report satisfaction after both treatments. Both treatments reported mean improvements in pain and functional outcome. Rates of complications were higher among fusion studies (13.7%) compared to denervation studies (7.3%). Only fusion studies reported infections (5.3%). No infections were reported among denervation patients. The evidence for all findings were very low to low; therefore, the relative efficacy or safety of one treatment over another cannot be established.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'Sacroiliac joint fusion or denervation can reduce pain for many patients. Whether a true arthrodesis of the joint is achieved by percutaneous techniques is open to question and whether denervation of the joint gives durable pain relief is not clear. Further comparative studies of these two techniques may provide the answers.'}]
|
[] |
other
|
PMC3427022
| null |
11
|
[
"{'Citation': 'Wise C L, Dall B E. Minimally invasive sacroiliac arthrodesis: outcomes of a new technique. J Spinal Disord Tech. 2008;21(8):579–584.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19057252'}}}",
"{'Citation': 'Schutz U, Grob D. Poor outcome following bilateral sacroiliac joint fusion for degenerative sacroiliac joint syndrome. Acta Orthop Belg. 2006;72:296–308.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16889141'}}}",
"{'Citation': 'Al-Khayer A, Hegarty J, Hahn D. et al.Percutaneous sacroiliac joint arthrodesis: a novel technique. J Spinal Disord Tech. 2008;21(5):359–363.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18600147'}}}",
"{'Citation': 'Buchowski J M Kebaish K M Sinkov V et al.2005Functional and radiographic outcome of sacroiliac arthrodesis for the disorders of the sacroiliac joint Spine J 55520–528.discussion 529', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16153580'}}}",
"{'Citation': 'Burnham R S, Yasui Y. An alternate method of radiofrequency neurotomy of the sacroiliac joint: a pilot study of the effect on pain, function, and satisfaction. Reg Anesth Pain Med. 2007;32(1):12–19.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17196487'}}}",
"{'Citation': 'Cohen S P, Hurley R W, Buckenmaier C C. et al.Randomized placebo-controlled study evaluating lateral branch radiofrequency denervation for sacroiliac joint pain. Anesthesiology. 2008;109(2):279–288.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC2666931'}, {'@IdType': 'pubmed', '#text': '18648237'}]}}",
"{'Citation': 'Cohen S P, Abdi S. Lateral branch blocks as a treatment for sacroiliac joint pain: a pilot study. Reg Anesth Pain Med. 2003;28(2):113–119.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '12677621'}}}",
"{'Citation': 'Vallejo R, Benyamin R M, Kramer J. et al.Pulsed radiofrequency denervation for the treatment of sacroiliac joint syndrome. Pain Med. 2006;7(5):429–434.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17014602'}}}",
"{'Citation': 'Khurana A, Guha A R, Mohanty K. et al.Percutaneous fusion of the sacroiliac joint with hollow modular anchorage screws: clinical and radiological outcome. J Bone Joint Surg Br. 2009;91(5):627–631.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '19407297'}}}",
"{'Citation': 'Yin W, Willard F, Carreiro J. et al.Sensory stimulation-guided sacroiliac joint radiofrequency neurotomy: technique based on neuroanatomy of the dorsal sacral plexus. Spine. 2003;28(20):2419–2425.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '14560094'}}}",
"{'Citation': 'Waisbrod H, Krainick J U, Gerbershagen H U. Sacroiliac joint arthrodesis for chronic lower back pain. Arch Orthop Trauma Surg. 1987;106(4):238–240.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '2956935'}}}"
] |
Oncogene. 2012 Oct 25; 31(43):4588-4598
|
NO-CC CODE
|
|
Reducing Pcdhg diversity disrupts heteroneuronal SAC interactionsa. Two nearby SACs from a wild-type mouse injected with contrasting fluorescent dyes. Right panel shows image of the green SAC flipped vertically.b. Overlap between red and green cells in a. First two bars are derived from the two panels in a. The green cell was rotated in 45° steps or flipped and then rotated; third and fourth bars show mean overlap ± s.e.m. derived from these images (n=7). All inversions and rotations decrease overlap, indicating that overlap in the real image is non-random.c-e. Tracings of SAC pairs, and versions flipped as in a, from wild-type, Pcdhgrko/rko and cA1;Pcdhgrko/rko mice. Overlap shown in black.f. Overlap between neighboring cells, expressed as ratio between overlap measured in real and flipped images. Bars show mean ± s.e.m. for 11, 9 and 8 pairs from wild-type, Pcdhgrko/rko and single isoform-expressing (cA1;Pcdhgrko/rko and cC3;Pcdhgrko/rko) animals. Expression of a single isoform in neighboring SACs decreases their interaction.g. Mean length of overlapping segments between SAC pairs. R, real image; F, flipped image. *P=0.05; **P<0.05; ***P<0.01. Error bars, s.e.m. N as in f. Scale bar, 50 μm.
|
nihms-383719-f0004
|
2
|
ad327b3695f718668726cb97625ab74c3684f748608fa662f874263785d4ec30
|
nihms-383719-f0004.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
423,
601
] |
[{'image_id': 'nihms-383719-f0001', 'image_file_name': 'nihms-383719-f0001.jpg', 'image_path': '../data/media_files/PMC3427422/nihms-383719-f0001.jpg', 'caption': 'Pcdhgs are required for self-avoidance of SAC dendritesa.\nPcdh locus comprises Pcdha, Pcdhb and Pcdhg subclusters. Pcdha and Pcdhg isoforms are assembled by splicing of 1 variable exon to 3 constant exons.b. SACs are present in both inner nuclear and ganglion cell layers (INL, GCL) and extend dendrites that form radially symmetrical arbors confined to thin sublaminae in the inner plexiform layer (IPL).c. SAC dendrites avoid isoneuronal dendrites but form synapses with dendrites of other SACs.d-i. Morphology of single SAC, labeled with membrane-Cherry, in the GCL in wild-type and Pcdhg mutant retinas. Wild-type SAC dendrites self-avoid. In Pcdhg mutants, self-avoidance defects include self-crossing and bundling of dendrites. Crossings are detected at 0.2 μm x-y resolution in single 0.8 μm optical sections (g,i magnified inset in f,h). Images with 0.2 μm z resolution are shown in Supplementary Fig. 1.j. SAC dendritic self-crossings in 1st-5th order branches per SAC. Graph underestimates difference between genotypes because the most severely affected mutant SACs could not be scored. ** P<0.01k,l. Number of terminal branches (i) and dendritic field diameter (j) do not differ between wild-type and mutant SACs. i-l show means ±s.e.m; n=8 cells from 5-6 animals per genotype.Scale bars, 50 μm (d,f) or 10 μm (e,g).', 'hash': '23fd200eb25379f5d7e8803605ecbfd76dea9893b20b42c23a21df9624a6cc29'}, {'image_id': 'nihms-383719-f0005', 'image_file_name': 'nihms-383719-f0005.jpg', 'image_path': '../data/media_files/PMC3427422/nihms-383719-f0005.jpg', 'caption': 'Purkinje cell dendrite self-avoidance requires Pcdhgsa-c. Control Purkinje cells labeled with Cre-dependent AAV-XFP in L7-cre transgenic mouse. Self-avoidance is clear in high magnification view in c (inset in b).d-f. Purkinje cells lacking Pcdhgs and labeled as in a-c have disorganized arbors marked by frequent self-crossing defects. f shows area boxed in e. g. Self-crossings detected in single confocal z-sections of 7225 μm2 unit area. *** P <0.001; n=8, 15 and 15 cells at P15, P21 and P35 respectively from ≥ 3 mice per genotype. h,i. Area of dendritic arbors (n=20 cells) and cell density (>40 regions) do not differ between control and mutant Purkinje cells. Bars show mean ±s.e.m. Scale bars, 50 μm in a, b, d, e; 10 μm in c, f.', 'hash': '2020fdc37cd35643cdfe0104916234d7c2c98826809692e1010e4397493c2234'}, {'image_id': 'nihms-383719-f0002', 'image_file_name': 'nihms-383719-f0002.jpg', 'image_path': '../data/media_files/PMC3427422/nihms-383719-f0002.jpg', 'caption': 'Pcdhgs pattern developing SAC dendrites in a cell-autonomous mannera-h. SACs in developing wild-type and Pcdhg mutant retinas. Wild-type SACs extend fine, exuberant branches (P3, P5) that make transient intradendritic contacts (P5, P8); by P12, excess branches and isoneuronal contacts are eliminated. Dendrites of mutant SACs display excessive self-crossing and bundling by P3; by P12, excess branches are eliminated, but crossing dendrites remain.i,j. Cultured Pcdhg mutant SACs exhibit loss of symmetric growth and uneven distribution of neurites.k. Histogram of fractal dimensions (Df, metric for space-filling) for 47 wild-type (black) and 47 mutant (grey) SACs. Wild-type SAC in i has Df of 1.61 and mutant SAC in j has Df of 1.53.l. Mean Df for cultured SACs (n=47 cells), SACs in vivo at P5 (n=6) and adult (n=9). *** P<0.001. Error bars, s.e.m. Scale bars, 50 μm except 20 μm in i,j.', 'hash': 'b4196ba274e6d88529a28a3a32e7b946613a519f8d01174c275905422d81e9c3'}, {'image_id': 'nihms-383719-f0003', 'image_file_name': 'nihms-383719-f0003.jpg', 'image_path': '../data/media_files/PMC3427422/nihms-383719-f0003.jpg', 'caption': 'No single Pcdhg isoform is necessary and any isoform is sufficient for dendrite self-avoidancea. SACs lacking Pcdhgc3-c5 (pcdhgtcko/fcon3;retina-cre) exhibit self-avoidance.b. Replacement of all 22 Pcdhgs by the PcdhgC3 isoform rescues SAC dendrite self-avoidance.c. SACs lacking Pcdhga1-a3 (pcdhgtako/tako) exhibit self-avoidance.d. Replacement of all 22 Pcdhgs by the PcdhgA1 isoform rescues SAC dendrite self-avoidance.e. Compared to mutants lacking all 22 isoforms, self-crossings in SACs expressing 19 or 1 isoform are restored to control levels. *** P<0.001; n.s., not significant. Bars are mean ± s.e.m, from 7 SACs from pcdhgtcko/fcon3;retina-cre retinas, 3 SACs from pcdhgtako/tako, and 9 from remaining genotypes. Scale bars, 50 μm.', 'hash': '51a043535e1656101234844492dad19875f140a710092b0e8bef939d48b4f8b5'}, {'image_id': 'nihms-383719-f0004', 'image_file_name': 'nihms-383719-f0004.jpg', 'image_path': '../data/media_files/PMC3427422/nihms-383719-f0004.jpg', 'caption': 'Reducing Pcdhg diversity disrupts heteroneuronal SAC interactionsa. Two nearby SACs from a wild-type mouse injected with contrasting fluorescent dyes. Right panel shows image of the green SAC flipped vertically.b. Overlap between red and green cells in a. First two bars are derived from the two panels in a. The green cell was rotated in 45° steps or flipped and then rotated; third and fourth bars show mean overlap ± s.e.m. derived from these images (n=7). All inversions and rotations decrease overlap, indicating that overlap in the real image is non-random.c-e. Tracings of SAC pairs, and versions flipped as in a, from wild-type, Pcdhgrko/rko and cA1;Pcdhgrko/rko mice. Overlap shown in black.f. Overlap between neighboring cells, expressed as ratio between overlap measured in real and flipped images. Bars show mean ± s.e.m. for 11, 9 and 8 pairs from wild-type, Pcdhgrko/rko and single isoform-expressing (cA1;Pcdhgrko/rko and cC3;Pcdhgrko/rko) animals. Expression of a single isoform in neighboring SACs decreases their interaction.g. Mean length of overlapping segments between SAC pairs. R, real image; F, flipped image. *P=0.05; **P<0.05; ***P<0.01. Error bars, s.e.m. N as in f. Scale bar, 50 μm.', 'hash': 'ad327b3695f718668726cb97625ab74c3684f748608fa662f874263785d4ec30'}]
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{'nihms-383719-f0001': ['The 58 genes of the mouse Pcdh locus are tandemly arranged in α, β and γ subclusters, called Pcdha, Pcdhb and Pcdhg, which encode 14, 22 and 22 cadherin-like proteins respectively8 (<xref ref-type="fig" rid="nihms-383719-f0001">Fig. 1a</xref>). In the ). In the Pcdha and g subclusters, single variable exons encoding extracellular, transmembrane and juxtamembrane domains are spliced to 3 constant exons, generating proteins with unique extracellular but common intracellular domains8. The complexity of this locus is reminiscent of that of Dscam1, which mediates self-avoidance in Drosophila4-7,15. Moreover, Pcdhs, like Dscam1, exhibit isoform-specific homotypic recognition and stochastic, combinatorial expression13,14. In contrast, the two vertebrate Dscams are not complex genes, so although they mediate both repulsive and attractive interactions among neurons16-19, they are unlikely to underlie self/non-self discrimination. We therefore investigated roles of Pcdhs in these processes.', 'Previous studies of mouse mutants lacking all 22 Pcdhg genes revealed that they are required for survival of multiple neuronal types20-23. To seek roles of Pcdhgs in self-avoidance, we focused on a retinal interneuron, the starburst amacrine cell (SAC), which expresses Pcdhgs22 and exhibits dramatic dendritic self-avoidance24. Radially symmetric SAC dendritic arbors are confined to narrow planes within the inner plexiform (synaptic) layer; SACs have no axons. Dendrites of a single SAC seldom cross one another, yet dendrites of neighboring SACs cross freely (<xref ref-type="fig" rid="nihms-383719-f0001">Fig. 1b, c</xref>; ; Supplementary Fig. 1) and even form synapses with each other24,25, suggesting that they can distinguish “self” from “non-self.”', 'We used a conditional mutant (Pcdhgfcon3)22 to bypass the neonatal lethality of constitutive Pcdhg mutants and employed Cre drivers that delete Pcdhgs from all or subsets of retinal cells. We visualized individual neurons by infection with recombinant adeno-associated virus (rAAV) expressing a fluorescent protein (XFP; <xref ref-type="fig" rid="nihms-383719-f0001">Fig. 1d</xref>), biolistic delivery of DNA encoding XFP, or intracellular injection of a fluorescent dye. We identified SACs, the sole cholinergic neurons in retina, with antibodies to choline acetytransferase (ChAT), which also demonstrated the association of XFP-positive SAC dendrites with dendrites from other (XFP-negative) SACs (), biolistic delivery of DNA encoding XFP, or intracellular injection of a fluorescent dye. We identified SACs, the sole cholinergic neurons in retina, with antibodies to choline acetytransferase (ChAT), which also demonstrated the association of XFP-positive SAC dendrites with dendrites from other (XFP-negative) SACs (Supplementary Figs. 1 and 2).', 'SAC morphology was profoundly altered in Pcdhg mutant retinas (Pcdhgfcon3/fcon3; retina-cre, called Pcdhgrko/rko here; see online Methods for genotypes). Dendrites arising from a single SAC frequently crossed each other and sometimes formed loose bundles (<xref ref-type="fig" rid="nihms-383719-f0001">Fig. 1f-i</xref> and and Supplementary Fig. 1). Crossing frequency was increased several-fold in both proximal and distal regions of the arbor (<xref ref-type="fig" rid="nihms-383719-f0001">Fig. 1j</xref>). These defects were highly specific in that the diameter of SAC arbors, the number of dendritic termini, the laminar targeting of SAC dendrites, heteroneuronal interactions among dendrites, and the mosaic arrangement of SAC bodies were all unaffected in ). These defects were highly specific in that the diameter of SAC arbors, the number of dendritic termini, the laminar targeting of SAC dendrites, heteroneuronal interactions among dendrites, and the mosaic arrangement of SAC bodies were all unaffected in Pcdhgrko/rko mutants (<xref ref-type="fig" rid="nihms-383719-f0001">Fig. 1k, l</xref> and and Supplementary Figs. 1 and 2). Thus, Pcdhgs are dispensable for many aspects of SAC morphogenesis but required for their self-avoidance.'], 'nihms-383719-f0002': ['We next asked whether Pcdhgs are required for the development of SAC arbors, or only for their maintenance. In wild-type neonates, SACs extended dendrites that branched profusely and contacted each other (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2a-c</xref>). By P12, however, excess neurites and isoneuronal contacts were eliminated, resulting in a radial arbor with evenly-spaced branches (). By P12, however, excess neurites and isoneuronal contacts were eliminated, resulting in a radial arbor with evenly-spaced branches (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2d</xref> and see and see ref. 24). Thus, self-avoidance arises rapidly following a short period of isoneuronal “sampling.” In Pcdhgrko/rko mice, SACs were clearly aberrant by P3, exhibiting excessive crossing and tangling of neurites (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2e-g</xref>). Excess branches were subsequently eliminated, but whereas most crossing branches were eliminated in controls, many persisted in mutants (). Excess branches were subsequently eliminated, but whereas most crossing branches were eliminated in controls, many persisted in mutants (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2h</xref>). Thus, Pcdhgs may lead to self-avoidance by mediating repulsive interactions that bias the rearrangement process to selectively eliminate contacts among isoneuronal branches.). Thus, Pcdhgs may lead to self-avoidance by mediating repulsive interactions that bias the rearrangement process to selectively eliminate contacts among isoneuronal branches.', 'To initiate analysis of the mechanism by which Pcdhgs mediate self-avoidance, we next asked whether they act cell-autonomously. We selectively removed Pcdhgs from SACs using a ChAT-Cre line. In this case, Pcdhg-negative SACs were surrounded by Pcdhg-positive neurons of other types. We also deleted Pcdhgs from individual SACs using a transgenic line that expressed tamoxifen-activated Cre recombinase in SACs; we activated Cre with a low dose of tamoxifen and introduced a Cre-dependent reporter to mark mutant SACs. In this case, Pcdhg-negative SACs were surrounded by Pcdhg-positive SACs. In both cases, SACs lacking Pcdhgs exhibited striking self-avoidance defects (Supplementary Fig.4). To test whether Pcdhgs can act in completely isolated SACs, we used fluorescence-activated cell sorting to purify SACs from a transgenic line in which they are selectively labeled by an orange fluorescent protein (Thy1-OFP3) and cultured them at low density. Isolated SACs extended dendrites that formed radial, web-like arbors (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2i</xref>) reminiscent of those observed at ~P5 in vivo () reminiscent of those observed at ~P5 in vivo (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2b</xref>). In contrast, SACs from ). In contrast, SACs from Pcdhgrko/rko; Thy1-OFP3 mice exhibited less symmetrical and unevenly spaced arbors, reminiscent of those observed in Pcdhgrko/rko retinas at P5 (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2j</xref> and and Supplementary Fig. 5). Analysis of space-filling capacity of dendritic arbors2,27 (see Methods) revealed that defects in vitro were similar in magnitude to those in vivo (<xref ref-type="fig" rid="nihms-383719-f0002">Fig. 2k,l</xref>). Thus, Pcdhgs do not depend on intercellular interactions to promote self-avoidance.). Thus, Pcdhgs do not depend on intercellular interactions to promote self-avoidance.'], 'nihms-383719-f0003': ['We next assessed the requirement for isoform diversity in Pcdhg-dependent self-avoidance. We used RT-PCR to survey expression of Pcdhg isoforms in whole retina, in amacrines generally, and in SACs specifically. All 22 Pcdhg variants were expressed in each preparation, with no indication of decreased diversity in purified subpopulations (Supplementary Fig. 6). We then analyzed a targeted mouse mutant, Pcdhgtcko, in which three contiguous Pcdhg variable exons, C3-C5, had been deleted. Expression of the remaining 19 Pcdhg isoforms is unperturbed in this allele 28. Because Pcdhgtcko homozygous mice die at birth 28}, we generated transheterozygous animals (Pcdhgtcko/fcon3;retina-cre) so that only retina lack both copies of Pcdhgc3-c5. In these retinas, neuronal death was as prevalent as in those of Pcdhgrko/rko mice29, yet SACs exhibited normal self-avoidance (<xref ref-type="fig" rid="nihms-383719-f0003">Fig. 3a,e</xref>).).', 'In a complementary approach, we generated a line in which the single PcdhgC3 isoform, fused to a fluorescent protein (mCherry), could be expressed in any cell in a Cre-dependent manner (ROSA26-CAG::lox-Stop-lox-Pcdhgc3-mCherry or cC3-mCherry). Thus, in cC3-mCherry;Pcdhgrko/rko mice, Cre both deletes all 22 endogenous Pcdhg genes and activates the single PcdhgC3-mCherry isoform throughout the retina. Analysis of mCherry fluorescence confirmed Cre-dependent expression of the transgene in all retinal cells and appropriate localization of the fusion protein to cell membranes and synaptic layers (Supplementary Fig. 7). Expression of Pcdhgc3 alone rescued self-avoidance defects of Pcdhg mutants (<xref ref-type="fig" rid="nihms-383719-f0003">Fig. 3b,e</xref>).).', 'To test the possibility that only some isoforms are dispensable for self-avoidance, we analyzed a second set of isoforms. We generated Pcdhgtako, which lacks the Pcdhga1-a3 variable exons28, and a line that expresses Pcdhga1-mCherry in a Cre-dependent manner (cA1-mCherry). Results were similar to those for the C3-5 group: self-avoidance persisted in the absence of PcdhgA1-A3 and was rescued by replacement of all Pcdhg isoforms with PcdhgA1 alone (<xref ref-type="fig" rid="nihms-383719-f0003">Figs. 3c-e</xref> and and Supplementary Fig. 7). From these results, we conclude that no single Pcdhg isoform is necessary but any single isoform is sufficient for dendritic self-avoidance.'], 'nihms-383719-f0004': ['Although Pcdhg isoform diversity is not required for isoneuronal self-avoidance, it may be required to ensure that dendrites of adjacent SACs do not avoid each other, which would prevent them from interacting. The ability to generate a SAC population expressing a single Pcdhg isoform (Pcdhga1 or Pcdhgc3) enabled us to test this idea. We injected closely spaced pairs of SACs with different fluorophores (<xref ref-type="fig" rid="nihms-383719-f0004">Fig. 4a</xref>) and measured the extent to which their dendrites overlapped. To determine whether this method reliably revealed interactions among SACs, we rotated, flipped or rotated and flipped the image of one of the cells, and recalculated overlap. Only the real image showed an overlap greater than that of the manipulated images () and measured the extent to which their dendrites overlapped. To determine whether this method reliably revealed interactions among SACs, we rotated, flipped or rotated and flipped the image of one of the cells, and recalculated overlap. Only the real image showed an overlap greater than that of the manipulated images (<xref ref-type="fig" rid="nihms-383719-f0004">Fig. 4b</xref>). We then measured overlap for pairs of SACs from wild-type, mutant and single isoform-expressing mice, normalizing for intercellular distance by comparing overlap to the value calculated from the flipped image (). We then measured overlap for pairs of SACs from wild-type, mutant and single isoform-expressing mice, normalizing for intercellular distance by comparing overlap to the value calculated from the flipped image (<xref ref-type="fig" rid="nihms-383719-f0004">Fig. 4c-e</xref> and and Supplementary Fig. 8). Overlap was equivalent in wild-type and mutant retina, but significantly decreased in retinas expressing a single isoform (<xref ref-type="fig" rid="nihms-383719-f0004">Fig. 4f</xref>); values for ); values for Pcdhga1 and Pcdhgc3 were similar (1.01 and 1.08). Likewise, the mean length of overlapping segments was greater than expected for random overlap in wild-type and mutant but not in single isoform-expressing pairs (<xref ref-type="fig" rid="nihms-383719-f0004">Fig. 4g</xref>). Thus, when all SACs express the same ). Thus, when all SACs express the same Pcdhg isoform, heteroneuronal dendrites avoid each other, just as isoneuronal dendrites do in control SACs. We conclude that isoform diversity enables SACs to distinguish isoneuronal from heteroneuronal dendrites.'], 'nihms-383719-f0005': ['Finally, we asked whether Pcdhgs mediate self-avoidance in areas other than retina. We examined cerebellar Purkinje cells, which have elaborate, planar dendritic arbors known to exhibit self-avoidance3 (<xref ref-type="fig" rid="nihms-383719-f0005">Fig. 5a-c</xref>). Importantly, stochastic and combinatorial expression, which underlies the ability of ). Importantly, stochastic and combinatorial expression, which underlies the ability of Drosophila Dscam1 to mediate self-avoidance4-6,12,14,15,29, has been documented for Pcdhgs in Purkinje cells10. We selectively deleted Pcdhgs from Purkinje neurons using an L7-cre transgene, marked cells with a vector that expresses fluorescent proteins in a Cre-dependent manner, and examined them at P15, P21 and at P35, after arbors have matured30. Deletion of Pcdhgs from Purkinje cells had no detectable effect on their survival, shape, size or branching pattern (<xref ref-type="fig" rid="nihms-383719-f0005">Fig. 5d,e,h,i</xref> and and Supplementary Fig. 9), but their arbors were disorganized and dendrites often crossed over each other (<xref ref-type="fig" rid="nihms-383719-f0005">Fig. 5f, g</xref>). Use of a Cre-dependent reporter revealed that deletion remained incomplete at P8, at which time Purkinje dendrite growth was already advanced (). Use of a Cre-dependent reporter revealed that deletion remained incomplete at P8, at which time Purkinje dendrite growth was already advanced (Supplementary Fig. 9). It is therefore possible that earlier deletion of Pcdhgs would lead to a more dramatic effect. Nonetheless, these results demonstrate a role for Pcdhgs in Purkinje cell self-avoidance.']}
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PROTOCADHERINS MEDIATE DENDRITIC SELF-AVOIDANCE IN THE MAMMALIAN NERVOUS SYSTEM
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Nature
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1345705200
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other
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PMC3427422
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0
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[] |
Nature. 2012 Aug 23; 488(7412):517-521
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NO-CC CODE
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TACC3 and Aurora A kinase interaction and localization in FTC-133 thyroid cells. (A) Mitotic FTC-133 cells were stained for TACC3 and β-tubulin (upper panel) or γ-tubulin (middle panel) or Aurora-A (lower panel) as described in Materials and methods. Scale bar, 10 μm. (B and C) Aurora-A or TACC3 were immunoprecipitated from FTC-133 cell extracts using either the anti-Aurora-A monoclonal antibody (B) or the anti-TACC3 polyclonal antibody (C). Aurora-A and TACC3 were then immunodetected with the anti-Aurora-A monoclonal antibody (upper panel) and the polyclonal anti-TACC3 antibody (lower panel) as described in Materials and methods.
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ERC070053f02
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2
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665f005f6ec77a67ae269b23a60596fc67d590135399f501bddc56d8f669bfc9
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ERC070053f02.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
415,
476
] |
[{'image_id': 'ERC070053f05', 'image_file_name': 'ERC070053f05.jpg', 'image_path': '../data/media_files/PMC2216418/ERC070053f05.jpg', 'caption': 'TACC3 and Aurora-A kinase expression in matched cancer and normal thyroid tissues. (A and B) Quantitative RT-PCR analysis of TACC3 (A) and Aurora-A kinase (B) mRNA levels in 13 papillary (PTC) and 3 follicular (FTC) thyroid cancer tissues compared with normal matched tissues obtained from the same patients. mRNA variations were relative to the TACC3/actin or Aurora A/actin ratios observed in normal thyroid tissues. The small bars represent the median values. (C) Correlation analysis between the variations of TACC3 and Aurora-A kinase mRNAs in the differentiated thyroid carcinomas (13 PTCs and 3 FTCs) analyzed.', 'hash': '1e2481fb7bb9514006f756e801501c227f9b8bc0261ab8f8f28eae06eab6991b'}, {'image_id': 'ERC070053f02', 'image_file_name': 'ERC070053f02.jpg', 'image_path': '../data/media_files/PMC2216418/ERC070053f02.jpg', 'caption': 'TACC3 and Aurora A kinase interaction and localization in FTC-133 thyroid cells. (A) Mitotic FTC-133 cells were stained for TACC3 and β-tubulin (upper panel) or γ-tubulin (middle panel) or Aurora-A (lower panel) as described in Materials and methods. Scale bar, 10\u200aμm. (B and C) Aurora-A or TACC3 were immunoprecipitated from FTC-133 cell extracts using either the anti-Aurora-A monoclonal antibody (B) or the anti-TACC3 polyclonal antibody (C). Aurora-A and TACC3 were then immunodetected with the anti-Aurora-A monoclonal antibody (upper panel) and the polyclonal anti-TACC3 antibody (lower panel) as described in Materials and methods.', 'hash': '665f005f6ec77a67ae269b23a60596fc67d590135399f501bddc56d8f669bfc9'}, {'image_id': 'ERC070053f03', 'image_file_name': 'ERC070053f03.jpg', 'image_path': '../data/media_files/PMC2216418/ERC070053f03.jpg', 'caption': 'Effects of Aurora kinase inhibition by VX-680 on FTC-133 cell ploidy, spindle formation and TACC3 subcellular localization. (A) FTC-133 cells were incubated for 24\u200ah with 500\u200anM VX-680 or DMSO used as vehicle (control) and pulse labeled with BrdU for 2\u200ah. The cells were then fixed and analyzed by flow as described in Materials and methods. (B) Fifty micrograms of cell protein extracts from control and VX-680-treated cells were analyzed by western blot with an anti-TACC3 antibody (diluted 1/250), anti-Aurora-A (diluted 1:500), or anti-actin antibody (1/500). (C) Subcellular localization of Aurora-A and TACC3 in control and VX-680-treated FTC-133 mitotic cells. Fixed cells were stained for TACC3 and β-tubulin or Aurora-A and β-tubulin as described in Materials and methods. Scale bar, 10\u200aμm.', 'hash': 'fd51d534bc82e0273a5dd5ae112b57caada25b73b72967a01374e1ca433eb41b'}, {'image_id': 'ERC070053f04', 'image_file_name': 'ERC070053f04.jpg', 'image_path': '../data/media_files/PMC2216418/ERC070053f04.jpg', 'caption': "Expression of TACC3 gene in normal human thyrocytes (HTU5) and cell lines derived from thyroid benign follicular adenoma (HTU42) and from papillary (B-CPAP), follicular (FTC-133), and anaplastic (8305C and CAL-62) thyroid carcinomas. (A) Quantitative RT-PCR analysis of TACC3 mRNA levels. Aliquots of 5\u200aμg RNA were analyzed by RT-PCR. Messenger RNA variations were relative to the TACC3/actin ratio observed in HTU5 cells. Data reported represent the mean±s.e.m. of three independent experiments. Statistical significance of data was assessed by Student's t-test. *P<0.01, **P<0.05. (B and C) Western blot analysis of TACC3 protein in the different cell lines as described above. Fifty micrograms of the cell protein extracts were analyzed for TACC3 and actin contents by immunoblotting (B). TACC3 variations (C) were relative to the TACC3/actin ratio observed in HTU5 cells. Data reported represent the mean±s.e.m. of three independent experiments. Statistical significance of data was assessed by Student's t-test. *P<0.01, **P<0.05.", 'hash': '74273dcf64c54637d0b0ad72a048d8547c012e58d23ef66196d7810f4a80a7a0'}, {'image_id': 'ERC070053f01', 'image_file_name': 'ERC070053f01.jpg', 'image_path': '../data/media_files/PMC2216418/ERC070053f01.jpg', 'caption': 'Expression, cell cycle regulation, and localization of TACC3 in normal human thyrocytes (HTU5). (A) Aliquots of 5\u200aμg DNA from HTU5 cells were used to prepare cDNA in the presence or absence of the reverse transcriptase. The cDNAs were amplified by quantitative RT-PCR using specific primers for TACC3 and actin as described in Materials and methods. (B) Fifty micrograms of cell protein extracts have been analyzed by western blot with an anti-TACC3 antibody (diluted 1/250) preincubated or not with the immunogenic peptide (upper panel) and anti-actin antibody (1/500; lower panel). (C and D) HTU5 cells have been treated for 4 days in the presence of 5% (C) or 0.3% (SF) FBS, some of the latter were then cultured for additional 24\u200ah in media containing 5% FBS. Quantitative RT-PCR (C) and western blot (D) analyses for TACC3 were performed on cell extracts as described in Materials and methods. Data reported are representative of one out of three experiments. (E) Interphasic (upper panel) and mitotic (middle and lower panels) HTU5 cells were stained for TACC3 and β-tubulin (upper and middle panels) as a marker of microtubules or γ-tubulin (lower panel) as a marker for centrosome as described in Material and methods. Scale bar, 10\u200aμm.', 'hash': '66e609a9dc4a0012d97e0d60caca563466686c35995eb84d5edb3433ab94efb1'}]
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{'ERC070053f01': ['We first evaluated the expression at the mRNA and protein level of the TACC3 gene in HTU5 cells. We demonstrated the presence of a specific TACC3 transcript in HTU5 cells. The omission of the reverse transcriptase (negative control) prevented the formation of amplicons (<xref ref-type="fig" rid="ERC070053f01">Fig. 1</xref>A). Western blot analysis of HTU5 cell protein extracts demonstrated the presence of an immunoreactive band of ∼100\u200akDa, which was completely abrogated when the anti-TACC3 antibody was preincubated with the immune peptide (A). Western blot analysis of HTU5 cell protein extracts demonstrated the presence of an immunoreactive band of ∼100\u200akDa, which was completely abrogated when the anti-TACC3 antibody was preincubated with the immune peptide (<xref ref-type="fig" rid="ERC070053f01">Fig. 1</xref>B).B).', 'We next determined whether TACC3 expression was cell cycle regulated in HTU5 cells. To this end, the cells were cultured for 4 days in the presence of full medium containing 5% FBS or in medium containing low serum concentration (0.3%). Some of the latter were then exposed to fresh medium containing 5% FBS for 24\u200ah. In these conditions, we previously shown, by means of flow cytometer analysis, that serum deprivation for 4 days reduced the number of proliferating cells (S+G2/M-phase) by more than 75%, while the exposure to fresh medium containing 5% FBS restored the number of proliferating cells to control level (Ulisse et al. 2006a). As shown in <xref ref-type="fig" rid="ERC070053f01">Fig. 1</xref>C and D, serum deprivation induced a marked reduction in the expression of C and D, serum deprivation induced a marked reduction in the expression of TACC3 gene, at both mRNA and protein level. The exposure to fresh full medium restored the expression of TACC3 gene at control level (<xref ref-type="fig" rid="ERC070053f01">Fig. 1</xref>C and D). As TACC3 is cell cycle regulated in thyrocytes, we next investigated the subcellular localization of TACC3 protein by indirect immunofluorescence microscopy in the HTU5 cells. In all interphasic cell lines tested, the TACC3 immunoreactivity was localized around the nuclei and decorated part of the interphasic microtubules (C and D). As TACC3 is cell cycle regulated in thyrocytes, we next investigated the subcellular localization of TACC3 protein by indirect immunofluorescence microscopy in the HTU5 cells. In all interphasic cell lines tested, the TACC3 immunoreactivity was localized around the nuclei and decorated part of the interphasic microtubules (<xref ref-type="fig" rid="ERC070053f01">Fig. 1</xref>E, upper panel). In mitotic cells, TACC3 localized on the spindle microtubules, where β-tubulin was also localized, and in the pericentrosomal material (PCM) area. No staining for TACC3 protein was observed onto the centrosome, as demonstrated by the double staining with γ-tubulin (E, upper panel). In mitotic cells, TACC3 localized on the spindle microtubules, where β-tubulin was also localized, and in the pericentrosomal material (PCM) area. No staining for TACC3 protein was observed onto the centrosome, as demonstrated by the double staining with γ-tubulin (<xref ref-type="fig" rid="ERC070053f01">Fig. 1</xref>E, middle and lower panels).E, middle and lower panels).'], 'ERC070053f02': ['Since Aurora-A has been reported to interact with TACC3 in other cellular systems, we analyzed their interaction in thyroid cells. To this end, the FTC-derived cell line, the FTC-133 was employed, since these cells express a high level of Aurora-A (Ulisse et al. 2006a) and, as described above, a relative high amount of TACC3. As in HTU5 cells, TACC3 co-localized with β-tubulin, but not γ-tubulin on the spindle microtubules, and was observed on the PCM (<xref ref-type="fig" rid="ERC070053f02">Fig. 2</xref>A, upper and middle panels). As expected, Aurora-A was found to localize on the centrosome and the microtubules of the spindle poles (A, upper and middle panels). As expected, Aurora-A was found to localize on the centrosome and the microtubules of the spindle poles (<xref ref-type="fig" rid="ERC070053f02">Fig. 2</xref>A, lower panel). TACC3 co-localized with Aurora-A solely on the PCM and the microtubules of the spindle poles. We thus investigated the interaction between TACC3 and Aurora-A by immunoprecipitating Aurora-A or TACC3 from FTC-133 cell extracts. The presence of TACC3 in the Aurora-A immunoprecipitate as well as that of Aurora-A in the TACC3 immunoprecipitate, demonstrated that Aurora-A and TACC3 interact, either directly or indirectly, in human thyroid cells (A, lower panel). TACC3 co-localized with Aurora-A solely on the PCM and the microtubules of the spindle poles. We thus investigated the interaction between TACC3 and Aurora-A by immunoprecipitating Aurora-A or TACC3 from FTC-133 cell extracts. The presence of TACC3 in the Aurora-A immunoprecipitate as well as that of Aurora-A in the TACC3 immunoprecipitate, demonstrated that Aurora-A and TACC3 interact, either directly or indirectly, in human thyroid cells (<xref ref-type="fig" rid="ERC070053f02">Fig. 2</xref>B).B).'], 'ERC070053f03': ['In the attempt to elucidate the functional role of Aurora-A and TACC3 interaction, the effects of FTC-133 treatment with the Aurora kinase inhibitor VX-680 were studied. In particular, FTC-133 cells were treated with VX-680 at 500\u200anM, a concentration previously shown on different cell types to elicit maximal response (Harrington et al. 2004). Analysis of cell DNA content after 24-h treatment, by flow cytometer analysis, showed the accumulation of cells with ≥4\u200aN DNA content (<xref ref-type="fig" rid="ERC070053f03">Fig. 3</xref>A). Cell treatment with VX-680 did not affect neither the levels of TACC3 or Aurora-A proteins (A). Cell treatment with VX-680 did not affect neither the levels of TACC3 or Aurora-A proteins (<xref ref-type="fig" rid="ERC070053f03">Fig. 3</xref>B). Immunofluorescence experiments demonstrated that 84.1% of VX-680-treated cells have more than two centrosomes compared with 4% in control cells (B). Immunofluorescence experiments demonstrated that 84.1% of VX-680-treated cells have more than two centrosomes compared with 4% in control cells (<xref ref-type="fig" rid="ERC070053f03">Fig. 3</xref>C). In the latter, all mitotic cells showed the presence of aberrant spindles characterized by shorter microtubules, or no spindle. In VX-680-treated cells, Aurora-A still present on the centrosomes, while TACC3 was missing on the spindle microtubules (C). In the latter, all mitotic cells showed the presence of aberrant spindles characterized by shorter microtubules, or no spindle. In VX-680-treated cells, Aurora-A still present on the centrosomes, while TACC3 was missing on the spindle microtubules (<xref ref-type="fig" rid="ERC070053f03">Fig. 3</xref>C).C).'], 'ERC070053f04': ['The above observations lead us to investigate the expression level of TACC3 gene in different human cell lines derived from benign follicular adenoma (HTU42), follicular (FTC-133), papillary (B-CPAP), and anaplastic (8305C and CAL-62) thyroid carcinomas. Quantitative RT-PCR analysis revealed that TACC3 mRNA levels were similar in the HTU42 and HTU5 cells, while a lower expression was observed in all the carcinoma-derived cell lines (<xref ref-type="fig" rid="ERC070053f04">Fig. 4</xref>A). In particular, the TACC3 mRNA level was reduced in the FTC-133 (0.72±0.06; A). In particular, the TACC3 mRNA level was reduced in the FTC-133 (0.72±0.06; P<0.01), the B-CPAP (0.64±0.01; P<0.01), the 8305C (0.39±0.04; P<0.01), and the CAL-62 (0.33±0.03; P<0.01) cell lines. Likewise, with respect to the HTU5 cells, the TACC3 protein was found significantly reduced in the FTC-133 (0.85±0.05; P<0.05), B-CPAP (0.53±0.05; P<0.01), 8305C (0.35±0.13; P<0.01), and CAL-62 (0.17±0.08; P<0.01), but similar in the HTU42 cells (0.98±0.29; <xref ref-type="fig" rid="ERC070053f04">Fig. 4</xref>B and C).B and C).'], 'ERC070053f05': ['The levels of the TACC3 mRNA in PTC and FTC carcinoma tissues were compared with those of the matched normal thyroid by means of quantitative RT-PCR. The results are shown in <xref ref-type="fig" rid="ERC070053f05">Fig. 5</xref>A. TACC3 mRNA levels were lower in 7 out of 13 PTCs and 2 out of 3 FTCs, but higher in 6 PTCs and 1 FTC than in the normal tissue. Altogether, TACC3 mRNA levels were reduced in 56% of the differentiated thyroid cancers (DTC; 0.50±0.07; A. TACC3 mRNA levels were lower in 7 out of 13 PTCs and 2 out of 3 FTCs, but higher in 6 PTCs and 1 FTC than in the normal tissue. Altogether, TACC3 mRNA levels were reduced in 56% of the differentiated thyroid cancers (DTC; 0.50±0.07; P<0.01) and increased in 44% of DTC (1.96±0.35; P<0.05) when compared with their normal matched tissues. Since TACC3 and Aurora-A interact in thyroid cells, the expression of Aurora-A was determined in the same tissues. The results are shown in <xref ref-type="fig" rid="ERC070053f05">Fig. 5</xref>B. Quantitative RT-PCR revealed that Aurora-A was upregulated in 5 out of 13 PTCs (2.75±0.44; B. Quantitative RT-PCR revealed that Aurora-A was upregulated in 5 out of 13 PTCs (2.75±0.44; P<0.01). Downregulation of Aurora-A was noted in five PTCs (0.51±0.13; P<0.01) and all three FTCs (0.41±0.03; P<0.01).', 'Using a linear regression analysis, we evidenced a significant correlation (r=0.717; P<0.01) between the variations of TACC3 and Aurora-A mRNAs in thyroid cancer tissues (<xref ref-type="fig" rid="ERC070053f05">Fig. 5</xref>C). We finally attempted to correlate the variation of TACC3 and Aurora-A expression in thyroid cancer tissues with the clinical and histological parameters, including patient\'s age, stage, tumor size, and histology. No correlation could be found with any of the parameters analyzed.C). We finally attempted to correlate the variation of TACC3 and Aurora-A expression in thyroid cancer tissues with the clinical and histological parameters, including patient\'s age, stage, tumor size, and histology. No correlation could be found with any of the parameters analyzed.']}
|
Transforming acidic coiled-coil 3 and Aurora-A interact in human thyrocytes and their expression is deregulated in thyroid cancer tissues
| null |
Endocr Relat Cancer
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1188630000
|
The present study represents an investigation of a novel stirred bioreactor for culture of a transformed cell line under defined hydrodynamic conditions in vitro. Cell colonies of the EL-4 mouse lymphoma cell line grown for the first time in a rotating disc bioreactor (RDB), were observed to undergo changes in phenotype in comparison to standard, static flask cultures. RDB cultures, with or without agitation, promoted the formation of adherent EL-4 cell plaques that merged to form contiguous tumor-like masses in longer-term cultures, unlike the unattached spheroid aggregates of flask cultures. Plaques grown under agitated conditions were further altered in morphology and distribution in direct response to fluid mechanical stimuli. Plaque colonies growth in RDBs with or without agitation also exhibited significant increases in production of interleukin-4 (IL-4) and lactate, suggesting an inducible "Warburg effect." Increases in cell biomass in RDB cultures were no different to flask cultures, though a trend toward a marginal increase was observed at specific rotational speeds. The RDB may therefore be a suitable alternative method to study mechanisms of tumor progression and invasiveness in vitro, under more complex physicochemical conditions that may approximate natural tissue environments.
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[] |
other
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PMC2216418
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44
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[
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] |
Endocr Relat Cancer. 2007 Sep; 14(3):827-837
|
NO-CC CODE
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|
Typical luminescence
confocal microscopy images of Jurkat cells that had been treated with
anti-CaM antibody–Alexa Fluor 594 conjugate, phalloidin–CruzFluor
555 conjugate, MitoTracker Red, LysoTracker Red, and ER-RFP in the
presence (10 μM) or absence of 4: (a, e, h, l,
o, s, v, z, ac) bright field of Jurkat cells; (b, i, p, w, ad) emission
images of 4; (c) emission images of the anti-CaM antibody;
(d) overlay image of (a)–(c); (f, j) emission images of phalloidin;
(g) overlay image of (e) and (f); (k) overlay images of (h)–(j);
(m, q) emission images of MitoTracker Red; (n) overlay images of (l)–(m);
(r) overlay images of (o)–(q); (t, x) emission images of LysoTracker
Red; (u) overlay images of (s)–(t); (y) overlay images of (v)–(x);
(aa, ae) emission images of ER-RFP; (ab) overlay images of (z)–(aa);
(af) overlay images of (ac)–(ae). Excitation at 405 nm for
(b), (i), (p), and (w) and at 559 nm for (c), (f), (j), (m), (q),
(t), and (x). Exposure time was 20 μs/pixel. Scale bar (black)
is 10 μm.
|
ao0c00337_0009
|
2
|
46bed229b5a936ec481357f14bece329954c4587ffd98ee7edc60b489ff1f624
|
ao0c00337_0009.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
826,
1708
] |
[{'image_id': 'ao0c00337_0002', 'image_file_name': 'ao0c00337_0002.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0002.jpg', 'caption': 'Effect of SCH772984\n(ERK inhibitor), SP600125 (JNK inhibitor), and U0126 (MEK inhibitor).\n(a) Typical luminescence microscopy images of Jurkat cells treated\nwith SCH772984, SP600125, and U0126. (b) MTT assays of Jurkat cells\nin the presence and/or absence of 4 and SCH772984, SP600125,\nor U0126. Scale bar (black) is 10 μm.', 'hash': 'fd2c94cdb12b5afc2ec93ba71534c7331e518b9940d826b24605426a69372ec5'}, {'image_id': 'ao0c00337_0005', 'image_file_name': 'ao0c00337_0005.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0005.jpg', 'caption': 'No caption found', 'hash': '59ec1d6ba8431177b793ed082a40bae3b4959026db61fff727f95b1228aa4a81'}, {'image_id': 'ao0c00337_0014', 'image_file_name': 'ao0c00337_0014.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0014.jpg', 'caption': 'No caption found', 'hash': '4d7fa7f39e56f1312de789fd160e644c7abfd556a16e9e06e919a8d1a476b91a'}, {'image_id': 'ao0c00337_0013', 'image_file_name': 'ao0c00337_0013.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0013.jpg', 'caption': '(a) Typical\nluminescence images (Biorevo, BZ-9000, Keyence) of Jurkat cells treated\nwith Rhod-4 (red emission), followed by 4 (50 μM).\nExcitation at 540 nm for Rhod-4. (b, c) Time-dependent changes in\nthe fluorescent intensity of Rhod-4 (F/F0) after the treatment with 4 (b) and 13 (c) (cell 1 (light green), 2 (orange), 3 (red), 4 (yellow),\n5 (blue), 6 (green), and 7 (black)). (d) Typical luminescence images\n(Biorevo, BZ-9000, Keyence) of Jurkat cells treated with Rhod-2 (red\nemission), followed by 4 (50 μM). Excitation at\n540 nm for Rhod-2. (e, f) Time-dependent fluorescent changes of Rhod-2\n(F/F0) after the treatment\nwith 4 (e) and 13 (f) (cell 1 (light green),\n2 (orange), 3 (red), 4 (yellow), 5 (blue), 6 (green), and 7 (black)).', 'hash': '5635dfc230da1f3eb706a6f8cb091a70a940a62e37d1ca94ebc1fad68f21de5b'}, {'image_id': 'ao0c00337_0004', 'image_file_name': 'ao0c00337_0004.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0004.jpg', 'caption': 'Transmission\nelectron microscopy (TEM) images of Jurkat cells treated with 4 (25 μM, 1 h) and celastrol (1 μM, 24 h). (a)\nControl, (b) Jurkat cells treated with 4 (25 μM,\nfor 1 h), and (c) Jurkat cells treated with celastrol (1 μM,\nfor 24 h). Arrows in (b) and (c) indicate cytoplasmic vacuolization\ninduced by 4 and celastrol, respectively. Scale bar (black)\nis 1 μm.', 'hash': '338378bcb8af47738e5ac798cbf58678dd011a8d2fc4e758154cf1e3d62d8a3c'}, {'image_id': 'ao0c00337_0003', 'image_file_name': 'ao0c00337_0003.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0003.jpg', 'caption': 'Typical\nmicroscopy images of Jurkat cells stained with methylene blue after\nthe treatment with 4 (25 μM) at 37 °C for\n1 h. (a) Control; (b, c) Jurkat cells treated with 4 (25\nμM, for 1 h); and (d, e) Jurkat cells treated with celastrol\n(1 μM, for 24 h). Arrows in (c) and (e) indicate cytoplasmic\nvacuolization induced by 4 and celastrol, respectively.\nScale bar (black) is 10 μm, and scale bar (white) is 5 μm.', 'hash': '2f1dff2858e2211722ff28c6935749cdaf5eb743364d4a2869ae3dddc9b061ab'}, {'image_id': 'ao0c00337_m001', 'image_file_name': 'ao0c00337_m001.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_m001.jpg', 'caption': 'No caption found', 'hash': '692f281600f2cf06c8d980deebf585d6fb167992bf7ca86bcf76a9ae3fb86172'}, {'image_id': 'ao0c00337_0012', 'image_file_name': 'ao0c00337_0012.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0012.jpg', 'caption': 'Our Assumption on the Complexation of 4 with Ca2+-CaM', 'hash': '2f3bc4b7773e42376d1a6dfef7cd29a33ad6d561136bfe8149d5cd9a3b7b2b09'}, {'image_id': 'ao0c00337_0015', 'image_file_name': 'ao0c00337_0015.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0015.jpg', 'caption': '(a) MTT assay of Jurkat\ncells with 4 (closed circles), 5 (closed\ndiamonds), and 3c (closed squares) in 10% FBS/RPMI medium\n(incubation at 37 °C for 24 h). (b) MTT assay of IMR90 cells\nwith 4 (closed circles) and 5 (closed diamonds)\nin 10% FBS/RPMI medium (incubation at 37 °C for 24 h). The net\ncharge of each complex is assumed to be +12 (4 and 5) and +9 (3c).', 'hash': '60abd196d496a4f9631d78ca779502a4710ddfac72a755fe75033b093faa9ee2'}, {'image_id': 'ao0c00337_0016', 'image_file_name': 'ao0c00337_0016.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0016.jpg', 'caption': 'Flow cytometry analysis of Jurkat cells after the treatment with\n(a, b) Rhod-4 (5 μM) or (c, d) Rhod-2 (5 μM) and then\nwith 4 (50 μM). Different colors indicate the incubation\ntimes of 4: control (black), 2 min (blue), 4 min (light\ngreen), 6 min (red), 8 min (sky blue), 10 min (violet), and 15 min\n(green) in (a) and (c), and control (black), 20 min (blue), 40 min\n(light green), and 60 min (red) in (b) and (d).', 'hash': '6132b3a3c63f73f1ce21f849461f23dcb853e08273d39075005b84470b0ac00b'}, {'image_id': 'ao0c00337_0011', 'image_file_name': 'ao0c00337_0011.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0011.jpg', 'caption': 'No caption found', 'hash': '9d56d75ae4e28eaf8d7cd54be0982fdfd9d747d438360ea14d740a4909b3a7c2'}, {'image_id': 'ao0c00337_0018', 'image_file_name': 'ao0c00337_0018.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0018.jpg', 'caption': 'No caption found', 'hash': 'f630b91706f07bef14b74c9807fb2a704b44c0d7c94e650af25a9e3864821adb'}, {'image_id': 'ao0c00337_0009', 'image_file_name': 'ao0c00337_0009.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0009.jpg', 'caption': 'Typical luminescence\nconfocal microscopy images of Jurkat cells that had been treated with\nanti-CaM antibody–Alexa Fluor 594 conjugate, phalloidin–CruzFluor\n555 conjugate, MitoTracker Red, LysoTracker Red, and ER-RFP in the\npresence (10 μM) or absence of 4: (a, e, h, l,\no, s, v, z, ac) bright field of Jurkat cells; (b, i, p, w, ad) emission\nimages of 4; (c) emission images of the anti-CaM antibody;\n(d) overlay image of (a)–(c); (f, j) emission images of phalloidin;\n(g) overlay image of (e) and (f); (k) overlay images of (h)–(j);\n(m, q) emission images of MitoTracker Red; (n) overlay images of (l)–(m);\n(r) overlay images of (o)–(q); (t, x) emission images of LysoTracker\nRed; (u) overlay images of (s)–(t); (y) overlay images of (v)–(x);\n(aa, ae) emission images of ER-RFP; (ab) overlay images of (z)–(aa);\n(af) overlay images of (ac)–(ae). Excitation at 405 nm for\n(b), (i), (p), and (w) and at 559 nm for (c), (f), (j), (m), (q),\n(t), and (x). Exposure time was 20 μs/pixel. Scale bar (black)\nis 10 μm.', 'hash': '46bed229b5a936ec481357f14bece329954c4587ffd98ee7edc60b489ff1f624'}, {'image_id': 'ao0c00337_0007', 'image_file_name': 'ao0c00337_0007.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0007.jpg', 'caption': 'Intracellular uptake of IPHs 3c, 4, 5, and 2c (25 μM)\ninto Jurkat cells at 37 °C for 1 h measured by ICP-MS.', 'hash': 'e529b148a492ef06743cf86e233f669a6354e4974d26f5a085d86db7c40b4c0f'}, {'image_id': 'ao0c00337_0019', 'image_file_name': 'ao0c00337_0019.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0019.jpg', 'caption': 'Typical luminescence microscopy images of (a–l)\nJurkat cells and (m–u) IMR90 cells treated with 4 (25 μM), 5 (25 μM), and 3c (25 μM) at 37 °C for 1 h. (a) Bright field, (b) emission,\nand (c) overlay images of the control; (d) bright field, (e) emission,\nand (f) overlay images with 4; (g) bright field, (h)\nemission, and (i) overlay images with 5; (j) bright field,\n(k) emission, and (l) overlay images with 3c of Jurkat\ncells; (m) bright field, (n) emission, and (o) overlay images of the\ncontrol; (p) bright field, (q) emission, and (r) overlay images with 4; (s) bright field, (t) emission, and (u) overlay images\nwith 5 of IMR90 cells. Scale bar (black) is 10 μm,\nand scale bar (white) is 50 μm.', 'hash': '5de002bdec90f4687cac3a1383b01bc602302f8c007c45cb6567eded4c9f710e'}, {'image_id': 'ao0c00337_0010', 'image_file_name': 'ao0c00337_0010.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0010.jpg', 'caption': 'Western blot\nanalysis of Jurkat cells treated with 4 (0–25\nμM). Proteins related to (a) autophagy, (b) MAPK signaling pathway,\nand (c) PI3K/Akt signaling pathway, (d) ER stress, (e) CaM, and (f)\napoptosis were investigated in a dose-dependent manner.', 'hash': '29cb3c27d56a45f45363c9b0ab590938541cd40c489a4df9d1b40886608c61d0'}, {'image_id': 'ao0c00337_0017', 'image_file_name': 'ao0c00337_0017.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0017.jpg', 'caption': 'No caption found', 'hash': '743e04477e1f7f84a3e8d8cb924952b441c7567052492cc01025dc67b4c35f79'}, {'image_id': 'ao0c00337_0006', 'image_file_name': 'ao0c00337_0006.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0006.jpg', 'caption': 'No caption found', 'hash': '271b13e8273dd3510a8025ecf80435be9753e2d2c25066ee17e4d9644a23c60e'}, {'image_id': 'ao0c00337_0001', 'image_file_name': 'ao0c00337_0001.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0001.jpg', 'caption': 'Typical luminescent\nconfocal microscopy images of Jurkat cells treated with DilC1(5) (500\nnM) in the presence of 4 (25 μM) and/or CCCP (40\nμM): (a, d, h, k) bright field images of Jurkat cells; (b, f,\ni, m) emission images of DilC1(5); (e, l) emission images of 4; (c) overlay image of (a) and (b); (g) overlay image of\n(d)–(f); (j) overlay image of (h) and (i); (n) overlay image\nof (k)–(m). Excitation at 405 nm for (e) and (l); 635 nm for\n(b), (f), (i), and (m). Exposure time is 20 μs/pixel. Scale\nbar (black) is 10 μm.', 'hash': 'fc4d8a932c44c5e736260677403dbae4afcf5a9276cb0a84f648f2e741aedd5a'}, {'image_id': 'ao0c00337_0008', 'image_file_name': 'ao0c00337_0008.jpg', 'image_path': '../data/media_files/PMC7114882/ao0c00337_0008.jpg', 'caption': 'Effect of Z-VAD-fmk (15 μM, an apoptosis inhibitor),\nnecrostatin-1 (30 μM, a necroptosis inhibitor), 3-MA (5 mM,\nan autophagy inhibitor), TFP (10 μM, a CaM-binding molecule),\nand CCCP (40 μM, an uncoupling reagent and an inhibitor of Ca2+ influx into mitochondria) on the cell death of Jurkat cells\ninduced by 4. (a, d, g, j, m, p, s, v, y) Bright field\nimages of Jurkat cells; (b, e, h, k, n, q, t) emission images of 4; (w, z) emission images of 5; (c) overlay image\nof (a) and (b); (f) overlay image of (d) and (e); (i) overlay image\nof (g) and (h); (l) overlay image of (j) and (k); (o) overlay image\nof (m) and (n); (r) overlay image of (p) and (q); (u) overlay image\nof (s) and (t); (x) overlay image of (v) and (w); (aa) overlay image\nof (y) and (z). Excitation at 377 nm. Scale bar (black) is 10 μm.', 'hash': 'c8158bab777b16e30152c0abb6ae226c4525642a92469be9225c658183570dbb'}]
|
{'ao0c00337_0015': ['The cytotoxicities of 4 and 5 containing the cationic KKKGG peptide\nsequence against Jurkat cells (T-lymphocyte leukemia) were assessed\nby means of an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium\nbromide (MTT) assay (<xref rid="ao0c00337_0015" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>a). Jurkat cells (2.0 × 10<xref rid="ao0c00337_0015" ref-type="fig">1</xref>a). Jurkat cells (2.0 × 104 cells/mL) were\nincubated at 37 °C for 24 h in 10% fetal bovine serum (FBS)/RPMI\n1640 medium containing 4 or 5 (0–25\nμM). The results of the MTT assay indicate that 4 had nearly the same cytotoxicity (EC50 = 1.5 μM)\nas 5 (EC50 = 1.5 μM) and is slightly\nmore cytotoxic than 3c (EC50 = 2.4 μM).\nOn the other hand, 4 and 5 (0–25\nμM) weakly induced cell death of IMR90 cells (human Caucasian\nfetal lung fibroblast), which are used as a model of normal cells\n(<xref rid="ao0c00337_0015" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>b). These\nresults suggest that Ir complexes <xref rid="ao0c00337_0015" ref-type="fig">1</xref>b). These\nresults suggest that Ir complexes 4 and 5 have the capacity to selectively kill cancer cells.', 'Another possibility is\nthat 4 binds to actin and promotes the degradation of\nactin filaments and the morphological changes in Jurkat cells (<xref rid="ao0c00337_0015" ref-type="fig">Figures <xref rid="fig1" ref-type="fig">1</xref></xref> and <xref rid="ao0c00337_0015" ref-type="fig">1</xref> and <xref rid="ao0c00337_0009" ref-type="fig">8</xref>e–k). It is also reported that the enhancement of intracellular\nCae–k). It is also reported that the enhancement of intracellular\nCa2+ collapses the bundling of actin filaments, resulting\nin morphological changes in the cells.64−66'], 'ao0c00337_0019': ['<xref rid="ao0c00337_0019" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>a–l\nshows fluorescence microscopic images of Jurkat cells treated with <xref rid="ao0c00337_0019" ref-type="fig">2</xref>a–l\nshows fluorescence microscopic images of Jurkat cells treated with 4, 5, and the reference compound 3c ([Ir complex] = 25 μM). Considerable morphological changes\nwere induced by 4, 5, and 3c, and the dead cells exhibited green emission arising from these\nIPHs. The cytotoxicities of 4 and 5 against\nIMR90 cells (human Caucasian fetal lung fibroblast), a normal cell\nline, were also evaluated. In <xref rid="ao0c00337_0019" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>p–u, morphological changes and emissions of <xref rid="ao0c00337_0019" ref-type="fig">2</xref>p–u, morphological changes and emissions of 4 and 5 from the cells\nwere both negligible or weak, indicating their lower cytotoxicity\nagainst IMR90 cells.', 'The cytotoxicity of 4 is more potent than\nthe reference compound 5, which could be attributed to\nits more efficient intracellular uptake than that of 5, partially due to the position of peptide units (<xref rid="ao0c00337_0019" ref-type="fig">Figures <xref rid="fig2" ref-type="fig">2</xref></xref> and <xref rid="ao0c00337_0019" ref-type="fig">2</xref> and <xref rid="ao0c00337_0007" ref-type="fig">3</xref>). The similar order of the EC). The similar order of the EC50 value of 4 to that of 5 is controlled by the affinity of these\nIPHs to the target biomolecules, whose affinity with these IPHs would\nbe on the order of micromolar.'], 'ao0c00337_0007': ['The intracellular uptake of IPHs 3c, 4, 5, and 2c in Jurkat cells was examined\nby inductively coupled plasma-mass spectrometer (ICP-MS). Jurkat cells\nwere incubated with 3c, 4, 5, and 2c (25 μM) for 1 h at 37 °C. After\ntreatment, the cells were washed three times with PBS and lysed by\nnitric acid. Then, lysed cells were incubated overnight at 4 °C,\nand the samples were analyzed on ICP-MS. Interestingly, it was found\nthat the intracellular uptake of 3c (6.26 ± 0.31\nfmol/cell) and 4 (7.72 ± 0.68 fmol/cell) was higher\nthan that of 5 (0.97 ± 0.18 fmol/cell) and 2c (0.34 ± 0.04 fmol/cell), as summarized in <xref rid="ao0c00337_0007" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>. We assume that\nthe three polycationic peptide parts of <xref rid="ao0c00337_0007" ref-type="fig">3</xref>. We assume that\nthe three polycationic peptide parts of 3c and 4 are assembled in the same direction of its structure and\nthat the position of cationic peptide parts on the IPHs is important\nfor their intracellular uptake.'], 'ao0c00337_0008': ['In our previous paper, it was reported that the induction of cell\ndeath by 2c is not inhibited by the broad caspase inhibitor\nbenzyloxycarbonyl-VAD(OMe)-fluoromethylketone (Z-VAD-fmk), indicating\nthat cell death does not involve apoptosis.24 In this study, the effects of Z-VAD-fmk, 3-chlorophenylhydrazone\n(CCCP, an uncoupling reagent and an inhibitor of mitochondrial Ca2+ uptake), necrostatin-1 (Nec-1, a specific RIPK-1 inhibitor\nand necroptosis inhibitor), 3-methyladenine (3-MA, an inhibitor of\nautophagosome formation that functions by inhibiting type III phosphatidylinositol\n3-kinases (PI3K), an autophagy inhibitor), and trifluoperazine (TFP,\na CaM-binding molecule) (for the structures of these inhibitors, see Charts S1 and S2 in the Supporting Information)\non the cytotoxicities of 4 and 5 were examined\n(<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>). Jurkat\ncells were preincubated with these inhibitors<xref rid="ao0c00337_0008" ref-type="fig">4</xref>). Jurkat\ncells were preincubated with these inhibitors31 for 30 min and then treated with 4 (25 μM) or 5 (25 μM) for 1 h. Morphological changes (and cell death)\nin the Jurkat cells caused by 4 and 5 were\ninhibited to a considerable extent by treatment with CCCP (40 μM),\nand the extent of IPH emission was negligible (<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>d–f,y-aa), indicating the existence\nof a relationship between the cell death and intracellular Ca<xref rid="ao0c00337_0008" ref-type="fig">4</xref>d–f,y-aa), indicating the existence\nof a relationship between the cell death and intracellular Ca2+ homeostasis and/or membrane potential of mitochondria. On\nthe other hand, the cell death induced by 4 and 5 was negligibly inhibited by Z-VAD-fmk (<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>g–i), Nec-1 (<xref rid="ao0c00337_0008" ref-type="fig">4</xref>g–i), Nec-1 (<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>j–l), 3-MA\n(<xref rid="ao0c00337_0008" ref-type="fig">4</xref>j–l), 3-MA\n(<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>m–o),\nTFP (<xref rid="ao0c00337_0008" ref-type="fig">4</xref>m–o),\nTFP (<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>p–r),\nand combinations thereof (Nec-1 + 3-MA) (<xref rid="ao0c00337_0008" ref-type="fig">4</xref>p–r),\nand combinations thereof (Nec-1 + 3-MA) (<xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>s–u). These results indicate that\ncell death induced by IPHs does not involve apoptosis, necroptosis,\nor autophagy alone.<xref rid="ao0c00337_0008" ref-type="fig">4</xref>s–u). These results indicate that\ncell death induced by IPHs does not involve apoptosis, necroptosis,\nor autophagy alone.'], 'ao0c00337_0013': ['Recently, the relationship of intracellular\nCa2+ signaling and CaM with cell survival and cell death\nwas well discussed.32−38 The enhancement in intracellular Ca2+ concentration was\nexamined by time-course fluorescent microscopy according to our previous\nreports.24,25 Jurkat cells were treated with Rhod-4/AM,\na cytosolic Ca2+ indicator, or Rhod-2/AM, a mitochondrial\nCa2+ indicator, as confirmed by costaining experiments\nwith MitoTracker Green (Figure S3 in the\nSupporting Information), whose red emission is enhanced upon complexation\nwith Ca2+ in cytosol and mitochondria, respectively. As\nshown in <xref rid="ao0c00337_0013" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>d,e,\nthe fluorescent intensity of Rhod-2 was enhanced in a few minutes\nafter the addition of <xref rid="ao0c00337_0013" ref-type="fig">5</xref>d,e,\nthe fluorescent intensity of Rhod-2 was enhanced in a few minutes\nafter the addition of 4 (50 μM) in mitochondria\nrather than in cytosol (<xref rid="ao0c00337_0013" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>a,b), and the morphology of Jurkat cells changed after ca.\n20 min. In contrast, <xref rid="ao0c00337_0013" ref-type="fig">5</xref>a,b), and the morphology of Jurkat cells changed after ca.\n20 min. In contrast, 13, which contains no peptide part\nand shows a very weak cytotoxicity, induces negligible Ca2+ response (<xref rid="ao0c00337_0013" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>c,f). These results suggest that <xref rid="ao0c00337_0013" ref-type="fig">5</xref>c,f). These results suggest that 4 initially induces\nan increase in the Ca2+ concentrations in mitochondria\nrather than in cytosol at the early stages of the cell death processes.', 'IPH 4 enhances the concentration of Ca2+ in mitochondria rather than in cytosol (<xref rid="ao0c00337_0013" ref-type="fig">Figures <xref rid="fig5" ref-type="fig">5</xref></xref> and <xref rid="ao0c00337_0013" ref-type="fig">5</xref> and <xref rid="ao0c00337_0016" ref-type="fig">6</xref>), possibly\nby Ca), possibly\nby Ca2+ release from the endoplasmic reticulum (ER), and\nthen induces the loss of the mitochondrial membrane potential (ΔΨm), which triggers the malfunction of mitochondria (<xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>o–r). Recently,\nit has been described that the excessive Ca<xref rid="ao0c00337_0009" ref-type="fig">8</xref>o–r). Recently,\nit has been described that the excessive Ca2+ release promotes\nPCD and proliferation via Ca2+-signaling events in the\nER and mitochondrion,32 which may support\nour hypothesis. This mechanism is also supported by the findings that\ncell death was considerably inhibited by CCCP, which is an inhibitor\nof mitochondrial Ca2+ influx and an uncoupling reagent\nthat compromises the mitochondrial membrane potential (ΔΨm) (<xref rid="ao0c00337_0008" ref-type="fig">Figures <xref rid="fig4" ref-type="fig">4</xref></xref>d–f and <xref rid="ao0c00337_0008" ref-type="fig">4</xref>d–f and <xref rid="ao0c00337_0001" ref-type="fig">7</xref>h–j). Further studies\nindicated that h–j). Further studies\nindicated that 4 induces mitochondrial Ca2+ overload within 15 min after the addition to Jurkat cells, as evidenced\nby <xref rid="ao0c00337_0016" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>c. In contrast,\ncelastrol, a naturally occurring triterpenoid, stimulates cytosolic\nCa<xref rid="ao0c00337_0016" ref-type="fig">6</xref>c. In contrast,\ncelastrol, a naturally occurring triterpenoid, stimulates cytosolic\nCa2+ overload in 1–5 h after the addition to Jurkat\ncells (Figure S7 in the Supporting Information).', 'It is strongly suggested that the cell death induced by 4 is a paraptosis-like cell death, as evidenced by cytoplasmic vacuolization,\nwhich was also observed by a treatment with celastrol, which had been\nreported to induce Ca2+ overload and paraptosis in the\nliterature.56,57 We therefore assessed the cytosolic\nand mitochondrial Ca2+ concentrations induced by celastrol\nby flow cytometric analysis (Figure S7 in\nthe Supporting Information). Interestingly, it was found that celastrol\ninduces considerable increase in cytosolic Ca2+ concentrations\nslowly (in ca. 1–5 h) with a small change in the mitochondrial\nCa2+ concentration. These findings suggest that 4 and celastrol induce paraptosis-like cell death via different responses\nof intracellular Ca2+. It is unlikely that the influx of\nCa2+ into mitochondria occurs from cytosol in the case\nof 4, according to negligible or weak enhancement of\nthe emission of a cytosolic Ca2+ probe, Rhod-4 (<xref rid="ao0c00337_0013" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>a,b). In addition,\nthe cell death induced by <xref rid="ao0c00337_0013" ref-type="fig">5</xref>a,b). In addition,\nthe cell death induced by 4 was negligibly inhibited\nby a Ca2+ chelator, BAPTA (Figure S4 in the Supporting Information). We assume that 4 induces\nthe direct transportation of Ca2+ from ER to mitochondria\nand then loss of ΔΨm, resulting in paraptosis-like\ncell death accompanied by their vacuolization, although the roles\nof the interaction of 4 with CaM in these processes are\nyet to be studied.'], 'ao0c00337_0016': ['The cytosolic and mitochondrial Ca2+ concentrations were also checked by flow cytometric analysis\n(<xref rid="ao0c00337_0016" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>). Jurkat\ncells were preincubated with Rhod-4/AM or Rhod-2/AM for 30 min, treated\nwith <xref rid="ao0c00337_0016" ref-type="fig">6</xref>). Jurkat\ncells were preincubated with Rhod-4/AM or Rhod-2/AM for 30 min, treated\nwith 4 (50 μM) for a given incubation time, and\nthen immediately analyzed by flow cytometry. <xref rid="ao0c00337_0016" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>a,b shows the emission intensity of Rhod-4\nfor 0–15 and 0–60 min after the addition of <xref rid="ao0c00337_0016" ref-type="fig">6</xref>a,b shows the emission intensity of Rhod-4\nfor 0–15 and 0–60 min after the addition of 4, respectively, and <xref rid="ao0c00337_0016" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>c,d shows the emission change of Rhod-2 for 0–15 and\n0–60 min after the addition of <xref rid="ao0c00337_0016" ref-type="fig">6</xref>c,d shows the emission change of Rhod-2 for 0–15 and\n0–60 min after the addition of 4, respectively.\nThe apparent increase in the emission from Rhod-2 was observed within\n15 min after the addition of 4 to Jurkat cells, supporting\nthe observations in <xref rid="ao0c00337_0013" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>.<xref rid="ao0c00337_0013" ref-type="fig">5</xref>.'], 'ao0c00337_0001': ['The mitochondrial membrane potential (ΔΨm) was measured using DilC1(5) (1,1′,3,3,3′,3′-hexamethylindodicarbocyanine\niodide)39 (its structure is shown in Chart S3 in the Supporting Information), which\naccumulates in mitochondria and responds to ΔΨm. In <xref rid="ao0c00337_0001" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>d–g,\nconsiderable decrease in the emission of DilC1(5) was observed in\nthe presence of <xref rid="ao0c00337_0001" ref-type="fig">7</xref>d–g,\nconsiderable decrease in the emission of DilC1(5) was observed in\nthe presence of 4 (25 μM), as compared with <xref rid="ao0c00337_0001" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>b,c, indicating the\nloss of ΔΨ<xref rid="ao0c00337_0001" ref-type="fig">7</xref>b,c, indicating the\nloss of ΔΨm. The decrease in ΔΨm was also observed by the treatment of CCCP, which prevents\nCa2+ influx into mitochondria and inhibits cell death induced\nby 4, in the absence (h–j) or presence (k–n)\nof 4 (25 μM). It has been well described that the\ninflux of Ca2+ into mitochondria induces the loss of ΔΨm, resulting in cell death.40,41 These results\nallowed us to conclude that 4 induces the influx of Ca2+ into mitochondria and then the loss of ΔΨm, which trigger the cell death signaling pathway.'], 'ao0c00337_0009': ['These\nfacts prompted us to conduct staining experiments of Jurkat cells\nby treating them with an anticalmodulin (CaM) antibody (+antirabbit\nIgG H&L conjugated with Alexa Fluor 594), phalloidin (a probe\nfor actin), MitoTracker Red (a probe for mitochondria), LysoTracker\nRed (a probe for lysosome), and ER-RFP (a probe conjugated with the\nred fluorescent protein (RFP) for the endoplasmic reticulum (ER))\n(<xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>). For staining\nwith an anti-CaM antibody, Jurkat cells were treated with <xref rid="ao0c00337_0009" ref-type="fig">8</xref>). For staining\nwith an anti-CaM antibody, Jurkat cells were treated with 4 (10 μM) at 37 °C for 1 h and then incubated with the\nanti-CaM antibody at room temperature for 2 h accompanied by the treatment\nwith the Alexa Fluor 594-conjugated secondary antibody. For the staining\nof actin, mitochondria, and lysosomes, the cells were treated with 4 for 1 h and then incubated with a phalloidin–CruzFluor\n555 conjugate (at a dilution of 1/1000), MitoTracker Red (100 nM),\nand LysoTracker Red (500 nM) for 1 h, sequentially. For the staining\nof ER, the cells were pretreated with ER-RFP for 24 h and then incubated\nwith 4 for 1 h. As shown in <xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>a–d, the overlapping of the green\nemission of <xref rid="ao0c00337_0009" ref-type="fig">8</xref>a–d, the overlapping of the green\nemission of 4 and the red emission of anti-CaM antibody\nwas negligible. This point will be discussed later.', 'Although actin constructs\ncytoskeletons in the cells in the absence of 4 (<xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>e–g), this\nnetwork underwent degradation after the treatment with <xref rid="ao0c00337_0009" ref-type="fig">8</xref>e–g), this\nnetwork underwent degradation after the treatment with 4, and the green emission of 4 and the red emission of\nphalloidin were overlapped to a considerable extent (<xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>h–k). The green emission\nof <xref rid="ao0c00337_0009" ref-type="fig">8</xref>h–k). The green emission\nof 4 and the red emission of MitoTracker Red were also\nconsiderably overlapped (<xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>l–r). On the other hand, the red emission of\nLysoTracker Red and ER-RFP was decreased by the treatment with <xref rid="ao0c00337_0009" ref-type="fig">8</xref>l–r). On the other hand, the red emission of\nLysoTracker Red and ER-RFP was decreased by the treatment with 4 (<xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>s–y,z–af), indicating the degradation of lysosome and\nER by <xref rid="ao0c00337_0009" ref-type="fig">8</xref>s–y,z–af), indicating the degradation of lysosome and\nER by 4. These results suggest that 4 interacts\nwith actin filaments and mitochondria and induces lysosome- and ER-related\ncell death accompanied by morphological changes, possibly through 4-(Ca2+-CaM) complexation.', 'In <xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>a–d, negligible\noverlap of the green emission from <xref rid="ao0c00337_0009" ref-type="fig">8</xref>a–d, negligible\noverlap of the green emission from 4 and the red emission\nfrom the anti-CaM antibody (+secondary IgG H&L-Alexa Fluor 594)\nwas observed. Then, the binding affinity of the anti-CaM antibody\n(Abcam) with CaM was checked by QCM analysis in the presence (80 μM)\nand absence of Ca2+ (Figure S6 and Table S3 in the Supporting Information). The Kapp and Kd values of the anti-CaM\nantibody were determined to be (4.22 ± 0.15) × 108 M–1 and 2.4 ± 0.1 nM, respectively, in the\nabsence of Ca2+. However, the interaction of the antibody\nwith CaM was very weak in the presence of Ca2+ (>1 mM),\nsuggesting that the anti-CaM antibody has weak affinity with the Ca2+ complex of CaM. It was assumed that this is the reason why\nnegligible overlap of the emission of 4 and the anti-CaM\nantibody was observed in <xref rid="ao0c00337_0009" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>a–d.<xref rid="ao0c00337_0009" ref-type="fig">8</xref>a–d.'], 'ao0c00337_0010': ['The aforementioned results strongly suggest that IPHs induce\nsome sort of programmed cell death (PCD), which can be categorized\ninto several types such as apoptosis, necroptosis, paraptosis, and\nautophagic cell death.51,55−63 For the further study of this issue, we checked the expression levels\nof proteins that are related to apoptosis, autophagy, and some signaling\npathway in Jurkat cells that had been treated with 4 by\nWestern blot analysis (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>). The degradation of caspase-3 was negligible, suggesting\nthat this is not an apoptosis process, as previously shown in <xref rid="ao0c00337_0010" ref-type="fig">9</xref>). The degradation of caspase-3 was negligible, suggesting\nthat this is not an apoptosis process, as previously shown in <xref rid="ao0c00337_0008" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>g–i.<xref rid="ao0c00337_0008" ref-type="fig">4</xref>g–i.', 'In <xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>a, LC3-I, LC3-II, Beclin-1, and Atg-12, which are autophagy markers,\nwere upregulated by <xref rid="ao0c00337_0010" ref-type="fig">9</xref>a, LC3-I, LC3-II, Beclin-1, and Atg-12, which are autophagy markers,\nwere upregulated by 4 in a dose-dependent manner. We\nfurther examined the autophagy signaling pathway such as mitogen-activated\nprotein kinase (MAPK) (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>b), the PI3K/Akt signaling pathway (<xref rid="ao0c00337_0010" ref-type="fig">9</xref>b), the PI3K/Akt signaling pathway (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>c), and ER stress (<xref rid="ao0c00337_0010" ref-type="fig">9</xref>c), and ER stress (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>d). In <xref rid="ao0c00337_0010" ref-type="fig">9</xref>d). In <xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>b, <xref rid="ao0c00337_0010" ref-type="fig">9</xref>b, p-p38 (phosphorylated form of p38), p-ERK-1, -2 (ERK: extracellular regulated kinase), p-JNK (JNK: c-jun N-terminal kinase 1), and CHOP, which\nare typical marker molecules of the MAPK signaling pathway, were upregulated\nafter the treatment by 4; in contrast, only negligible\nchanges were observed in the expression levels of proteins related\nto the PI3K/Akt signaling pathway (Akt and p-Akt)\nand ER stress (IRE1α, p-eIF2a, and Bip) (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>c,d). Change in the\nexpression levels of CaM was also negligible (<xref rid="ao0c00337_0010" ref-type="fig">9</xref>c,d). Change in the\nexpression levels of CaM was also negligible (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>e). In addition, negligible effect of <xref rid="ao0c00337_0010" ref-type="fig">9</xref>e). In addition, negligible effect of 4 was observed in the apoptosis signaling pathway in Jurkat\ncells (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>f).<xref rid="ao0c00337_0010" ref-type="fig">9</xref>f).', 'The results\nof Western blot analysis revealed that 4 induces the\nupregulation of typical marker proteins of paraptosis and autophagy\n(LC3-II, Beclin-1, and Atg-12) through the MAPK signaling pathway\n(phosphorylation of p38, ERKs, and JNK 1), possibly by CaMKK and CaMKII\nactivated by a (Ca2+-CaM)–4 complex,\nrather than the PI3K/Akt signaling pathway and ER stress (<xref rid="ao0c00337_0010" ref-type="fig">Figure <xref rid="fig9" ref-type="fig">9</xref></xref>). However, the cell\ndeath of Jurkat cells by <xref rid="ao0c00337_0010" ref-type="fig">9</xref>). However, the cell\ndeath of Jurkat cells by 4 was negligibly inhibited by\nan ERK inhibitor (SCH772984), a JNK inhibitor (SP600125), and an MEK\ninhibitor (U0126) (<xref rid="ao0c00337_0002" ref-type="fig">Figure <xref rid="fig10" ref-type="fig">10</xref></xref>), indicating that autophagy-mediated cell death is not the\nmain pathway of cell death.<xref rid="ao0c00337_0002" ref-type="fig">10</xref>), indicating that autophagy-mediated cell death is not the\nmain pathway of cell death.'], 'ao0c00337_0002': ['To evaluate the role of the MAPK signaling pathway in this process,\nthe effects of SCH772984, SP600125, and U0126, which are ERK, JNK,\nand MEK inhibitors (see Chart S4 in the\nSupporting Information for their chemical structures), respectively,\nwere assessed (<xref rid="ao0c00337_0002" ref-type="fig">Figure <xref rid="fig10" ref-type="fig">10</xref></xref>). In <xref rid="ao0c00337_0002" ref-type="fig">10</xref>). In <xref rid="ao0c00337_0002" ref-type="fig">Figure <xref rid="fig10" ref-type="fig">10</xref></xref>a,\nmorphological changes (and cell death) of Jurkat cells by <xref rid="ao0c00337_0002" ref-type="fig">10</xref>a,\nmorphological changes (and cell death) of Jurkat cells by 4 were observed, and the dead cells exhibited a strong green emission\nfrom 4. The results of MTT assays indicate that negligible\ninhibition of cell death induced by 4 (25 μM, 1\nh) was observed by SCH772984, SP600125, or U0126 (<xref rid="ao0c00337_0002" ref-type="fig">Figure <xref rid="fig10" ref-type="fig">10</xref></xref>b), suggesting that autophagy-mediated\ncell death through the MAPK signaling pathway is not a major pathway\nin <xref rid="ao0c00337_0002" ref-type="fig">10</xref>b), suggesting that autophagy-mediated\ncell death through the MAPK signaling pathway is not a major pathway\nin 4-induced cell death.', '<xref rid="ao0c00337_0002" ref-type="fig">Figure <xref rid="fig10" ref-type="fig">10</xref></xref>b,c shows images of Jurkat cells that were\ntreated with <xref rid="ao0c00337_0002" ref-type="fig">10</xref>b,c shows images of Jurkat cells that were\ntreated with 4 (25 μM) for 1 h, fixed with glutaraldehyde\nand osmium tetroxide (OsO4), embedded into Poly 812 resin,\nsliced, and then stained with methylene blue. These pictures clearly\nshow vacuolization of the cytoplasm and intracellular organelle (it\nhas been described in the literature that cytoplasmic vacuolization\nis triggered by the damage of mitochondria and ER)55−58 in Jurkat cells and morphological\nchanges. The same intracellular morphological changes were observed\nby the treatment with celastrol (1 μM and incubation for 24\nh) (<xref rid="ao0c00337_0003" ref-type="fig">Figure <xref rid="fig11" ref-type="fig">11</xref></xref>d,e).<xref rid="ao0c00337_0003" ref-type="fig">11</xref>d,e).'], 'ao0c00337_0004': ['In addition, transmission electron microscopy (TEM)\nimages of Jurkat cells treated with 4 and celastrol were\nalso undertaken. Pictures in <xref rid="ao0c00337_0004" ref-type="fig">Figure <xref rid="fig12" ref-type="fig">12</xref></xref> display the vacuolization in Jurkat cells, similar\nto <xref rid="ao0c00337_0004" ref-type="fig">12</xref> display the vacuolization in Jurkat cells, similar\nto <xref rid="ao0c00337_0003" ref-type="fig">Figure <xref rid="fig11" ref-type="fig">11</xref></xref>, strongly\nimplying that <xref rid="ao0c00337_0003" ref-type="fig">11</xref>, strongly\nimplying that 4 and celastrol induce similar PCD, which\ncan be classified into paraptosis although their PCD-inducing mechanisms\nare somewhat different, as described in <xref rid="ao0c00337_0016" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref> in the text and <xref rid="ao0c00337_0016" ref-type="fig">6</xref> in the text and Figure S4 in the Supporting Information. These results also suggest\nthat the staining experiments of dead cells with glutaraldehyde, OsO4, and methylene blue shown in <xref rid="ao0c00337_0003" ref-type="fig">Figure <xref rid="fig11" ref-type="fig">11</xref></xref> may be a more convenient and cheaper method\nthan TEM experiments to characterize paraptosis and the related PCD.<xref rid="ao0c00337_0003" ref-type="fig">11</xref> may be a more convenient and cheaper method\nthan TEM experiments to characterize paraptosis and the related PCD.'], 'ao0c00337_0003': ['Jurkat cells (3.0 × 106 cells)\nwere incubated with 4 (25 μM) in an RPMI 1640 medium\nwith 10% FBS at 37 °C under 5% CO2 for 1 h. After\nthe incubation, the cells were collected by centrifugation and washed\nwith ice-cold PBS containing 0.1% NaN3 and 0.5% FBS twice\nand then prefixed with glutaraldehyde (2.5%) at 4 °C for 40 min\nand washed with ice-cold PBS twice. Postfixing was conducted with\nosmium tetroxide (1%) at 4 °C for 30 min. After washing, the\ncells were included\nin an agarose gel and dehydrated with 50–100% anhydrous EtOH.\nEmbedding of the cells in Poly 812 resin (Nisshin EM Co. Ltd.) was\nconducted at 60 °C for 3 days. The resin was sliced with a glass\nknife (150 nm thickness) on an ultramicrotome (EM UC6, Leica), and\nthe sections were stained with methylene blue for microscopic observation\non a microscope (BX51, Olympus) (<xref rid="ao0c00337_0003" ref-type="fig">Figure <xref rid="fig11" ref-type="fig">11</xref></xref>). For transmission electron microscopy\n(TEM) observation, the sliced samples (ca. 100 nm thickness) were\nstained with an EM stainer (Nisshin EM Co. Ltd.) and observed on a\nTEM instrument (H-7650, HITACHI) with electron irradiation at 100\nkV (<xref rid="ao0c00337_0003" ref-type="fig">11</xref>). For transmission electron microscopy\n(TEM) observation, the sliced samples (ca. 100 nm thickness) were\nstained with an EM stainer (Nisshin EM Co. Ltd.) and observed on a\nTEM instrument (H-7650, HITACHI) with electron irradiation at 100\nkV (<xref rid="ao0c00337_0004" ref-type="fig">Figure <xref rid="fig12" ref-type="fig">12</xref></xref>).<xref rid="ao0c00337_0004" ref-type="fig">12</xref>).']}
|
2+ Amphiphilic Cationic Triscyclometalated
Iridium(III) Complex–Peptide Hybrids Induce Paraptosis-like
Cell Death of Cancer Cells via an Intracellular Ca-Dependent
Pathway
| null |
ACS Omega
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1584428400
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We report on the design and synthesis of a green-emitting iridium complex-peptide hybrid (IPH) , which has an electron-donating hydroxyacetic acid (glycolic acid) moiety between the Ir core and the peptide part. It was found that is selectively cytotoxic against cancer cells, and the dead cells showed a green emission. Mechanistic studies of cell death indicate that induces a paraptosis-like cell death through the increase in mitochondrial Ca concentrations via direct Ca transfer from ER to mitochondria, the loss of mitochondrial membrane potential (ΔΨ), and the vacuolization of cytoplasm and intracellular organelle. Although typical paraptosis and/or autophagy markers were upregulated by through the mitogen-activated protein kinase (MAPK) signaling pathway, as confirmed by Western blot analysis, autophagy is not the main pathway in -induced cell death. The degradation of actin, which consists of a cytoskeleton, is also induced by high concentrations of Ca, as evidenced by costaining experiments using a specific probe. These results will be presented and discussed.
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other
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PMC7114882
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81
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[
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"{'Citation': 'Tardito S.; Bassanetti I.; Bignardi C.; Elviri L.; Tegoni M.; Mucchino C.; Bussolati O.; Franchi-Gazzola R.; Marchiò L. Copper binding agents acting as copper ionophores lead to caspase inhibition and paraptotic cell death in human cancer cells. J. Am. Chem. Soc. 2011, 133, 6235–6242. 10.1021/ja109413c.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1021/ja109413c'}, {'@IdType': 'pubmed', '#text': '21452832'}]}}",
"{'Citation': 'Marzano C.; Gandin V.; Pellei M.; Colavito D.; Papini G.; Lobbia G. G.; Del Giudice E.; Porchia M.; Tisato F.; Santini C. In vitro antitumor activity of the water soluble copper (I) complexes bearing the tris (hydroxymethyl) phosphine ligand. J. Med. Chem. 2008, 51, 798–808. 10.1021/jm701146c.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1021/jm701146c'}, {'@IdType': 'pubmed', '#text': '18251492'}]}}",
"{'Citation': 'Li C.; Ip K. W.; Man W. L.; Song D.; He M. L.; Yiu S. M.; Lau T. C.; Zhu G. Cytotoxic (salen) ruthenium(III) anticancer complexes exhibit different modes of cell death directed by axial ligands. Chem. Sci. 2017, 8, 6865–6870. 10.1039/C7SC02205K.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1039/C7SC02205K'}, {'@IdType': 'pmc', '#text': 'PMC5632802'}, {'@IdType': 'pubmed', '#text': '29147511'}]}}",
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"{'Citation': 'Giorgi C.; Romagnoli A.; Pinton P.; Rizzuto R. Ca2+ signaling, mitochondria and cell death. Curr. Mol. Med. 2008, 8, 119–130. 10.2174/156652408783769571.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.2174/156652408783769571'}, {'@IdType': 'pubmed', '#text': '18336292'}]}}",
"{'Citation': 'Contreras L.; Drago I.; Zampese E.; Pozzan T. Mitochondria: the calcium connection. Biochim. Biophys. Acta 2010, 1797, 607–618. 10.1016/j.bbabio.2010.05.005.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.bbabio.2010.05.005'}, {'@IdType': 'pubmed', '#text': '20470749'}]}}",
"{'Citation': 'Rizzuto R.; Marchi S.; Bonora M.; Aguiari P.; Bononi A.; Stefani D. D.; Giorgi C.; Leo S.; Rimessi A.; Siviero R.; Zecchii E.; Pinton P. Ca2+ transfer from the ER to mitochondria: when, how and why. Biochim. Biophys. Acta 2009, 1787, 1342–1351. 10.1016/j.bbabio.2009.03.015.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.bbabio.2009.03.015'}, {'@IdType': 'pmc', '#text': 'PMC2730423'}, {'@IdType': 'pubmed', '#text': '19341702'}]}}",
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"{'Citation': 'Rizzuto R.; Pinton P.; Carrington W.; Fay F. S.; Fogarty K. E.; Lifshitz L. M.; Tuft R. A.; Pozzan T. T. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 1998, 280, 1763–1766. 10.1126/science.280.5370.1763.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1126/science.280.5370.1763'}, {'@IdType': 'pubmed', '#text': '9624056'}]}}",
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] |
ACS Omega. 2020 Mar 17; 5(12):6983-7001
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NO-CC CODE
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(a) Schematics of the FRAP experiments. (b) Confocal images of
the recovery of fluorescence of bilayers after photobleaching. Insets
in panel b showing the fluorescence recovery in the inner water droplet.
(c) Fluorescence intensity–distance profiles along the line
in panel b. Scale bars, 50 μm.
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ja-2018-03123h_0003
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125fa559c5a598e0a805b4ca7930556b20a4cfe5c37b1cf182621cae7ad26b8b
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ja-2018-03123h_0003.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
666,
402
] |
[{'image_id': 'ja-2018-03123h_0006', 'image_file_name': 'ja-2018-03123h_0006.jpg', 'image_path': '../data/media_files/PMC6016064/ja-2018-03123h_0006.jpg', 'caption': 'No caption found', 'hash': '42aa6e3f6cbf41129ea9691ef4b58edaef599de9984adf09d4ef267c2ff50109'}, {'image_id': 'ja-2018-03123h_0001', 'image_file_name': 'ja-2018-03123h_0001.jpg', 'image_path': '../data/media_files/PMC6016064/ja-2018-03123h_0001.jpg', 'caption': '(a) Schematics and (b)\nconfocal images of diverse configurations\nof protocells prepared from partial dewetting of W/O/W emulsion templates\nwith 0.00–0.20 wt % F-68 in the W2 phase. (c) Reconstructed\nconfocal image showing the 3D structure of protocells. (d) As-formed\nmodel protocells with different-sized oil organelles (in green). Scale\nbars, 100 μm.', 'hash': 'c69d5ab5c93d7a8c38b75e7284006ea3caf8138326ae9a15325bba03058c73f6'}, {'image_id': 'ja-2018-03123h_0002', 'image_file_name': 'ja-2018-03123h_0002.jpg', 'image_path': '../data/media_files/PMC6016064/ja-2018-03123h_0002.jpg', 'caption': '(a–c) Schematics, confocal and optical\nimages of the shrinking\nprocess of protocells in response to hypertonic shock. (d,e) Schematics\nand confocal images of the swelling process of protocells when in\nhypotonic solution. Scale bars, 100 μm.', 'hash': 'b8ad104ab9c5f2580a3542c01e753d791c0b7a90e932eb420506e50adea02ce6'}, {'image_id': 'ja-2018-03123h_0005', 'image_file_name': 'ja-2018-03123h_0005.jpg', 'image_path': '../data/media_files/PMC6016064/ja-2018-03123h_0005.jpg', 'caption': '(a,b)\nCoacervate formation in liposomes induced by decrease of\nvolume. (c) Optical images of liquid–liquid phase separation\nprocess in protocells. Scale bars, 50 μm.', 'hash': 'dee001e92ec5579f952c902288c22618a29d9ee3082f5dbd8890b160bc4b0611'}, {'image_id': 'ja-2018-03123h_0004', 'image_file_name': 'ja-2018-03123h_0004.jpg', 'image_path': '../data/media_files/PMC6016064/ja-2018-03123h_0004.jpg', 'caption': '(a,b) Illustration and images of macromolecularly crowded\nprotocells.\nInset in panel b1 is the sample before shrinking. (c) Fluorescence\nrecovery of eGFP in liposomes before (upper sequence) and after (lower\nsequence) shrinking after photobleaching. (d) Expression profiles\nof mRFP in normal and shrunk liposomes and confocal images of liposomes\nafter expression for 6 h. Scale bars: 50 μm in panels b, c;\n100 μm in panel d.', 'hash': 'acdf8fa345626f1b508eafc0b2083494e77c832e69d102623dd93a40dd4e9525'}, {'image_id': 'ja-2018-03123h_0003', 'image_file_name': 'ja-2018-03123h_0003.jpg', 'image_path': '../data/media_files/PMC6016064/ja-2018-03123h_0003.jpg', 'caption': '(a) Schematics of the FRAP experiments. (b) Confocal images of\nthe recovery of fluorescence of bilayers after photobleaching. Insets\nin panel b showing the fluorescence recovery in the inner water droplet.\n(c) Fluorescence intensity–distance profiles along the line\nin panel b. Scale bars, 50 μm.', 'hash': '125fa559c5a598e0a805b4ca7930556b20a4cfe5c37b1cf182621cae7ad26b8b'}]
|
{'ja-2018-03123h_0001': ['We used microfluidic emulsification\nto produce monodisperse water-in-oil-in-water\n(W/O/W) double emulsion droplets as templates (<xref rid="ja-2018-03123h_0001" ref-type="fig">Figures <xref rid="fig1" ref-type="fig">1</xref></xref>a, S1 in <xref rid="ja-2018-03123h_0001" ref-type="fig">1</xref>a, S1 in Supporting Information (SI)), which subsequently undergo partial dewetting (<xref rid="ja-2018-03123h_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>a). The dewetting process is\ndetermined by the spreading coefficient defined as <xref rid="ja-2018-03123h_0001" ref-type="fig">1</xref>a). The dewetting process is\ndetermined by the spreading coefficient defined as Si = γjk – (γij + γik), where γij is\nthe interfacial tension between fluids i and j.17 Only if Sw1 < 0, So < 0 and Sw2 < 0, will the emulsion templates form\na stable partial dewetting configuration (see SI Experimental Section for details of the typically used\ninner, middle and outer fluids).17a The\nsuccess of partial dewetting relies on careful control of the concentration\nof surfactant F-68 which tunes the interfacial energies. Without addition\nof F-68, the emulsion templates will keep the core–shell structure\ndue to a positive So (<xref rid="ja-2018-03123h_0001" ref-type="fig">Figures <xref rid="fig1" ref-type="fig">1</xref></xref>a,b, first panel).<xref rid="ja-2018-03123h_0001" ref-type="fig">1</xref>a,b, first panel).1b When F-68 is added into W2, γw1w2 and γow2 will decrease and So becomes negative, triggering the dewetting process. A lipid\nbilayer is formed via combining two lipid monolayers at the two water–oil\ninterfaces (<xref rid="ja-2018-03123h_0001" ref-type="fig">Figures <xref rid="fig1" ref-type="fig">1</xref></xref>a and <xref rid="ja-2018-03123h_0001" ref-type="fig">1</xref>a and S1). Higher concentrations of F-68\npromote the formation of more bilayer until an intact liposome is\ngenerated (C(F-68) > 0.2 wt\n%),1b as F-68 in W2 reduces γw1w2 and γow2, which make the adhesion energy\nΔF (ΔF = γow2 + γw1w2 – γow1)18 smaller, resulting in less contact\narea between oil droplet and\nliposome. Other combinations of liquids and surfactants reported in\nthe literature enable tuning of the structures of double emulsion\ndroplets17,20 and these also appear promising for making\nvesicles by the partial dewetting method.', 'Our approach yields excellent\ncontrol over the liposomal structures\nby adjusting concentrations of F-68 or the flow rates (<xref rid="ja-2018-03123h_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>b,c). As the concentrations\nof F-68 increase from 0%, 0.01%, 0.02%, 0.075% to 0.20%, the bilayer\narea gradually increases from 0%, 41%, 49%, 72% to 84% of the surface\narea of inner droplets (<xref rid="ja-2018-03123h_0001" ref-type="fig">1</xref>b,c). As the concentrations\nof F-68 increase from 0%, 0.01%, 0.02%, 0.075% to 0.20%, the bilayer\narea gradually increases from 0%, 41%, 49%, 72% to 84% of the surface\narea of inner droplets (D = 69 μm), respectively.\nMoreover, the sizes of attached oil droplets can be easily tuned (<xref rid="ja-2018-03123h_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>d), and complex structures\nwith multiple compartments (so-called multisomes<xref rid="ja-2018-03123h_0001" ref-type="fig">1</xref>d), and complex structures\nwith multiple compartments (so-called multisomes19) were also easily prepared in our method (Figure S2). Importantly, the as-formed liposomes are stable\n(Figures S3b,c), with no obvious loss in\nnumbers after storage for 4 days (Figure S4).'], 'ja-2018-03123h_0002': ['Next, we studied the swelling and shrinking of the liposomes\n(<xref rid="ja-2018-03123h_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>). As <xref rid="ja-2018-03123h_0002" ref-type="fig">2</xref>). As <xref rid="ja-2018-03123h_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>a–c shows,\nwhen the\nprotocells consisting of 0.05 wt % PEG were dispersed in a hypertonic\nsolution of 2 M sucrose, they rapidly lost water and shrunk to balance\nthe osmotic difference (<xref rid="ja-2018-03123h_0002" ref-type="fig">2</xref>a–c shows,\nwhen the\nprotocells consisting of 0.05 wt % PEG were dispersed in a hypertonic\nsolution of 2 M sucrose, they rapidly lost water and shrunk to balance\nthe osmotic difference (Movies S1, S2).\nWe did not observe any lipid spots, vesicles or tubule formation in\nthe bilayers during the shrinking process, indicating the extra lipids\nfrom the bilayers were collected into the attached oil droplets. Remarkably,\nthe membrane surface and volume are only 1/77 and 1/670, respectively,\nof their initial states (Figure S5, Movie S2). In contrast, liposomes without LDs collapsed and burst immediately\nunder the same conditions (Figure S6).\nInversely, LDs also allow the supply of lipids to the bilayers to\ninduce membrane growth (<xref rid="ja-2018-03123h_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>d,e), because a negative pressure outside the liposomes reverses\nthe water flux, leading to the growth of surface area and volume.\nThe shrinking process is reversible, as demonstrated in <xref rid="ja-2018-03123h_0002" ref-type="fig">2</xref>d,e), because a negative pressure outside the liposomes reverses\nthe water flux, leading to the growth of surface area and volume.\nThe shrinking process is reversible, as demonstrated in Figure S7. Liposomes with an inner phase of 1.7\nwt % PEG and 170 mM sucrose were shrunk from 72 to 47 μm in\n750 mM sucrose solution with 0.05 wt % F-68 added. Subsequently, an\naqueous solution of 0.05 wt % F-68 was carefully added and the liposomes\nswelled to 60 μm.'], 'ja-2018-03123h_0003': ['To verify the lipid exchange between the bilayers and the\nLDs,\nwe performed fluorescence recovery after photobleaching (FRAP) experiments\nvia labeling the bilayers and the inner water droplets with two different\nfluorophores (<xref rid="ja-2018-03123h_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>a, see <xref rid="ja-2018-03123h_0003" ref-type="fig">3</xref>a, see SI for details). As <xref rid="ja-2018-03123h_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>b,c shows, the fluorescence\nof the whole bilayers recovered gradually in about 2 min after photobleaching\n(<xref rid="ja-2018-03123h_0003" ref-type="fig">3</xref>b,c shows, the fluorescence\nof the whole bilayers recovered gradually in about 2 min after photobleaching\n(Movie S3), but the fluorescence in the\ninterior did not recover due to shortage of dye supply (Figure S9). This experiment directly demonstrates\nthe successful lipid exchange between the bilayers and the attached\nLDs.'], 'ja-2018-03123h_0004': ['Cells are densely packed with macromolecules (total macromolecule\nconcentrations in excess of 300 g·L–1 in E. coli),21 which influences\nbiochemical kinetics.22 However, no method\nenables the production of liposomes with levels of crowding found\nin cells, because high concentrations of macromolecules are too viscous\nto encapsulate. To solve this issue and to reconstitute a realistic\ncell-like internal environment, we encapsulated cell lysate (60 g·L–1), nucleoids into E. coli lipid liposomes of 88 μm in diameter (see SI for details). We then shrunk them to 54 μm in diameter\nto form protocells with a concentration of macromolecules at about\n260 g·L–1 (<xref rid="ja-2018-03123h_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>a,b). To illustrate the dense interior, FRAP\nexperiments were performed to probe the diffusion of enhanced green\nfluorescent protein (eGFP) encapsulated. As <xref rid="ja-2018-03123h_0004" ref-type="fig">4</xref>a,b). To illustrate the dense interior, FRAP\nexperiments were performed to probe the diffusion of enhanced green\nfluorescent protein (eGFP) encapsulated. As <xref rid="ja-2018-03123h_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>c shows, the fluorescence recovers within\n1 s in liposomes before shrinking, while it takes more than 40 s to\nrecover after shrinking, which demonstrates the crowded interior of\nthe protocells.<xref rid="ja-2018-03123h_0004" ref-type="fig">4</xref>c shows, the fluorescence recovers within\n1 s in liposomes before shrinking, while it takes more than 40 s to\nrecover after shrinking, which demonstrates the crowded interior of\nthe protocells.', 'We then performed IVTT in both crowded and noncrowded protocells\nto investigate the influence of crowded interiors on gene expression.\nWe encapsulated a mix of cell lysate, feeding buffers and plasmids\ncoding for monomeric red fluorescent protein (mRFP) (total concentration\nis about 40 g L–1) into l-α-phosphatidylcholine\n(eggPC) liposomes (see SI Experimental\nSection for details), then collected them into two containers (one\nwith hypertonic solution, the other without) to form crowded protocells\n(diameter: 48 μm) and noncrowded protocells (diameter: 91 μm)\n(<xref rid="ja-2018-03123h_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>d). In crowded\nprotocells, the concentration of the interior solution increases approximately\n6.8 times, yielding a concentration of IVTT mix of about 272 g L<xref rid="ja-2018-03123h_0004" ref-type="fig">4</xref>d). In crowded\nprotocells, the concentration of the interior solution increases approximately\n6.8 times, yielding a concentration of IVTT mix of about 272 g L–1. The expression of mRFP in shrunk liposomes is notably\nenhanced compared to expression in normal liposomes which is very\nslow and barely detectable after 6 h (Movie S4). We postulate that the rate enhancement in gene expression is not\nonly due to increased concentration of key components such as DNA\nor ribosomes in the IVTT mixture but also because of the molecularly\ncrowded interior.5,6c The slightly increased ratio\nof the surface to volume of the bilayer membrane is not expected to\nalter gene expression significantly.23'], 'ja-2018-03123h_0005': ['To extend the technological scope for constructing protocells with\nmore synthetic complexity, we induced complex coacervation of the\ncell lysates to create subcompartments in protocells. Complex coacervation\nis a form of liquid–liquid phase separation of oppositely charged\npolyelectrolytes, and provides powerful means of membrane-free compartmentalization.\nCoacervation has been explored extensively in protocell models for\nthe construction of artificial cells or organelles.6,7 The\nphase separation of cell lysates to form crowded coacervates has been\naccomplished in water-in-oil droplets recently,6c but it has not been demonstrated in biological vesicles\nbecause of the use of concentrated salt solution (as high as 6 M)\nand fragile nature of vesicles. To address this problem, we shrunk\nthe liposomes containing cell lysate, feeding buffers and 8 g·L–1 PEG via multistep osmotic shocks (<xref rid="ja-2018-03123h_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>a, see <xref rid="ja-2018-03123h_0005" ref-type="fig">5</xref>a, see SI Experimental Section for details). Meanwhile, plasmids\ncoding for eGFP were also encapsulated into the protocells to perform\nIVTT. As <xref rid="ja-2018-03123h_0005" ref-type="fig">Figures <xref rid="fig5" ref-type="fig">5</xref></xref>b,c\nand <xref rid="ja-2018-03123h_0005" ref-type="fig">5</xref>b,c\nand S10 show, shrinking the volume of liposomes\ninduced coacervate droplet formation with cell lysate and PEG in the\nliposomes (Movie S5), due to the phase\ntransition of salt and PEG as well as partitioning of cell lysate\ninto the PEG phase. Notably, the expressed eGFP also prefers to partition\ninto the innermost coacervate droplet (bright core in <xref rid="ja-2018-03123h_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>b).<xref rid="ja-2018-03123h_0005" ref-type="fig">5</xref>b).6c']}
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Macromolecularly
Crowded Protocells from Reversibly
Shrinking Monodisperse Liposomes
| null |
J Am Chem Soc
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1529478000
|
[
"DNA, Fungal",
"Humans",
"Pneumocystis carinii",
"Pneumonia, Pneumocystis",
"Respiratory System"
] |
other
|
PMC6016064
| null |
2
|
[
"{'Citation': 'Schildgen V, Mai S, Khalfaoui S, Lüsebrink J, Pieper M, Tillmann RL, Brockmann M, Schildgen O. 2014. Pneumocystis jirovecii can be productively cultured in differentiated CuFi-8 airway cells. mBio 5:e01186-14. doi:10.1128/mBio.01186-14.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1128/mBio.01186-14'}, {'@IdType': 'pmc', '#text': 'PMC4030487'}, {'@IdType': 'pubmed', '#text': '24825015'}]}}",
"{'Citation': 'Larsen HH, Masur H, Kovacs JA, Gill VJ, Silcott VA, Kogulan P, Maenza J, Smith M, Lucey DR, Fischer SH. 2002. Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 40:490–494. doi:10.1128/JCM.40.2.490-494.2002.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1128/JCM.40.2.490-494.2002'}, {'@IdType': 'pmc', '#text': 'PMC153364'}, {'@IdType': 'pubmed', '#text': '11825961'}]}}"
] |
J Am Chem Soc. 2018 Jun 20; 140(24):7399-7402
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MAP was immunolocalized inside macrophages. a–d GFP-MAP (A1 157) (green) (MOI 5) was identified by confocal immunofluorescence in murine macrophages at 3 h in comparison to control MOI 0. Macrophages were counterstained with phalloidin, which binds actin cytoskeleton (red) and DAPI as a nucleus maker (blue). Representative images for 3 independent experiments. Scale bar = 50 μm. e–i Internalization of MAP within macrophages. The enclosed dashed rectangle represents a higher magnification of a x-z-plane image (a stacks of 10 images 1 mm apart). Scale bar = 50 μm
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441_2019_3098_Fig1_HTML
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2
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4e7e5d060bbde51af87e61b4a506ad8be96eb7f86b6bb173c1a60e2a452040c4
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441_2019_3098_Fig1_HTML.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[{'image_id': '441_2019_3098_Fig4_HTML', 'image_file_name': '441_2019_3098_Fig4_HTML.jpg', 'image_path': '../data/media_files/PMC7224033/441_2019_3098_Fig4_HTML.jpg', 'caption': 'Cathelicidin LL-37 modulated the production of TNF-α and IL-8 on macrophages infected by MAP. Transcriptional gene expression and secretion of TNF-α (a–d) and IL-8 (e–h) were determined in MAP-infected macrophages pretreated with LL-37 (2\xa0μM; 1\xa0h). Expression of mRNA was quantified with qRT-qPCR. Secreted proteins were determined with ELISA. Means ± SEM are shown (n\u2009=\u20093 independent experiments done in triplicate). *p\u2009<\u20090.05 compared to the untreated control at same MOI. #p\u2009<\u20090.05 compared with MOI 0', 'hash': 'b9c6f0fc64b23734a731ec010064267b558165447859d1ac02bd51edf2385c8e'}, {'image_id': '441_2019_3098_Fig3_HTML', 'image_file_name': '441_2019_3098_Fig3_HTML.jpg', 'image_path': '../data/media_files/PMC7224033/441_2019_3098_Fig3_HTML.jpg', 'caption': 'Cathelicidin LL-37 reduced quantitative burden of MAP. (a, b) MAP DNA was quantified by qPCR in macrophages stimulated with synthetic LL-37 (10\xa0μM; 1\xa0h) and challenged with MAP (MOIs 1 and 5) for up to 24\xa0h. The estimated values represent the averages for two separate qPCR experiments targeting MAP F57 DNA. (c–c”) Live MAP (105\xa0CFU/mL) was directly incubated with synthetic LL37 (up to 50\xa0μM; 2\xa0h) or inert control solution. Fluorescent microscopic images were taken from representative cathelicidin treated and untreated bacteria subjected to live (green) or dead/damaged (red) by a bacterial viability assay (Live/Dead Backlight © stain). Scale bar\u2009=\u2009100\xa0μm. Relative ratio of live/dead bacteria were quantified from 5 different images using ImageJ', 'hash': '174d83d44cf4d0444a7403480890526663073d70e6dc4b0e61f63392ff062c1e'}, {'image_id': '441_2019_3098_Fig5_HTML', 'image_file_name': '441_2019_3098_Fig5_HTML.jpg', 'image_path': '../data/media_files/PMC7224033/441_2019_3098_Fig5_HTML.jpg', 'caption': 'Expression of IFN-γ, TLR-2, and TLR-4 on macrophages infected by MAP pretreated with cathelicidin LL-37. Transcriptional gene/protein expression of IFN-γ (a, b), TLR-2 (c–e), and TLR-4 (f, g) was determined in MAP-infected macrophages (for 3 and 24\xa0h postinfection) pretreated with LL-37 (2\xa0μM; 1\xa0h). Expression of mRNA was quantified with RT-qPCR. Proteins were determined with western blotting for TLR-2. Means + SEM are shown (n\u2009=\u20093 independent experiments done in triplicate). *p\u2009<\u20090.05 compared to the untreated control at same MOI. #p\u2009<\u20090.05 compared with MOI 0', 'hash': 'dd36d593cfca54f5e29dada5bc9ea000f9325a2cb7d6c4e21c6a0e76d16cb8da'}, {'image_id': '441_2019_3098_Fig2_HTML', 'image_file_name': '441_2019_3098_Fig2_HTML.jpg', 'image_path': '../data/media_files/PMC7224033/441_2019_3098_Fig2_HTML.jpg', 'caption': '(a–d”’) Pretreatment with LL-37 cathelicidins reduced GFP-MAP burden in macrophages. GFP-MAP (K10) (green) was cultured with macrophages pretreated with LL-37 (up to 10\xa0μM; 1\xa0h) for 3\xa0h. Macrophages were counterstained with phalloidin, which binds actin (red) and DAPI as a nucleus marker (blue). Representative images for 3 independent experiments. Scale bar\u2009=\u2009100\xa0μm. (e) Mean fluorescence intensity (MFI) of MAP internalized in macrophages pretreated with cathelicidins. MFI was calculated as the ration between GFP-MAP (green) fluorescence MAP intensity/number of macrophages (5 fields/treatment; n\u2009=\u20093 independent experiments done in triplicate). *p\u2009<\u20090.05', 'hash': '3d85e92387f4749d938fdad7915b556e8bc0e2472bd74868f5d7f79ff59615de'}, {'image_id': '441_2019_3098_Fig1_HTML', 'image_file_name': '441_2019_3098_Fig1_HTML.jpg', 'image_path': '../data/media_files/PMC7224033/441_2019_3098_Fig1_HTML.jpg', 'caption': 'MAP was immunolocalized inside macrophages. a–d GFP-MAP (A1 157) (green) (MOI 5) was identified by confocal immunofluorescence in murine macrophages at 3\xa0h in comparison to control MOI 0. Macrophages were counterstained with phalloidin, which binds actin cytoskeleton (red) and DAPI as a nucleus maker (blue). Representative images for 3 independent experiments. Scale bar\u2009=\u200950\xa0μm. e–i Internalization of MAP within macrophages. The enclosed dashed rectangle represents a higher magnification of a x-z-plane image (a stacks of 10 images\xa01\xa0mm apart). Scale bar\u2009=\u200950\xa0μm', 'hash': '4e7e5d060bbde51af87e61b4a506ad8be96eb7f86b6bb173c1a60e2a452040c4'}]
|
{'441_2019_3098_Fig1_HTML': ['Rapid internalization of MAP was shown in macrophages challenged by MAP that expressed green fluorescent protein (GFP). The A1 157 GFP-MAP was identified in the cytoplasm of macrophages, contained within actin boundaries, as early as 3\xa0h (Fig.\xa0<xref rid="441_2019_3098_Fig1_HTML" ref-type="fig">1a–h</xref>) and persisted until 24\xa0h postinfection. An intracellular MAP distribution throughout the cell cytoplasm as intact bacillus once engulfed by macrophages was confirmed by X-Z-stack images (Fig. ) and persisted until 24\xa0h postinfection. An intracellular MAP distribution throughout the cell cytoplasm as intact bacillus once engulfed by macrophages was confirmed by X-Z-stack images (Fig. <xref rid="441_2019_3098_Fig1_HTML" ref-type="fig">1</xref> h and i). A quickly (3\xa0h) internalization of MAP into J774.A1 cells was further verified in a genetical similar MAP strain (K-10; pWes4) (Suppl Fig. h and i). A quickly (3\xa0h) internalization of MAP into J774.A1 cells was further verified in a genetical similar MAP strain (K-10; pWes4) (Suppl Fig. 1). Taken togheter, we confirmed this murine phagocytic cell line is susceptible to MAP infection and that A1 157 MAP transformation with GFP plasmid did not affects its invasive virulence.Fig. 1MAP was immunolocalized inside macrophages. a–d GFP-MAP (A1 157) (green) (MOI 5) was identified by confocal immunofluorescence in murine macrophages at 3\xa0h in comparison to control MOI 0. Macrophages were counterstained with phalloidin, which binds actin cytoskeleton (red) and DAPI as a nucleus maker (blue). Representative images for 3 independent experiments. Scale bar\u2009=\u200950\xa0μm. e–i Internalization of MAP within macrophages. The enclosed dashed rectangle represents a higher magnification of a x-z-plane image (a stacks of 10 images\xa01\xa0mm apart). Scale bar\u2009=\u200950\xa0μm'], '441_2019_3098_Fig2_HTML': ['It has been demonstrated that exogenous cathelicidin (LL-37) reduces intracellular survival of M. smegmatis, M. tuberculosis, and M. bovis BCG in macrophages (Sonawane et al. 2011); however, antibacterial effect of cathelicidin on MAP has not been reported. We found macrophage pretreated with increased doses of synthetic recombinant LL-37 showed decreased intracellular presence of MAP (MOI 1) at 3 (Fig.\xa0<xref rid="441_2019_3098_Fig2_HTML" ref-type="fig">2</xref> a–a”’, b–b”’, c–c”’, and d–d”’) and 24\xa0h postinfection. This reduced MAP presence into macrophages stimulated by cathelicidin (i.e., reduced MFI green fluorescence MAP intensity per macrophage) was significant with higher doses of LL-37 (5 and 10\xa0μM) at 3\xa0h postinfecction ( a–a”’, b–b”’, c–c”’, and d–d”’) and 24\xa0h postinfection. This reduced MAP presence into macrophages stimulated by cathelicidin (i.e., reduced MFI green fluorescence MAP intensity per macrophage) was significant with higher doses of LL-37 (5 and 10\xa0μM) at 3\xa0h postinfecction (p\u2009<\u20090.05) (Fig. <xref rid="441_2019_3098_Fig2_HTML" ref-type="fig">2e</xref>). Reduced intracellular MAP in macrophages stimulated by cathelicidin was confirmed by the quantification of MAP (F57) DNA. MAP burden was lesser in macrophages pretreated with LL-37 with significant differences for high MAP challenge (MOI 5) at 3 and 24\xa0h postinfection (). Reduced intracellular MAP in macrophages stimulated by cathelicidin was confirmed by the quantification of MAP (F57) DNA. MAP burden was lesser in macrophages pretreated with LL-37 with significant differences for high MAP challenge (MOI 5) at 3 and 24\xa0h postinfection (p\u2009<\u20090.05) (Fig.\xa0<xref rid="441_2019_3098_Fig3_HTML" ref-type="fig">3a</xref>).).Fig. 2(a–d”’) Pretreatment with LL-37 cathelicidins reduced GFP-MAP burden in macrophages. GFP-MAP (K10) (green) was cultured with macrophages pretreated with LL-37 (up to 10\xa0μM; 1\xa0h) for 3\xa0h. Macrophages were counterstained with phalloidin, which binds actin (red) and DAPI as a nucleus marker (blue). Representative images for 3 independent experiments. Scale bar\u2009=\u2009100\xa0μm. (e) Mean fluorescence intensity (MFI) of MAP internalized in macrophages pretreated with cathelicidins. MFI was calculated as the ration between GFP-MAP (green) fluorescence MAP intensity/number of macrophages (5 fields/treatment; n\u2009=\u20093 independent experiments done in triplicate). *p\u2009<\u20090.05Fig. 3Cathelicidin LL-37 reduced quantitative burden of MAP. (a, b) MAP DNA was quantified by qPCR in macrophages stimulated with synthetic LL-37 (10\xa0μM; 1\xa0h) and challenged with MAP (MOIs 1 and 5) for up to 24\xa0h. The estimated values represent the averages for two separate qPCR experiments targeting MAP F57 DNA. (c–c”) Live MAP (105\xa0CFU/mL) was directly incubated with synthetic LL37 (up to 50\xa0μM; 2\xa0h) or inert control solution. Fluorescent microscopic images were taken from representative cathelicidin treated and untreated bacteria subjected to live (green) or dead/damaged (red) by a bacterial viability assay (Live/Dead Backlight © stain). Scale bar\u2009=\u2009100\xa0μm. Relative ratio of live/dead bacteria were quantified from 5 different images using ImageJ'], '441_2019_3098_Fig3_HTML': ['To understand the reduced MAP burden in macrophages stimulated by cathelicidins, a direct microbicidal effect of the peptide was investigated by live/dead bacterial viability staining. Synthetic LL-37 at a dose of 10\xa0μM did not kill MAP whereas high dosage of cathelicidin (50\xa0μM) revealed some bactericidal effect on MAP (≈\u200950%) (Fig. <xref rid="441_2019_3098_Fig3_HTML" ref-type="fig">3b</xref>). Same cathelicidin doses (10 and 50\xa0μM) killed (>\u200990%) ). Same cathelicidin doses (10 and 50\xa0μM) killed (>\u200990%) E. coli, a known cathelicidin susceptible bacteria (data not shown). The doses used of synthetic LL-37 (up to 50\xa0μM) did not cause macrophage cell damage or death as determined by the microscopical observation of healthy macrophages (Suppl Fig.\xa02a–b”’) and a insignificant LDH release compared to control (Suppl Fig.\xa02c) after infection with MAP (A1 157, MOIs 1 and 5) for up to 24\xa0h. These studies confirmed cathelicidin reduces the MAP load in macrophages, albeit without killing the host cell or directly killing the bacterium.'], '441_2019_3098_Fig4_HTML': ['Macrophages exposed to inflammatory stimuli secrete a signature array of pro-inflammatory cytokines, including IFN-γ, IL-8, TNF-α, and IL-1β, which increase vascular permeability and recruit inflammatory cells to the site of infection (Janeway and Medzhitov 2002; Boucher et al. 2018). MAP infection induces in vitro strong production of IFN-γ and TNF-α in human (THP-1) macrophages (Wang et al. 2014). We showed that whereas MAP induced transcriptional mRNA synthesis of TNF-α in murine macrophages (peak with MOI 5; 3\xa0h) (p\u2009<\u20090.05, Fig.\xa0<xref rid="441_2019_3098_Fig4_HTML" ref-type="fig">4a, b</xref>), pretreatment with synthetic LL-37 reduced TNF-α synthesis in response to live MAP (MOI 5; 3 and 24\xa0h) (), pretreatment with synthetic LL-37 reduced TNF-α synthesis in response to live MAP (MOI 5; 3 and 24\xa0h) (p\u2009<\u20090.05, Fig. <xref rid="441_2019_3098_Fig4_HTML" ref-type="fig">4a, b</xref>). Moreover, concentrations of secreted TNF-α were lower in macrophages exposed to MAP and pretreated with LL-37 (385 and 957\xa0pg/mL at 3 and 24\xa0h, respectively) than in challenged macrophages without cathelicidins (464 and 1193\xa0pg/mL; Fig. ). Moreover, concentrations of secreted TNF-α were lower in macrophages exposed to MAP and pretreated with LL-37 (385 and 957\xa0pg/mL at 3 and 24\xa0h, respectively) than in challenged macrophages without cathelicidins (464 and 1193\xa0pg/mL; Fig. <xref rid="441_2019_3098_Fig4_HTML" ref-type="fig">4c, d</xref>).).Fig. 4Cathelicidin LL-37 modulated the production of TNF-α and IL-8 on macrophages infected by MAP. Transcriptional gene expression and secretion of TNF-α (a–d) and IL-8 (e–h) were determined in MAP-infected macrophages pretreated with LL-37 (2\xa0μM; 1\xa0h). Expression of mRNA was quantified with qRT-qPCR. Secreted proteins were determined with ELISA. Means ± SEM are shown (n\u2009=\u20093 independent experiments done in triplicate). *p\u2009<\u20090.05 compared to the untreated control at same MOI. #p\u2009<\u20090.05 compared with MOI 0', 'Directional movement of leukocytes into the site of infection is mostly regulated by a C-X-C motif containing chemokine ligand 8 (CXCL8; IL-8) (Janeway and Medzhitov 2002). Thus, we determined whether exogenous cathelicidins modulated neutrophil factor IL-8 synthesis in macrophages as a defensive mechanism. MAP induced a delayed IL-8 transcriptional gene response (MOI 5; 24\xa0h); this was further increased by pretreatment with synthetic LL-37 (MOI 5, mostly at 24\xa0h) as compared to untreated MAP-infected macrophages (p\u2009<\u20090.05, Fig. <xref rid="441_2019_3098_Fig4_HTML" ref-type="fig">4e, f</xref>). LL-37 induced slight decrease in IL-8 transcriptional gene in macrophages challenged with low MAP doses (MOI 1) (). LL-37 induced slight decrease in IL-8 transcriptional gene in macrophages challenged with low MAP doses (MOI 1) (p\u2009>\u20090.05; Fig. <xref rid="441_2019_3098_Fig4_HTML" ref-type="fig">4e, f</xref>). The levels of secreted IL-8 were not significant different among treatments (Fig. ). The levels of secreted IL-8 were not significant different among treatments (Fig. <xref rid="441_2019_3098_Fig4_HTML" ref-type="fig">4g, h</xref>).).'], '441_2019_3098_Fig5_HTML': ['Cellular responses induced by IFN-γ (e.g., autophagy, lysosomal degradation) are indispensable for resistance to intracelullar pathogens, including MAP (Burger et al. 2018). In the present study, pretreatment with LL-37 in MAP-infected macrophages reduced IFN-γ transcriptional gene expression to baseline levels (MOI 1; 3\xa0h; p\u2009<\u20090.05, Fig.\xa0<xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5a</xref>). Only a slight transcriptional IFN-γ gene downregulation was observed in macrophages pre treated with LL-37 (2\xa0μM) and challenged with high MAP doses (MOI 5 at 3\xa0h and 24\xa0h) (). Only a slight transcriptional IFN-γ gene downregulation was observed in macrophages pre treated with LL-37 (2\xa0μM) and challenged with high MAP doses (MOI 5 at 3\xa0h and 24\xa0h) (p\u2009>\u20090.05; Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5a, b</xref>). The levels of secreted IFN-γ were undectable (data not shown).). The levels of secreted IFN-γ were undectable (data not shown).Fig. 5Expression of IFN-γ, TLR-2, and TLR-4 on macrophages infected by MAP pretreated with cathelicidin LL-37. Transcriptional gene/protein expression of IFN-γ (a, b), TLR-2 (c–e), and TLR-4 (f, g) was determined in MAP-infected macrophages (for 3 and 24\xa0h postinfection) pretreated with LL-37 (2\xa0μM; 1\xa0h). Expression of mRNA was quantified with RT-qPCR. Proteins were determined with western blotting for TLR-2. Means + SEM are shown (n\u2009=\u20093 independent experiments done in triplicate). *p\u2009<\u20090.05 compared to the untreated control at same MOI. #p\u2009<\u20090.05 compared with MOI 0', 'Signaling through TLR-2 and TLR-4 regulates phagosome trafficking and antimicrobial and inflammatory responses in mononuclear phagocytes infected by MAP (Lee et al. 2014; Weiss et al. 2008). Herein, MAP quickly (MOI 5; 3\xa0h) induced higher transcriptional gene expression of TLR-2 in macrophages (p\u2009<\u20090.05, Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5c, d</xref>). This TLR-2 gene response was abolished by pretreatment with synthetic LL-37 (). This TLR-2 gene response was abolished by pretreatment with synthetic LL-37 (p\u2009<\u20090.05; Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5c</xref>). No TLR-2 response or cathelicidin effect was noticed at later points after MAP challenge (MOI 1, 24\xa0h) (Fig. ). No TLR-2 response or cathelicidin effect was noticed at later points after MAP challenge (MOI 1, 24\xa0h) (Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5d</xref>). Effects on TLR-2 in macrophages by MAP or LL-37 did not correspond to significant variations in the total level TLR-2 protein (). Effects on TLR-2 in macrophages by MAP or LL-37 did not correspond to significant variations in the total level TLR-2 protein (p\u2009>\u20090.05 Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5e</xref>). For TLR4, mRNA transcription was reduced in macrophages during early MAP infection (MOI 5, 3\xa0h) (). For TLR4, mRNA transcription was reduced in macrophages during early MAP infection (MOI 5, 3\xa0h) (p\u2009<\u20090.05, Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5f, g</xref>) and only a slight TLR4 gene upregulation was observed in LL-37 pretreate cells MAP (MOI 1 and 5, at 3 and 24\xa0h) () and only a slight TLR4 gene upregulation was observed in LL-37 pretreate cells MAP (MOI 1 and 5, at 3 and 24\xa0h) (p\u2009>\u20090.05; Fig. <xref rid="441_2019_3098_Fig5_HTML" ref-type="fig">5f, g</xref>).).']}
|
Mycobacterium avium paratuberculosis Synthetic cathelicidin LL-37 reduces subsp. internalization and pro-inflammatory cytokines in macrophages
|
[
"{'italic': ['Mycobacterium avium', 'paratuberculosis'], '#text': 'subsp.'}",
"Macrophages",
"Cathelicidin",
"LL-37",
"IL-8"
] |
Cell Tissue Res
|
1599030000
|
Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1β cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.
|
[
"Animals",
"Anti-Bacterial Agents",
"Antimicrobial Cationic Peptides",
"Cattle",
"Cell Line",
"Cytokines",
"Gene Expression",
"Humans",
"Macrophages",
"Mice",
"Mycobacterium avium subsp. paratuberculosis",
"Paratuberculosis",
"Cathelicidins"
] |
other
|
PMC7224033
| null |
65
|
[
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] |
Cell Tissue Res. 2020 Sep 2; 379(1):207-217
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NO-CC CODE
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Chemokines produced by NLCs impact B cell motility. (A) Graph comparing the mean speed of B cells loaded on NLC monolayers in 14-day culture-conditioned medium (cdt) or fresh medium. B cells were left untreated or incubated with anti-CCR7 (10 μg/mL), anti-CXCR4 (30 μg/mL), and anti-CXCR5 antibodies (20 μg/mL) before loading. CD68+ NLCs were incubated or not with anti-CCL21 antibody (4 μg/mL) before addition of untreated B cells. Motility was analyzed using Trackmate in ImageJ software. Only displacement lengths >10 μm were included in the analysis (n = 12). (B) Representative images of interactions between CLL B cells (green) and CD68+ NLCs (red) after 4 hours of coculture in conditions identical to panel A and vigorous washing (objective ×20; scale bar represents 10 μm). The areas in the white dotted boxes in the upper panel are ×3.5 zoomed in the lower panels to show B/NLC interactions. Green arrows show noninteracting B cells. (C) Graph comparing the ratios of colocalized B cells/NLCs detected in the different conditions. Postacquisition data analysis of images in panel B was performed using ImageJ software. To quantify B cells in contact with NLCs, a threshold was applied to obtain a mask of NLCs, and the number of B cells present in this mask was evaluated using the “analyze particles” tool and represented in the graph as number of B cells per NLC. Statistical analysis was carried out by Student t-test (***P < .001; ****P < .0001; n = 12).
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advancesADV2021006169f5
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16723cd0cffbdc67cfe9aaaf50ee433a6d79f414e7873f608ba0a33581aafc09
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
1485
] |
[{'image_id': 'advancesADV2021006169f1', 'image_file_name': 'advancesADV2021006169f1.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169f1.jpg', 'caption': 'Disorganization of FRC/FDC networks and increase of CD68+ macrophages in CLL lymph nodes. (A) Representative images from nontumoral (panels a-d) and CLL (panels e-h) lymph node tissue sections. Immunohistochemical staining was used on samples (anti-CCL21 and anti-αSMA or anti-CXCL13 and anti-CD21 antibodies), representative of FRC and FDC (original magnification ×200). (B) Representative images from nontumoral (left panels) and CLL (right panels) lymph node tissue sections stained with anti-CD68 antibody (original magnification ×200 and ×400, respectively). (C) Quantification of CD68+ cells per 550 μm2 section in nontumoral (n = 10) and CLL lymph nodes (n = 10); statistical analysis was carried out by Student t-test (*P < .005). Images were acquired using a DFC 300 FX Leica microscope with a ×10 objective.', 'hash': 'af7ae373e733f686b75716748719e0eef7010a96fa60a57c1d2db34429e48d0b'}, {'image_id': 'advancesADV2021006169f6', 'image_file_name': 'advancesADV2021006169f6.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169f6.jpg', 'caption': 'Ibrutinib impacts B cells/NLC interaction. (A) Graph shows the CCL21mRNA MFI values detected in NLC cells exposed or not to ibrutinib 24 hours before analysis (P = ns; n = 16) or in NLCs derived from patients treated with ibrutinib (n = 4). (B) Image galleries of single macrophages or aggregates (as defined in Figure 3A) exposed or not to ibrutinib for 24 hours (upper panels) or derived from patients treated with ibrutinib (lower panels) showing CD19 (purple), CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF) in permeabilized conditions and in presence of BFA (a) or nonpermeabilized conditions and no BFA treatment (b). (C) Graph comparing the ratios of colocalized B cells/NLCs after 4 hours of coculture exposed (+) or not (−) to ibrutinib for 24 hours (n = 6) or in cocultures derived from patients treated long-term with ibrutinib (n = 3). Statistical analysis was carried out by Student t-test (***P < .001; ****P < .0001) comparing untreated and treated samples. (D) Representative images of interactions between CLL B cells (green) and CD68+ NLCs (red) after 4 hours of coculture priorly treated or not with ibrutinib (24 hours) or in cocultures derived from patients treated with ibrutinib (objective ×10; scale bar represents 10 μm). The areas in the white dotted boxes in the upper panel are ×2.5 zoomed in the lower panels to show B cell/NLC interactions.', 'hash': 'b5bb56bfe4b567e4bcd597722b8473c86e148eea6c44bde7a7a44ae53fbc247d'}, {'image_id': 'advancesADV2021006169absf1', 'image_file_name': 'advancesADV2021006169absf1.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169absf1.jpg', 'caption': 'No caption found', 'hash': 'd1fd4be438767596ead5fe5d6334813bfde2866497958e1cad2f8d7f9bec1df3'}, {'image_id': 'advancesADV2021006169f3', 'image_file_name': 'advancesADV2021006169f3.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169f3.jpg', 'caption': 'CD68+ NLCs produce CCL21. (A) Assay outline and gating strategy applied to identify single cells and aggregates. Cells in camera focus were selected from all events on the basis of gradient RMS of the bright field image. Single B cells, macrophages, and cellular aggregates were identified by plotting “area” vs “aspect ratio” in which events with higher aspect ratio are assigned to single cells while those with lower aspect ratio are aggregates. (B) Image gallery of single macrophages showing different subpopulations based on CD14 (purple), CD206 (green), CD163 (yellow), CD19 (red), and CD68 (cyan) fluorescent signals as well as Brightfield (BF). (C) Detection of CCL21 mRNA and protein in CD68+ CD163+ or CD68+ CD163− subpopulations. Galleries show CCL21 protein (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals as well as Brightfield (BF). (D) Image gallery of aggregates of macrophages and B cells showing CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals as well as Brightfield (BF). Arrows show B cells present in the aggregates. Merged images show that B cells are negative for CCL21 and CCL21 mRNA signals. All the experiments were performed in permeabilized cells after brefeldin (BFA) treatment.', 'hash': 'f5671cc0bf644d34ed9c340d2831192fc5440a6881de56ab5a1a2fede87d276b'}, {'image_id': 'advancesADV2021006169f4', 'image_file_name': 'advancesADV2021006169f4.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169f4.jpg', 'caption': 'CCL21 is retained on cell membrane and not detected in the supernatant. (A) Image galleries of single macrophages (as defined in panel 3A) showing CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF) in permeabilized (upper and middle) and nonpermeabilized (lower) conditions in presence (upper) or absence (middle and lower) of BFA treatment. (B) Confocal images of adherent CD68+ cells stained with anti-CCL21 Ab in permeabilized (left) or nonpermeabilized (right) conditions (objective ×63; scale bar represents 12 μm). (C) CCL21, CXCL13, and CXCL12 protein levels secreted by adherent NLCs after 14 days of culture detected by Luminex technology. Dots represent individual protein levels from 14 patients with CLL. (D) Detection of CXCL13 mRNA in single macrophages. Image galleries of single macrophages showing RPL13 mRNA (green), CD163 (yellow), CXCL13 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF). RPL13 mRNA of ribosomal protein L13A was used as housekeeping control. (E) Image gallery of aggregates showing macrophages and B cells based on CD206 (green) and CD68 (cyan) or CCR7 (yellow) and CD19 (red) fluorescent signals, respectively, as well as Brightfield (BF) in permeabilized conditions. Arrows show B cells present in the aggregates. (F) Graph shows the CCR7 MFI (Mean Fluorescence Intensity) values detected on B cells in suspension or remaining in the adherent fraction after 14 days of culture.', 'hash': 'aec821a7507eaa6e021233c921c6b31699bfe09286e23988fe60a723119a8491'}, {'image_id': 'advancesADV2021006169f5', 'image_file_name': 'advancesADV2021006169f5.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169f5.jpg', 'caption': 'Chemokines produced by NLCs impact B cell motility. (A) Graph comparing the mean speed of B cells loaded on NLC monolayers in 14-day culture-conditioned medium (cdt) or fresh medium. B cells were left untreated or incubated with anti-CCR7 (10 μg/mL), anti-CXCR4 (30 μg/mL), and anti-CXCR5 antibodies (20 μg/mL) before loading. CD68+ NLCs were incubated or not with anti-CCL21 antibody (4 μg/mL) before addition of untreated B cells. Motility was analyzed using Trackmate in ImageJ software. Only displacement lengths >10 μm were included in the analysis (n = 12). (B) Representative images of interactions between CLL B cells (green) and CD68+ NLCs (red) after 4 hours of coculture in conditions identical to panel A and vigorous washing (objective ×20; scale bar represents 10 μm). The areas in the white dotted boxes in the upper panel are ×3.5 zoomed in the lower panels to show B/NLC interactions. Green arrows show noninteracting B cells. (C) Graph comparing the ratios of colocalized B cells/NLCs detected in the different conditions. Postacquisition data analysis of images in panel B was performed using ImageJ software. To quantify B cells in contact with NLCs, a threshold was applied to obtain a mask of NLCs, and the number of B cells present in this mask was evaluated using the “analyze particles” tool and represented in the graph as number of B cells per NLC. Statistical analysis was carried out by Student t-test (***P < .001; ****P < .0001; n = 12).', 'hash': '16723cd0cffbdc67cfe9aaaf50ee433a6d79f414e7873f608ba0a33581aafc09'}, {'image_id': 'advancesADV2021006169f2', 'image_file_name': 'advancesADV2021006169f2.jpg', 'image_path': '../data/media_files/PMC9631672/advancesADV2021006169f2.jpg', 'caption': 'CCL21 is produced by FRCs and CD68+ cells. (A) Representative images from nontumoral (upper panels) and CLL (lower panels) lymph node sections stained with anti-CD68 (green), anti-ER-TR7 (blue), or anti-CCL21 (red) antibodies (objective ×63; scale bar represents 12 μm) and merged images in pink (FRC/CCL21) or yellow (CD68/CCL21). Images were acquired using a DMI6000 Leica Spinning Disk microscope with a ×40 1.25NA objective (Leica Microsystems, Wetzlar, Germany) and a Photometrics Coolsnap HQ CCD, driven by Metamorph software (Molecular Devices, Sunnyvale, CA) at the Imaging Facility of Cochin Institute, Paris, France. (B) Masks created from the images in panel A by combining FRC and CCL21 signals or CD68 and CCL21 signals. Pixels are white when both channels are positive and pink when CCL21 is alone. (C) Graph comparing the ratios between colocalized CCL21/CD68 or CCL21/FRC pixels and the total number of CCL21+ cells in nontumoral and CLL samples. Measure of the colocalization was obtained by computing the total number of positive pixels. Means were compared in a Student t-test (**P = .0026; n = 8).', 'hash': '5a0b3bcf94cff303145b9553ca52a18367c5a9044c7983e21ce28da399a16914'}]
|
{'advancesADV2021006169f1': ['Stromal cell organization in lymph node biopsies from patients with CLL was compared with biopsies from nontumoral vascular surgery. As expected, in nontumoral lymph nodes the distribution of the CCL21 chemokine positive cells corresponded to α smooth muscle actin (αSMA) positive network of FRC. Meanwhile CXCL13+ cells were superimposable to the CD21+ network of FDCs (<xref rid="advancesADV2021006169f1" ref-type="fig">Figure 1A</xref>, panels a-d). In almost every tumoral CLL lymph node, this subtissular organization was lost. Several isolated αSMA, panels a-d). In almost every tumoral CLL lymph node, this subtissular organization was lost. Several isolated αSMA+ cells persisted without distinguished organization as an FRC network (<xref rid="advancesADV2021006169f1" ref-type="fig">Figure 1A</xref>, panel f), whereas CD21, panel f), whereas CD21+ FDCs completely disappeared (<xref rid="advancesADV2021006169f1" ref-type="fig">Figure 1A</xref>, panel h). Consequently, CCL21, panel h). Consequently, CCL21+ and CXCL13+ cells were randomly distributed (<xref rid="advancesADV2021006169f1" ref-type="fig">Figure 1A</xref>, panels e,g) over a diffuse lymphoid proliferation in CLL in contrast with a strong localization in the germinal center in nontumoral samples (supplemental Figure 1A, panels a,d). Specifically, a massive infiltration of CD20, panels e,g) over a diffuse lymphoid proliferation in CLL in contrast with a strong localization in the germinal center in nontumoral samples (supplemental Figure 1A, panels a,d). Specifically, a massive infiltration of CD20+ cells and a dispersed and reduced distribution of CD3+ cells were detected in CLL biopsies (supplemental Figure 1A, panels e-f).', 'We also observed an enrichment in CD68+ cells in all tumoral cases of our series compared with nontumoral biopsies (<xref rid="advancesADV2021006169f1" ref-type="fig">Figure 1B</xref>). An evaluation of the CD68). An evaluation of the CD68+ cells showed a significantly higher density of these cells in the tumor infiltrate compared with the nontumoral compartment (outside sinuses) for the various biopsies tested in the study (n = 10 of each case; P < .005) (<xref rid="advancesADV2021006169f1" ref-type="fig">Figure 1C</xref>).).'], 'advancesADV2021006169f2': ['In line with this relevant disorganization of lymph nodes, we addressed the identification of CCL21+ cells in the tumor microenvironment and asked whether they might correspond at least partly to CD68+ cells or were only representative of residual FRCs. Therefore, we compared the distribution of CCL21 expressing cells among CD68+ cells and ER-TR7–labeled FRCs in lymph node sections from nontumoral and CLL samples (n = 8 from each one) using immunofluorescence methodologies. Confocal images confirmed the increase of CD68+ cells and the loss of an organized FRC network in CLL samples as previously observed by immunohistochemistry. Merged images showed that CCL21 colocalized almost exclusively with FRC staining in control samples whereas CD68 labeling remained isolated (<xref rid="advancesADV2021006169f2" ref-type="fig">Figure 2A</xref>, FRC/CCL21 magenta and CD68 green, respectively, in nontumoral merge panel). Conversely, in CLL samples, CCL21 signal colocalized with both CD68, FRC/CCL21 magenta and CD68 green, respectively, in nontumoral merge panel). Conversely, in CLL samples, CCL21 signal colocalized with both CD68+ cells and FRCs (<xref rid="advancesADV2021006169f2" ref-type="fig">Figure 2A</xref>, CD68/CCL21 yellow and FRC/CCL21 magenta, respectively, in CLL merge panel). The colocalization was further quantified using an image processing tool. Upon image capture, a mask for each fluorescence signal was developed. A significantly increased number of CD68-specific pixels was detected in CLL samples compared with nontumoral samples, confirming the increase of CD68, CD68/CCL21 yellow and FRC/CCL21 magenta, respectively, in CLL merge panel). The colocalization was further quantified using an image processing tool. Upon image capture, a mask for each fluorescence signal was developed. A significantly increased number of CD68-specific pixels was detected in CLL samples compared with nontumoral samples, confirming the increase of CD68+ cells. Conversely, some nonsignificant decreases were observed for FRC or CCL21 signals between samples (supplemental Figure 1B). Next, additional masks combining CCL21 with either FRC+ (ER-TR7) or CD68+ signals were created. White pixels matched with positivity in both channels whereas isolated CCL21+ labeling remained red (<xref rid="advancesADV2021006169f2" ref-type="fig">Figure 2B</xref>). Indeed, white pixels were mainly observed for FRC in nontumoral tissues and were randomly distributed between FRC and CD68). Indeed, white pixels were mainly observed for FRC in nontumoral tissues and were randomly distributed between FRC and CD68+ cells in CLL samples. Importantly, the specificity of the staining was proved with a strong inverse exclusion for CCL21 expression between CD68+ cells and FRCs (<xref rid="advancesADV2021006169f2" ref-type="fig">Figure 2B</xref>). A measure of the percentage of colocalization was obtained by computing the number of merged pixels. We then compared the ratios between merged CCL21/FRC and CCL21/CD68 pixels relative to the total number of CCL21). A measure of the percentage of colocalization was obtained by computing the number of merged pixels. We then compared the ratios between merged CCL21/FRC and CCL21/CD68 pixels relative to the total number of CCL21+ cells in nontumoral or CLL samples. The colocalization CCL21/CD68 was significantly higher in CLL samples and was concomitant with a decreased CCL21/FRC colocalization (<xref rid="advancesADV2021006169f2" ref-type="fig">Figure 2C</xref>, left graph) This result indicated that in CLL lymph nodes an important proportion of CCL21 was produced by CD68, left graph) This result indicated that in CLL lymph nodes an important proportion of CCL21 was produced by CD68+ cells whereas the remaining FRCs could still produce CCL21 (<xref rid="advancesADV2021006169f2" ref-type="fig">Figure 2C</xref>, right graph)., right graph).'], 'advancesADV2021006169f3': ['Previous studies indicated a prosurvival role for peripheral blood–derived CD68+ macrophages in coculture with CLL B cells. These so-called NLCs mimic the protective niches observed in lymph nodes.21 We investigated the production of CCL21 by NLCs in vitro differentiated from PBMCs of patients with CLL (supplemental Figure 2A). After removal of the nonadherent cells, the remaining cells were harvested and analyzed by imaging flow cytometry. This approach allowed not only a quantitative analysis but also a visualization of interacting cells. Interestingly, the interaction between macrophages and B cells persisted even after a detaching intensive washing, and the aggregates could be distinguished from single macrophages in “area” versus “aspect ratio” plotting (<xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3A</xref>). All the single CD68). All the single CD68+ macrophages expressed CD206, a typical marker of TAMs (<xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3B</xref>). Conversely, expression of another M2-TAM typical marker, CD163, revealed highly heterogeneous proportions of positive cells among different patients, suggesting variable degrees of maturation (). Conversely, expression of another M2-TAM typical marker, CD163, revealed highly heterogeneous proportions of positive cells among different patients, suggesting variable degrees of maturation (<xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3B;</xref> supplemental Figure 2B). supplemental Figure 2B).', 'We further used the single-cell approach, referred to as the Prime Flow RNA assay, which allowed simultaneous detection of CCL21 mRNA and protein in combination with immunophenotyping for cell surface and intracellular proteins.22 First, the specificity of the mRNA probe was confirmed using both primary FRC cells issued from nontumoral lymph nodes and the CCL21+ MDA-MB231 cell line23 (supplemental Figure 2C). In CD68+CD163+ macrophages from 6 different patients, both CCL21 mRNA and protein were detected in most cells. However, an mRNA−/protein+ subpopulation was also observed, likely reflecting cells that no longer transcribe CCL21 but still hold substantial amounts of protein due to the presence of brefeldin in the procedure (<xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3C</xref>, upper panel; supplemental Figure 2D, upper graph). Several CD68, upper panel; supplemental Figure 2D, upper graph). Several CD68+CD163− macrophages presented with an additional subset of cells in which only CCL21 mRNA was observed (<xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3C</xref>, lower panel; supplemental Figure 2D, lower graph), further arguing for different degrees of maturation among the subpopulation. Imaging of the aggregates composed of CD68, lower panel; supplemental Figure 2D, lower graph), further arguing for different degrees of maturation among the subpopulation. Imaging of the aggregates composed of CD68+ and B cells demonstrated the production of CCL21 mRNA and protein in macrophages exclusively (<xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3D</xref>). Accordingly, CCL21 mRNA was not detected in B cells issued from the suspension of PBMCs in culture for 14 days (supplemental Figure 2E).). Accordingly, CCL21 mRNA was not detected in B cells issued from the suspension of PBMCs in culture for 14 days (supplemental Figure 2E).', 'Ibrutinib impacts B cells/NLC interaction. (A) Graph shows the CCL21mRNA MFI values detected in NLC cells exposed or not to ibrutinib 24 hours before analysis (P = ns; n = 16) or in NLCs derived from patients treated with ibrutinib (n = 4). (B) Image galleries of single macrophages or aggregates (as defined in <xref rid="advancesADV2021006169f3" ref-type="fig">Figure 3A</xref>) exposed or not to ibrutinib for 24 hours (upper panels) or derived from patients treated with ibrutinib (lower panels) showing CD19 (purple), CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF) in permeabilized conditions and in presence of BFA (a) or nonpermeabilized conditions and no BFA treatment (b). (C) Graph comparing the ratios of colocalized B cells/NLCs after 4 hours of coculture exposed (+) or not (−) to ibrutinib for 24 hours (n = 6) or in cocultures derived from patients treated long-term with ibrutinib (n = 3). Statistical analysis was carried out by Student ) exposed or not to ibrutinib for 24 hours (upper panels) or derived from patients treated with ibrutinib (lower panels) showing CD19 (purple), CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF) in permeabilized conditions and in presence of BFA (a) or nonpermeabilized conditions and no BFA treatment (b). (C) Graph comparing the ratios of colocalized B cells/NLCs after 4 hours of coculture exposed (+) or not (−) to ibrutinib for 24 hours (n = 6) or in cocultures derived from patients treated long-term with ibrutinib (n = 3). Statistical analysis was carried out by Student t-test (***P < .001; ****P < .0001) comparing untreated and treated samples. (D) Representative images of interactions between CLL B cells (green) and CD68+ NLCs (red) after 4 hours of coculture priorly treated or not with ibrutinib (24 hours) or in cocultures derived from patients treated with ibrutinib (objective ×10; scale bar represents 10 μm). The areas in the white dotted boxes in the upper panel are ×2.5 zoomed in the lower panels to show B cell/NLC interactions.'], 'advancesADV2021006169f4': ['Interestingly, in these imaging experiments, CCL21 protein was also stained at the membrane of nonpermeabilized cells and in absence of brefeldin treatment (<xref rid="advancesADV2021006169f4" ref-type="fig">Figure 4A</xref>). We confirmed the presence of CCL21 protein at the cell surface of adherent cells after 2 weeks of culture in both permeabilized and nonpermeabilized conditions by immunofluorescence and confocal microscopy (). We confirmed the presence of CCL21 protein at the cell surface of adherent cells after 2 weeks of culture in both permeabilized and nonpermeabilized conditions by immunofluorescence and confocal microscopy (<xref rid="advancesADV2021006169f4" ref-type="fig">Figure 4B</xref>). This observation suggested that the chemokine could be captured on the cell surface upon secretion, as described for unpermeabilized lymphatic endothelium. Indeed, CCL21 appeared to be randomly distributed on CD68). This observation suggested that the chemokine could be captured on the cell surface upon secretion, as described for unpermeabilized lymphatic endothelium. Indeed, CCL21 appeared to be randomly distributed on CD68+ cell membrane, as observed in endothelial cells.24 Accordingly, when we analyzed chemokine secretion by the adherent NLCs after 14 days of culture, CCL21 was barely present above the detection limit whereas both CXCL13 (112.2 ± 150\xa0pg/mL, mean ± SD; n = 14) and CXCL12 (54.2 ± 33\xa0pg/mL, mean ± SD; n = 14) chemokines were present at various extents (<xref rid="advancesADV2021006169f4" ref-type="fig">Figure 4C</xref>), as previously described.), as previously described.15 Correspondingly, CXCL13 mRNA was detected in adherent CD68+ cells (<xref rid="advancesADV2021006169f4" ref-type="fig">Figure 4D</xref>).).', 'Interestingly, gating of the aggregates showed that CD19+ B cells surrounding CD68+ macrophages expressed CCR7, the CCL21 receptor (<xref rid="advancesADV2021006169f4" ref-type="fig">Figure 4E</xref>). Furthermore, CCR7 expression levels were significantly higher in CD19). Furthermore, CCR7 expression levels were significantly higher in CD19+ cells still present in the adherent fraction compared with those in suspension (<xref rid="advancesADV2021006169f4" ref-type="fig">Figure 4F</xref>). Also, we observed that CD19). Also, we observed that CD19+CD5+ cells interacting with CD68+ cells showed a significantly increased level of activated pY759 PCLγ2 compared with CD19+CD5+ cells still present in the adherent fraction but separated from NLCs or CD19+CD5+ cells in suspension (supplemental Figure 2F).'], 'advancesADV2021006169f5': ['To investigate the impact of CD68+ NLCs in CLL B cell trafficking, we analyzed the mobility of B cells cocultured with NLCs. After 14 days of PBMC differentiation, B cells in suspension were recovered, fluorescently stained, and plated onto their autologous NLC monolayer. We first recorded their motility using time-lapse microscopy for 20 minutes. B cells that were resuspended in the 14-day culture medium (conditioned medium) showed sustained motility and random displacements near the adherent cells (supplemental Video 1; <xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5A</xref>, gray area). Conversely, a reduction of the mean speed was observed when B cells were resuspended in fresh medium, namely deprived of any factor produced during the 14 days of culture (supplemental Video 2; , gray area). Conversely, a reduction of the mean speed was observed when B cells were resuspended in fresh medium, namely deprived of any factor produced during the 14 days of culture (supplemental Video 2; <xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5A</xref>, yellow area). Indeed, analysis of the supernatants by Luminex showed heterogenous levels of CXCL13 in all samples whereas CXCL12 was detected only in several samples. As previously observed, CCL21 was not detected above threshold (supplemental Figure 3A). The presence of CXCR4, CXCR5, and CCR7 on B cells was also measured, showing heterogenous expression levels (supplemental Figure 3B). The impact of their specific blocking by inhibitory antibodies was then analyzed. Prevention of MAPK/ERK activation upon binding of the respective chemokines attested to inhibition in the presence of the antibodies (supplemental Figure 3C). We then simultaneously blocked CXCR4 and CXCR5 receptors on B cells before loading them in conditioned medium, and a substantial reduction of their velocity was observed comparable to loading in fresh medium (supplemental Video 3; , yellow area). Indeed, analysis of the supernatants by Luminex showed heterogenous levels of CXCL13 in all samples whereas CXCL12 was detected only in several samples. As previously observed, CCL21 was not detected above threshold (supplemental Figure 3A). The presence of CXCR4, CXCR5, and CCR7 on B cells was also measured, showing heterogenous expression levels (supplemental Figure 3B). The impact of their specific blocking by inhibitory antibodies was then analyzed. Prevention of MAPK/ERK activation upon binding of the respective chemokines attested to inhibition in the presence of the antibodies (supplemental Figure 3C). We then simultaneously blocked CXCR4 and CXCR5 receptors on B cells before loading them in conditioned medium, and a substantial reduction of their velocity was observed comparable to loading in fresh medium (supplemental Video 3; <xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5A</xref>, blue area). On the contrary, mean speed did not significantly decrease after blocking of CCR7 receptor on B cells and increased after blocking of CCL21 on NLC cells (supplemental Videos 4 and 5; , blue area). On the contrary, mean speed did not significantly decrease after blocking of CCR7 receptor on B cells and increased after blocking of CCL21 on NLC cells (supplemental Videos 4 and 5; <xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5A</xref>, pink and green areas, respectively)., pink and green areas, respectively).', 'We then analyzed B cell interaction with NLC cells. After image recording, cocultures in the various experimental settings were incubated for 4 hours and the presence of remaining B cells was evaluated after vigorous washing. In untreated conditions, the presence of B cells interacting with NLCs was detected (<xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5B</xref>, panel a). In contrast, a substantial reduction of B cells, still interacting with NLCs, was observed when cells were resuspended in fresh media as well as upon CXCR4/CXCR5 before blocking (, panel a). In contrast, a substantial reduction of B cells, still interacting with NLCs, was observed when cells were resuspended in fresh media as well as upon CXCR4/CXCR5 before blocking (<xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5B</xref>, panels b-c). More importantly, few B cells could be detected upon CCR7 blocking on B cells or CCL21 blocking on NLCs. Interestingly, in the latest conditions, the residual B cells present did not directly interact with NLCs (, panels b-c). More importantly, few B cells could be detected upon CCR7 blocking on B cells or CCL21 blocking on NLCs. Interestingly, in the latest conditions, the residual B cells present did not directly interact with NLCs (<xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5B</xref>, panels d-e). These interactions were quantified by creating masks to detect B- and NLC cell signals and combine them when overlapping. The B cell/NLC ratio represented\xa0colocalization of B cells with adherent NLCs. The quantification of 12 samples confirmed the significative differences observed, particularly confirming the loss of interaction after CCL21 inhibition (, panels d-e). These interactions were quantified by creating masks to detect B- and NLC cell signals and combine them when overlapping. The B cell/NLC ratio represented\xa0colocalization of B cells with adherent NLCs. The quantification of 12 samples confirmed the significative differences observed, particularly confirming the loss of interaction after CCL21 inhibition (<xref rid="advancesADV2021006169f5" ref-type="fig">Figure 5C</xref>).).'], 'advancesADV2021006169f6': ['We finally investigated the impact of ibrutinib on CD68+ NLC/CLL B cell direct interaction by exposing the coculture to ibrutinib for 24 hours before analysis or using a coculture with NLC/B cells derived from patients treated with ibrutinib. In these experiments, ibrutinib in vitro exposure did not alter CCL21 mRNA expression in NLC cells, as shown by similar CCL21 mRNA MFI (Mean Fluorescence Intensity) in untreated and treated samples, nor did it alter samples from long-term treated patients (<xref rid="advancesADV2021006169f6" ref-type="fig">Figure 6A-B</xref>, upper panel a). Also, CCL21 proteins were detected on the membrane of nonpermeabilized cells in both untreated and ibrutinib-treated samples as well as on cells from long-term treated patients (, upper panel a). Also, CCL21 proteins were detected on the membrane of nonpermeabilized cells in both untreated and ibrutinib-treated samples as well as on cells from long-term treated patients (<xref rid="advancesADV2021006169f6" ref-type="fig">Figure 6B</xref>, lower panel b). These results suggest that Ibrutinib does not directly target CCL21 production by NLCs. The quantification of B/NLC interactions after 4-hour incubation of the different cocultures showed that 4 out of 6 ibrutinib-treated samples and 2 out of 3 long-term treated patient samples displayed a significant reduction of colocalization (, lower panel b). These results suggest that Ibrutinib does not directly target CCL21 production by NLCs. The quantification of B/NLC interactions after 4-hour incubation of the different cocultures showed that 4 out of 6 ibrutinib-treated samples and 2 out of 3 long-term treated patient samples displayed a significant reduction of colocalization (<xref rid="advancesADV2021006169f6" ref-type="fig">Figure\xa06C-D</xref>). On the other hand, B cells still able to interact with NLCs were observed in the various experimental settings (). On the other hand, B cells still able to interact with NLCs were observed in the various experimental settings (<xref rid="advancesADV2021006169f6" ref-type="fig">Figure 6D</xref>), and CCR7 expression levels at their surface were not affected by ibrutinib treatment (supplemental Figure 4F).), and CCR7 expression levels at their surface were not affected by ibrutinib treatment (supplemental Figure 4F).']}
|
Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21
| null |
Blood Adv
|
1660287600
|
[
"Antigens, CD19",
"Feasibility Studies",
"Immunotherapy, Adoptive",
"Salvage Therapy",
"Stem Cells"
] |
other
|
PMC9631672
| null |
25
|
[
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"{'Citation': 'Bento L, Gutiérrez A, Novelli S, et al. ; Grupo Español de Trasplante Hematopoyético (GETH) and Grupo Español de Linfoma y Trasplante Autólogo (GELTAMO) . Allogeneic stem cell transplantation as a curative option in relapse/refractory diffuse large B cell lymphoma: Spanish multicenter GETH/GELTAMO study. Bone Marrow Transplant. 2021;56(8): 1919-1928.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '33767400'}}}",
"{'Citation': 'Tian C, Li Y, Liu S, et al. . Modified conditioning regimen with idarubicin followed by autologous hematopoietic stem cell transplantation for invasive B-cell non-Hodgkin’s lymphoma patients. Sci Rep. 2021;11(1):4273.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7895978'}, {'@IdType': 'pubmed', '#text': '33608570'}]}}",
"{'Citation': 'Kamezaki K, Kikushige Y, Numata A, et al. . Rituximab does not compromise the mobilization and engraftment of autologous peripheral blood stem cells in diffuse-large B-cell lymphoma. Bone Marrow Transplant. 2007;39(9):523-527.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17369863'}}}",
"{'Citation': 'Fenske TS, Hari PN, Carreras J, et al. . Impact of pre-transplant rituximab on survival after autologous hematopoietic stem cell transplantation for diffuse large B cell lymphoma. Biol Blood Marrow Transplant. 2009;15(11):1455-1464.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC2913553'}, {'@IdType': 'pubmed', '#text': '19822306'}]}}",
"{'Citation': 'Young NS. Aplastic anemia. N Engl J Med. 2018;379(17): 1643-1656.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC6467577'}, {'@IdType': 'pubmed', '#text': '30354958'}]}}",
"{'Citation': 'Zhang H, Rodriguez S, Wang L, et al. . Sepsis induces hematopoietic stem cell exhaustion and myelosuppression through distinct contributions of TRIF and MYD88. Stem Cell Reports. 2016;6(6):940-956.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4911503'}, {'@IdType': 'pubmed', '#text': '27264973'}]}}",
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"{'Citation': 'Fajgenbaum DC, June CH. Cytokine storm. N Engl J Med. 2020;383(23):2255-2273.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7727315'}, {'@IdType': 'pubmed', '#text': '33264547'}]}}"
] |
Blood Adv. 2022 Aug 12; 6(16):4691-4704
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Cooperation between RAG1, RAG2 and AID promotes clonal evolution towards pre-B ALL(a) Immunoblot depicting protein expression of AID, RAG1 and RAG2 after retroviral overexpression of these vectors in EBV-immortalized human cord blood B cells. (b) Verification of Aicda (iRFP670), Rag1 (eGFP) and Rag2 (dsRedE2) overexpression in EBV transformed human B cells by flow cytometry (top) and verification of Rag1 and Rag2 overexpression in in these cells by fluorescence microscopy (bottom). (c) Whole exome sequencing analyses to compare the total counts of chromosomal abnormalities in empty vector (EV), Aicda, Rag1-Rag2, and Aicda Rag1-Rag2 transduced EBV-transformed human cord blood B cells.
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a9b2fe31bc5f7a49a165384ad804096afe15f44c52263f03c9797ca0f234bef7
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multiple panels: images & plots
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[
"Microscopy",
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"Immuno Assays"
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[{'image_id': 'emss-62816-f0006', 'image_file_name': 'emss-62816-f0006.jpg', 'image_path': '../data/media_files/PMC4475638/emss-62816-f0006.jpg', 'caption': 'Cooperation between RAGs and AID is required for clonal evolution of pre-leukemic ETV6-RUNX1 B cell precursors(a) Luciferase bioimaging of NOD-SCID mice injected with Aicda+/+\nRag1+/+ +IL-7, Aicda+/+\nRag1+/+ No IL-7+LPS, Aicda−/− +IL-7, Aicda−/− No IL-7+LPS, Rag1−/− +IL-7 and Rag1−/− No IL-7+LPS, with all cell types overexpressing ETV6-RUNX1GFP. IL-7 dependent pre-B cells from each group were taken through 5 rounds of IL-7 withdrawal and LPS treatment. 7 mice were used per group. Luciferase bioimages are shown for five mice in each group. (b) Kaplan Meier curves comparing the overall survival percentage of mice in all 6 groups. (c) Verification of pre-B ALL development in the Aicda+/+\nRag1+/+ No IL-7+LPS group by immunohistochemistry. Haematoxylin and Eosin (H&E) staining was carried out to verify lymphocyte infiltration into the liver and spleen of sick mice. Additionally, CD19 immunohistochemistry was carried out to verify that the infiltrating cells represent B-lineage ALL. Immunohistochemistry pictures are shown for one representative mouse out of 7.', 'hash': 'e8cf0eefbab7277d7e50f62dfb80453b415cb68a7898b8b9d9fa3b14362cdb33'}, {'image_id': 'emss-62816-f0001', 'image_file_name': 'emss-62816-f0001.jpg', 'image_path': '../data/media_files/PMC4475638/emss-62816-f0001.jpg', 'caption': 'Expression and activity of AID in human B cell precursors and B-lineage ALL(a) Plot showing correlation between common somatic hypermutation targets of mAID and their alteration status in childhood ALL. On plotting the genes which are frequently bound and targeted by mAID24, against genes which are commonly deleted and amplified in childhood ALL (data from clinical trial P9906), a positive correlation was observed using a 2×2 contingency table analysis by Chi-square test with Yates’ correction. (b) SHM frequency of IGH VH region genes in single pre-B cell clones isolated from human bone marrow (n=1) and human fetal liver (n=3). SHM of variable regions of TCRVG and TCRVB measured in these cells were used as negative controls. p-values were calculated using student t-test. (c) A schematic depicting CSR in early human B cells derived from three fetal liver donors.', 'hash': 'ac6692f41365b251e2cecd9a6007b00cb8798ccd794bd8353627193175170bcf'}, {'image_id': 'emss-62816-f0002', 'image_file_name': 'emss-62816-f0002.jpg', 'image_path': '../data/media_files/PMC4475638/emss-62816-f0002.jpg', 'caption': 'Late pre-B cells (small pre-BII) represent a natural subset of increased genetic vulnerability(a) Wildtype mouse bone marrow was sorted into different fractions of early B cell development20. Quantitative RT-PCR showing Aicda mRNA abundance in each fraction of early B cell development (n=3; mean ± s.d.), as compared to splenic B cells induced with LPS and IL-4 (positive control) and Aicda−/− pre-B cells (negative control). (b) FACS analysis of surface IL-7Rα in Blnk−/− pre-B cells expressing empty GFP vector, Blnk/GFP or BlnkY96F/GFP. (c) Immunoblot depicting increase in mAID protein level upon differentiation of Blnk−/− pre-B cells from large pre-BII to small pre-BII by Blnk reconstitution. (d) mAID protein levels measured by western blotting before (large pre-BII) and after (small pre-BII) IL-7 withdrawal from mouse pre-B cell cultures. (e) Plots showing AICDA mRNA levels (left) and VH mutation frequency (right) in large pre-BII and small pre-BII human pre-B cells, in children with mutation in one of the chains of the IL-7R. Normal human bone marrows were used as negative controls. All p-values were calculated using student t-test. One representative of three experiments is shown for FACS plots and immunoblots.', 'hash': 'effc171e74015825984f1b558d008817c1233a25cd95f2e8f2b1f4c1f3e429e9'}, {'image_id': 'emss-62816-f0005', 'image_file_name': 'emss-62816-f0005.jpg', 'image_path': '../data/media_files/PMC4475638/emss-62816-f0005.jpg', 'caption': 'Cooperation between RAG1, RAG2 and AID promotes clonal evolution towards pre-B ALL(a) Immunoblot depicting protein expression of AID, RAG1 and RAG2 after retroviral overexpression of these vectors in EBV-immortalized human cord blood B cells. (b) Verification of Aicda (iRFP670), Rag1 (eGFP) and Rag2 (dsRedE2) overexpression in EBV transformed human B cells by flow cytometry (top) and verification of Rag1 and Rag2 overexpression in in these cells by fluorescence microscopy (bottom). (c) Whole exome sequencing analyses to compare the total counts of chromosomal abnormalities in empty vector (EV), Aicda, Rag1-Rag2, and Aicda Rag1-Rag2 transduced EBV-transformed human cord blood B cells.', 'hash': 'a9b2fe31bc5f7a49a165384ad804096afe15f44c52263f03c9797ca0f234bef7'}, {'image_id': 'emss-62816-f0004', 'image_file_name': 'emss-62816-f0004.jpg', 'image_path': '../data/media_files/PMC4475638/emss-62816-f0004.jpg', 'caption': 'Evidence for concurrent activities of RAGs and AID in single pre-B cell clones(a) Aicda-GFP pre-B cells37 upregulate expression of AID, RAG1 and RAG2 at small pre-BII in the context of inflammatory signals like LPS (GFP+κLC+ cells) (b) Immunoblot comparing mAID protein levels in large pre-BII and small pre-BII mouse pre-B cells, following LPS exposure. Splenocytes from a wildtype mouse induced with LPS and IL-4 were used as a positive control for mAID expression. (c) SHM frequencies of IGH VH regions in pediatric ALL cases were compared against SHM of TCRG and TCRB in these cases. Leukemia patients displaying ongoing VH replacements are represented by solid black circles. (d-e) Effects of ETV6-RUNX1GFP and its mutant ETV6-RUNX1-ΔRHDGFP on Rag1 and Rag2 expression in mouse pre-B cells were evaluated by overexpression of retroviral vectors encoding these fusion proteins. Empty vector (EV) transduced mouse pre-B cells served as negative control. (d) Representative FACS plots showing the percentages of GFP+ cells in each case on day 2 post transduction. One representative of three replicates is shown. (e) GFP+ cells in (d) were sorted and used to measure Rag1 and Rag2 mRNA levels by qRT-PCR (n=3, mean ± s.d.). All p-values were calculated using student t-test.', 'hash': '10c0dfd0dce64b76d3cf8c92125708338adeb07177d3f1c38bf1bc553b0887eb'}, {'image_id': 'emss-62816-f0003', 'image_file_name': 'emss-62816-f0003.jpg', 'image_path': '../data/media_files/PMC4475638/emss-62816-f0003.jpg', 'caption': 'Aicda and Rag1-Rag2 are regulated by the same transcriptional control elements in pre-B cells(a) AID protein levels in Stat5fl/fl IL-7-dependent cells transduced with empty ERT2 (no Stat5 deletion) and Cre-ERT2 (with Stat5 deletion) vectors were compared by immunoblotting. (b) Immunoblot for active nuclear FOXO4 after Blnk−/− IL-7-dependent pre-B cells are differentiated from large pre-BII to small pre-BII stage by Blnk reconstitution. (c) Aicda mRNA levels were compared between the empty vector (EV) transduced and Pten deleted Ptenfl/fl pre-B cells 48 hours after tamoxifen induction, by qRT-PCR (n=3, mean ± s.d.). (d-e) Rag1 and Rag2 mRNA levels were measured by qRT-PCR following inducible deletion of Pten in Ptenfl/fl pre-B cells (n=3, mean ± s.d.). (f-h) Mouse IL-7-dependent pre-B cells were retrovirally transduced with either an empty vector or a constitutively active form of Foxo1 (Foxo1CA). The 2 groups of cells were then subject to two conditions each, either they were retained in the presence of IL-7 (large pre-BII) or IL-7 withdrawal was carried out for 24 hours to differentiate them to small pre-BII. (f) Aicda mRNA level was then measured in each case by qRT-PCR (n=3, mean ± s.d.). (g-h) Rag1 and Rag 2 mRNA levels measured by qRT-PCR after retroviral expression of a constitutively active form of Foxo1 (Foxo1CA) or empty vector (EV) in mouse pre-B cells, in the presence or absence of IL-7 (n=3, mean ± s.d.). All p-values were calculated using student t-test.', 'hash': '666a19d72c9c1e5d5209d51e2f57c6ff4ee86a85f55bf40380280aec21337e6a'}]
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{'emss-62816-f0001': ['To test the plausibility of AID’s contribution to RAG1-RAG2-mediated deletions and rearrangements, we measured whether lesions in childhood ALL (data from clinical trial P9906) occur preferentially at genes bound by AID in normal B cells. We identified 40 genes that were recurrently mutated, deleted or rearranged in childhood ALL (207 patients in COG P9906) and plotted the frequency of these lesions against mouse AID (mAID) targeting of these genes in mouse B cells. mAID target genes (4,167) were identified in a previous ChIP-seq study based on increased mAID ChIP-seq tags compared to Aicda−/− B cells24. 18,248 genes (non-mAID targets) did not have significantly higher mAID ChIP-seq tag counts compared to Aicda−/− B cells. Interestingly, frequencies of recurrent genetic lesions correlated significantly with mAID ChIP-seq counts (<xref ref-type="fig" rid="emss-62816-f0001">Fig. 1a</xref>). In this analysis, 34 of 40 genes with recurrent lesions in childhood ALL are mAID-targets, thereby pointing to AID’s possible contribution to frequent genetic lesions in childhood ALL. Of the remaining six genes that are non-mAID targets, five (). In this analysis, 34 of 40 genes with recurrent lesions in childhood ALL are mAID-targets, thereby pointing to AID’s possible contribution to frequent genetic lesions in childhood ALL. Of the remaining six genes that are non-mAID targets, five (CDKN2A, TBL1XR1, NR3C1, NR3C2 and PBX1)23,25 have breakpoints at known RSS motifs with characteristic N-nucleotide addition at the break junctions, suggesting that lesions at these loci were caused by RAG1-RAG2 activity alone. It is important to note that not all AID targets are syntenic between human and mouse. While this represents a limitation to our approach, disparity between AID targets in humans and mice will only lead to an underestimation of the correlation we observed. Moreover, a recent study identifying hAID targets in B cell lymphoma26, although not quantitative, includes multiple genes from our analysis (PAX5, BTG1, BTG2, BACH2, RHOH, EBF1, TCF3). These studies confirm that AID may be responsible for mutating a substantial percentage of genes found frequently altered in pre-B ALLs.', 'B-ALL arises from sites of early B-lymphopoiesis- the fetal liver and the bone marrow during pre- and postnatal development, respectively. RAG enzymes are active in B cell precursors in both sites. Studies in identical twins with concordant ETV6-RUNX1 pre-B ALL indicate clonal origin in cells undergoing RAG-dependent IGH variable region rearrangement and continued recombinant activity during subsequent clonal evolution13,27. To measure AID-activity in human fetal liver and bone marrow B cell precursors, we sorted CD19+ pre-B cells that lack κ and λ immunoglobulin light chains, from fetal liver tissues of three donors and bone marrow from one healthy adult donor. We then cloned and sequenced the IGH variable regions for SHM and constant regions for CSR (Supplementary Tables 1,2). Interestingly, most bone marrow pre-B cell clones expressed IGH with mutated VH regions (26×10−3 bp, <xref ref-type="fig" rid="emss-62816-f0001">Fig. 1b</xref>, , Supplementary Table 1). Likewise, fetal liver pre-B cells of three donors carried mutated VH regions (14×10−3 bp, <xref ref-type="fig" rid="emss-62816-f0001">Fig. 1b</xref>, , Supplementary Table 2). To control for reverse transcriptase and Pfu DNA polymerase errors, peripheral blood-derived CD3+ T cells lacking AICDA expression were sorted and, rearranged TCRB and TCRG variable regions were amplified and analyzed in parallel with IGH VH regions of bone marrow and fetal liver pre-B cells. Overall, IGH VH regions from sorted bone marrow and fetal liver pre-B cells carried a significant frequency of somatic mutations (P=3.3×10−18), relative to TCRB and TCRG variable regions from peripheral blood T-lymphocytes (<xref ref-type="fig" rid="emss-62816-f0001">Fig. 1b</xref>). We also found a significant fraction of the sorted CD19). We also found a significant fraction of the sorted CD19+ pre-B cells sorted from fetal liver tissues expressing class-switched IGH, containing Cγ3, Cγ1 and Cα constant regions (<xref ref-type="fig" rid="emss-62816-f0001">Fig. 1b,c</xref>, , Supplementary Table 2). It is important to note that our method of IGH cloning and sequencing was not strictly quantitative. Therefore, we are not in a position to predict the exact fraction of fetal liver pre-B cells that express class-switched IGH constant regions. These findings suggested that AID might be prematurely activated in human bone marrow and fetal liver pre-B cells. While we found genetic traces of AID activity in RAG1-RAG2-expressing pre-B cells, these findings did not establish that AID and RAG1-RAG2 are concurrently active in the same cells.'], 'emss-62816-f0002': ['To elucidate whether and how premature activity of mAID is regulated during early B-lymphopoiesis, we sorted B cell precursors from normal mouse bone marrow as described previously20 by flow cytometry. Pro-B cells (c-kit+ B220+), large pre-BII (CD25+ B220+ FSChi SSClo) and small pre-BII (CD25+ B220+ FSClo SSClo) were isolated. As large and small pre-BII cells carry similar surface markers we used their size differences (FSC) to separate them. We then measured Aicda mRNA abundance in each fraction of early B cell development (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2a</xref>). In comparison to activated mature splenic B cells, ). In comparison to activated mature splenic B cells, Aicda mRNA in bone marrow B cell precursors was generally low or absent. However, Aicda mRNA was increased ~21-fold at the small pre-BII as compared to the large pre-BII stage of early B-lymphopoiesis (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2a</xref>). This transition is characterized by signals from the pre-B cell receptor (pre-BCR) and its intracellular signaling components that are assembled by the pre-BCR linker BLNK). This transition is characterized by signals from the pre-B cell receptor (pre-BCR) and its intracellular signaling components that are assembled by the pre-BCR linker BLNK28,29. One consequence of pre-BCR signaling at the large to small pre-BII transition is the downregulation of interleukin-7 receptor (IL-7R) surface expression by BLNK activation (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2b</xref>). Attenuation of IL-7R expression reduces downstream STAT5 signaling in small pre-BII cells). Attenuation of IL-7R expression reduces downstream STAT5 signaling in small pre-BII cells30. We therefore tested whether Aicda mRNA abundance was increased in small pre-BII cells by abrogating IL-7R-STAT5 signaling using two methods. First, we reconstituted the pre-BCR linker Blnk in Blnk−/− pre-B cells to induce IL-7R downregulation, and measured Aicda mRNA (Supplementary Fig. 2a) and, mAID protein abundance (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2c</xref>). Second, we removed IL-7 from pre-B cell cultures (). Second, we removed IL-7 from pre-B cell cultures (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2d</xref>, , Supplementary Fig. 2b) to force the transition from large pre-BII to small pre-BII stage. In vitro differentiation by both methods induced mAID protein upregulation, demonstrating that active IL-7R signaling in mouse large pre-BII cells safeguards against pre-mature mAID expression.', 'We next investigated whether IL-7R signaling protects against pre mature AID expression in human pre-B cells. While IL-7R signaling is essential for B cell development in mice, humans with inherited IL7R deficiency have almost normal B cell counts31, which allows experimental analysis of human B-lymphopoiesis in the absence of IL-7R signaling. We isolated large and small pre-BII cells from bone marrow samples of three patients with biallelic germline mutations in either gene that encode the IL-7R, namely IL7R and IL2RG. One patient harbored an IL7RG28X mutation, the two other patients carried IL2RGQ144X and IL2RGR289X mutations, respectively (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2e</xref>). As controls, we studied sorted large and small pre-BII cells from bone marrow of three healthy donors. As in mouse B-lymphopoiesis, we observed that human pre-B cells upregulate ). As controls, we studied sorted large and small pre-BII cells from bone marrow of three healthy donors. As in mouse B-lymphopoiesis, we observed that human pre-B cells upregulate AICDA mRNA by 10- to 25-fold at the large pre-BII to small pre-BII transition. In the three patients lacking functional IL-7R signaling, pre-B cells expressed more AICDA mRNA as compared to the healthy controls (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2e</xref>). No significant difference was observed between large and small pre-BII cell subsets obtained from IL-7R-deficient patients. Amplification and sequencing of ). No significant difference was observed between large and small pre-BII cell subsets obtained from IL-7R-deficient patients. Amplification and sequencing of IGH VH regions revealed a low frequency of SHM in normal small pre-BII cells. Frequencies of somatic IGH VH region mutations were ~5-fold higher, in both large and small pre-BII cells isolated from patients lacking functional IL-7R (<xref ref-type="fig" rid="emss-62816-f0002">Fig. 2e</xref>). Thus, IL-7R safeguards human pre-B cells from premature AID activation.). Thus, IL-7R safeguards human pre-B cells from premature AID activation.'], 'emss-62816-f0003': ['IL-7R-STAT5 signaling in early pre-B cells32 prevents upregulation of Rag1-Rag2 mRNA and recombination of Igk V and J gene segments32,33. Downregulation of IL-7R expression attenuates activities of STAT5 and Akt, and induces activation of Rag1-Rag2 mRNA (Supplementary Fig. 2c). STAT5 recruits the polycomb repressor EZH2 to Rag1-Rag2, while Akt phosphorylates and inactivates FOXO1, a potent transcriptional activator of Rag1 and Rag234. Here we show that inducible, Cre-mediated deletion of Stat5a and Stat5b in large pre-BII cells not only results in activation of RAG1-RAG2 but also upregulation of AID (<xref ref-type="fig" rid="emss-62816-f0003">Fig. 3a</xref>). The mechanistic role of Akt-Foxo1 signaling in regulation of ). The mechanistic role of Akt-Foxo1 signaling in regulation of Aicda mRNA abundance was tested using genetic loss of function of PTEN, a negative regulator of Akt, and gain-of-function studies for Foxo1. Inducible deletion of Pten results in hyperactivation of Akt and, hence, phosphorylation and inactivation of Foxo transcription factors35. Pre-BCR signaling via BLNK not only decreased IL-7R surface expression but also caused activation of Foxo factors (<xref ref-type="fig" rid="emss-62816-f0003">Fig. 3b</xref>). CRE-mediated, inducible ablation of ). CRE-mediated, inducible ablation of Pten caused near-complete loss of Rag1, Rag2 and Aicda mRNA expression (<xref ref-type="fig" rid="emss-62816-f0003">Fig. 3c-e</xref>). In agreement with these findings, a constitutively active form of Foxo1 that is protected against Akt-mediated phosphorylation strongly activates ). In agreement with these findings, a constitutively active form of Foxo1 that is protected against Akt-mediated phosphorylation strongly activates Rag1, Rag2 and Aicda transcription. Interestingly, these effects were further enhanced by removal of IL-7 from cell culture medium (<xref ref-type="fig" rid="emss-62816-f0003">Fig. 3f-h</xref>, , Supplementary Fig. 2d). In summary, IL-7R signaling protects pre-B cells from premature activation of Aicda through STAT5- and Akt-dependent pathways.'], 'emss-62816-f0004': ['AID is upregulated in mature B cells upon antigen encounter in germinal center, where it introduces SHM in Igh VH regions and promotes CSR of constant regions16,36. Antigen encounter in germinal centers can be mimicked by treatment of mature splenic B cells with lipopolysaccharide (LPS) and IL-4. We tested whether small pre-BII cells respond to inflammatory agents like LPS in a manner similar to mature B cells, by upregulating mAID. To measure mAID-activation in pre-B cells at the single cell level, we studied pre-B cells from an Aicda-GFP reporter mouse strain where GFP gene was fused in frame to Aicda exon 537. Aicda-GFP pre-B cells were propagated in IL-7 and then treated with or without LPS, and in the presence or absence of IL-7 (<xref ref-type="fig" rid="emss-62816-f0004">Fig. 4a</xref>, , Supplementary Fig. 3a,b). Treatment with LPS induced minimal increases of mAID protein expression. However, when LPS-treatment was combined with IL-7-withdrawal, upregulation of mAID was increased by 20-fold compared to LPS-treatment in the presence of IL-7 (<xref ref-type="fig" rid="emss-62816-f0004">Fig. 4a,b</xref>). To measure mAID and RAG1-RAG2 activities in parallel at the single-cell level, we monitored mAID-GFP and ). To measure mAID and RAG1-RAG2 activities in parallel at the single-cell level, we monitored mAID-GFP and de novo surface expression of Igκ light chains after RAG-mediated Vκ-Jκ gene rearrangement (<xref ref-type="fig" rid="emss-62816-f0004">Fig.4a</xref>, , Supplementary Fig. 3b). Treatment of Aicda-GFP pre-B cells with LPS in the presence of IL-7 induced activation of the Aicda-GFP reporter in 2.8% of cells as compared to 0.01% in LPS-untreated condition. However, combination of LPS-treatment with removal of IL-7 from the cell culture medium increased the fraction of mAID-expressing cells to 42% from 2.8% in the presence of IL-7. Interestingly, majority of Aicda-GFP+ cells also expressed de novo κ light chains (<xref ref-type="fig" rid="emss-62816-f0004">Fig. 4a</xref>, , Supplementary Fig. 3b), providing direct evidence that mAID expression and RAG-mediated recombination of Vκ- and Jκ-gene segments can occur in the same cells. These findings were confirmed in a second Aicda reporter mouse model, in which mAID drives expression of Cre for excision of a loxP-flanked Stop cassette of an eYPF marker that is located in the Rosa26 locus37 (Supplementary Fig. 3c,d).', 'Studying IGH VH genes amplified from 72 cases of childhood ALL (13 ETV6-RUNX1, 2 TCF3-PBX1, 3 MLL-rearranged, 1 MYC-IGH, 17 hyperdiploid, 26 with normal or undetermined karyotype and 10 with sporadic chromosomal translocations), we found evidence of ongoing RAG1-RAG2 and AID activity in all subgroups (Supplementary Tables 3-5). Pre-B ALL clones consistently carried somatically mutated VH and JH gene segments, indicative of AID activity (Supplementary Tables 3-5). Interestingly, in 10 of 72 cases, we found subclones in each patient that carried the same defining D-JH junction (Supplementary Tables 3-5). Multiple distinct VH segments diversified these subclones through sequential RAG-mediated VH replacement38. The process of leukemic clonal evolution by cooperative activities of AID (SHM) and RAGs (VH replacement) in a pediatric pre-B ALL patient is shown (Supplementary Fig. 4, Supplementary Table 5). Comparing mutation frequencies in VH and JH segments, we found that JH segments carry a significantly higher load of somatic mutations (102×10−3 bp in JH, 16×10−3 bp in VH; P=0.0007; Supplementary Tables 3-5, <xref ref-type="fig" rid="emss-62816-f0004">Fig. 4c</xref>). Multiple rounds of V). Multiple rounds of VH replacement, where acquired mutations in VH segments are erased but D-JH junctions remain constant, could explain this unexpected difference. In 7 of 13 ETV6-RUNX1 pre-B ALL cases, we found multiple distinct subclones that had undergone VH replacement, suggesting that RAG enzymes are particularly active in this subset (Supplementary Table 3). This finding is in agreement with a recent study highlighting aberrant activation of RAG-mediated recombination as the primary driver of ETV6-RUNX1 leukemogenesis23. To test if ETV6-RUNX1 activity is linked to deregulated RAG expression, we transduced murine pre-B cells with vectors encoding full-length ETV6-RUNX1, a mutant ETV6-RUNX1 lacking the DNA binding Runt Homology Domain (ΔRHD) and an empty vector (EV) control. Interestingly, full-length ETV6-RUNX1 but not ΔRHD ETV6-RUNX1 induced expression of Rag1-Rag2 in mouse pre-B cells, suggesting that abnormally high recombinase activity in this ALL subset is caused by the ETV6-RUNX1 fusion (<xref ref-type="fig" rid="emss-62816-f0004">Fig. 4d,e</xref>). Hence, cooperative AID and RAG activities may explain the mechanism of clonal evolution in the ). Hence, cooperative AID and RAG activities may explain the mechanism of clonal evolution in the ETV6-RUNX1 leukemias.'], 'emss-62816-f0005': ['Based on our earlier findings, we propose that pre-mature AID expression in small pre-BII cells represents a vulnerability that exposes early B lymphocyte development to genetic lesions in the context of repeated exposure to inflammatory stimuli (Supplementary Fig. 5). We therefore tested the relevance of RAG and AID cooperation in clonal evolution in normal cord blood-derived human B cells through a genetic gain-of-function experiment. Normal CD19+ B cells were sorted from human cord blood (CB) and immortalized by EBV-containing supernatants from B98-5 cultures. Proliferating CB B cells were then transduced with vectors encoding Aicda (iRFP670), Rag1 (eGFP) and Rag2 (dsRedE2), either alone or in combination or empty vector controls for Aicda, Rag1 and Rag2 vectors (<xref ref-type="fig" rid="emss-62816-f0005">Figs. 5a,b</xref>; ; Supplementary Figs. 6,7). Transduced CB B cells were sorted in each condition and expression of AID, RAG1 and RAG2 and lack thereof was confirmed by immunoblotting (<xref ref-type="fig" rid="emss-62816-f0005">Fig. 5a</xref>). Our finding, that AID expression is absent in EBV-infected B cells is consistent with the published literature. EBV suppresses AID expression). Our finding, that AID expression is absent in EBV-infected B cells is consistent with the published literature. EBV suppresses AID expression41, which is the reason why EBV+ B cell lymphomas do not exhibit ongoing SHM and CSR. EBV-mediated suppression of AID is induced by the EBV oncoprotein EBNA242.', 'To be able to study genetic lesions at the level of individual clones, single transduced human B cell clones were plated into individual wells on 96-well plates. CB B cell clones were harvested and subjected to whole exome sequencing. CB B cells carrying three empty vector controls (iRFP670, eGFP, dsRedE2), Aicda and two empty vector controls, Rag1, Rag2 and one empty vector control and, Aicda, Rag1 and Rag2, were analyzed. The analysis focused on structural genetic lesions: insertions, deletions, inversions and translocations. Compared to empty vector transduced CB B cells, overexpression of either Aicda alone or Rag1-Rag2 alone only increased the frequency of interchromosomal translocations. However, CB B cells expressing Aicda in combination with Rag1-Rag2 had increased counts of insertions, deletions, inversions and intrachromosomal translocations (<xref ref-type="fig" rid="emss-62816-f0005">Fig. 5c</xref>).).'], 'emss-62816-f0006': ['After five cycles, firefly luciferase-labeled pre-B cells were injected intravenously into NOD/SCID mice. All mice receiving wildtype pre-B cells with IL-7 withdrawal and LPS treatment died of leukemia within three weeks. Multiple mice (3-4) in the Aicda+/+\nRag1+/+ -IL-7+LPS group were sacrificed on the same day because they were terminally ill with leukemia. This phenomenon is reflected in the rapid drops of the Kaplan Meier curve for this group. In contrast, the absence of Aicda or Rag1 delayed and abrogated leukemia development respectively, demonstrating that both proteins are required for clonal evolution of ETV6-RUNX1 pre-B cells towards leukemia (<xref ref-type="fig" rid="emss-62816-f0006">Fig. 6a,b</xref>, , Supplementary Figs. 8,9). In the absence of Aicda, two mice developed leukemia, albeit after a substantially prolonged latency period (<xref ref-type="fig" rid="emss-62816-f0006">Fig. 6a,b</xref>). We were able to verify that the sick mice injected with stimulated ). We were able to verify that the sick mice injected with stimulated Aicda+/+\nRag1+/+ pre-B cells indeed died of pre-B ALL (<xref ref-type="fig" rid="emss-62816-f0006">Fig. 6c</xref>, , Supplementary Figs. 8,9). These findings provide genetic evidence that clonal evolution of pre-leukemic ETV6-RUNX1 pre-B cells in the context of inflammatory/repetitive infectious stimulation requires both AID and RAG activities.']}
|
Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia
| null |
Nat Immunol
|
1437202800
|
Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.
|
[
"Adolescent",
"Animals",
"Antibody Diversity",
"B-Lymphocytes",
"Child",
"Child, Preschool",
"Clonal Evolution",
"Cytidine Deaminase",
"DNA-Binding Proteins",
"Female",
"Flow Cytometry",
"Homeodomain Proteins",
"Humans",
"Immunoblotting",
"Infant",
"Male",
"Mice, Inbred NOD",
"Mice, Knockout",
"Mice, SCID",
"Mice, Transgenic",
"Microscopy, Fluorescence",
"Precursor Cell Lymphoblastic Leukemia-Lymphoma",
"Precursor Cells, B-Lymphoid",
"Reverse Transcriptase Polymerase Chain Reaction",
"Tumor Cells, Cultured"
] |
other
|
PMC4475638
| null |
54
|
[
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] |
Nat Immunol. 2015 Jul 18; 16(7):766-774
|
NO-CC CODE
|
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Impact of remdesivir (RDV) on cell survival. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5. A. Representative images of RDV-treated embryos at E4.5 that are stained for live (green) and dead (red) cells by the LIVE/DEAD Cell Viability Assay. Binary images (see Materials and methods) are also shown for dead cells. Scale bar = 100 µm. B. The areas occupied by dead cells in RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ± 95% CI, n = 24 or 25 for each treatment group, one-way ANOVA followed by t-test, p < 0.01).
|
gr3_lrg
|
2
|
db3609ea4cc338c89840ad58023bed6df3c4d7ebe881b0d8e9fc10500b8fb663
|
gr3_lrg.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
727,
430
] |
[{'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr3_lrg.jpg', 'caption': 'Impact of remdesivir (RDV) on cell survival. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5. A. Representative images of RDV-treated embryos at E4.5 that are stained for live (green) and dead (red) cells by the LIVE/DEAD Cell Viability Assay. Binary images (see Materials and methods) are also shown for dead cells. Scale bar =\u2009100\u2009µm. B. The areas occupied by dead cells in RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200924 or 25 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01).', 'hash': 'db3609ea4cc338c89840ad58023bed6df3c4d7ebe881b0d8e9fc10500b8fb663'}, {'image_id': 'gr7_lrg', 'image_file_name': 'gr7_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr7_lrg.jpg', 'caption': 'Impact of early exposure to remdesivir (RDV) on the expansion and maintenance of the blastocyst cavity. Embryos are treated with indicated concentrations of RDV from E1.5 to E4.5. A. Representative images of RDV-treated embryos at E2.5, E3.5, and E4.5. Scale bar =\u2009100\u2009µm. B. The size of RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200936–40\u2009for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01). No significant difference is observed among treatments at E3.5.', 'hash': 'f140512491db295ed4de1e1b1259e639cf0f0f7b915417f425dffc5630d56065'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr2_lrg.jpg', 'caption': 'Adverse effects of remdesivir (RDV) on the expansion and maintenance of the blastocyst cavity. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5. A. Representative images of RDV-treated embryos at E2.5 (before treatment), E3.5, and E4.5. Scale bar =\u2009100\u2009µm. B. The size of RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200927 or 28 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01). No significant difference is observed among treatments at E3.5.', 'hash': '4d530691b8c744afbe42619899fbeb52a02294153e051b087542086d7393643e'}, {'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr6_lrg.jpg', 'caption': 'Impact of remdesivir (RDV) on the epiblast (EPI) and the primitive endoderm (PrE) cells. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5, and immunohistochemically examined for SOX2 (EPI marker), NANOG (EPI marker), SOX17 (PrE marker), and GATA4 (PrE marker). A. Representative images of embryos that are stained for the nuclei (DAPI), SOX2 (green) and SOX17 (red). B. Representative images of embryos stained for the nuclei (DAPI), NANOG (green) and GATA4 (red). Scale bars =\u200950\u2009µm. C. Numbers of nuclei that are positive for the EPI marker and PrE marker per embryo. Red lines indicate mean values (n\u2009=\u200919 or 20 for each treatment group). Statistically significant differences are marked with horizontal bars and p-values (t-test) between two groups.', 'hash': '3ea9969d3797647d18fce91c2c78ab3df89ebb918ac6ffff6eb7b0b78ce65a1f'}, {'image_id': 'ga1_lrg', 'image_file_name': 'ga1_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/ga1_lrg.jpg', 'caption': 'No caption found', 'hash': 'dfd554cffce011cfdb5e18906e0a031ed6d29dddd46dc74b1b406ce71f1e9b96'}, {'image_id': 'gr8_lrg', 'image_file_name': 'gr8_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr8_lrg.jpg', 'caption': 'Duration- and timing-dependent impact of remdesivir (RDV) exposure on the blastocyst morphology and the gene expression profiles. A. A diagram depicting the RDV treatment regimen. B. Representative images of treated embryos at E4.5. Scale bar =\u2009100\u2009µm. C. The size of treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200930–54\u2009for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01). D. The expression levels of the markers for the trophectoderm (TE), the epiblast (EPI), and the primitive endoderm (PrE) at E4.5. Graphs are qRT-PCR data, showing relative expression levels of each lineage marker, normalized against housekeeping gene Gapdh. C, C-R, and R-C correspond to the treatment groups CON, CON-RDV, and RDV-CON, depicted in A, respectively. The RDV group is not analyzed due to extensive cell death. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20093 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.05). No significant difference is observed for Cdx2, Pou5f1, and Nanog.', 'hash': '77e1b5ab65812ecee4dc7f4af17ce89d8c337119442cad840de39d945f0ffc2b'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr4_lrg.jpg', 'caption': 'Effects of GS-441524, a major metabolite of remdesivir, on the expansion and maintenance of the blastocyst cavity. Embryos are treated with indicated concentrations of GS-441524 from E2.5 to E4.5. A. Representative images of GS-441524-treated embryos at E3.5 and E4.5. Scale bar =\u2009100\u2009µm. B. The size of GS-441524-treated embryos. No significant difference is found at E3.5 or E4.5 among treatments (mean ±\u200995% CI, n\u2009=\u200930–32\u2009for each treatment group).', 'hash': 'c3ee91825e7910916f9c93cecfdacaa7a246a416c439aa9378f081eca1f37e3b'}, {'image_id': 'gr10_lrg', 'image_file_name': 'gr10_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr10_lrg.jpg', 'caption': 'Effects of remdesivir (RDV) on the trophectoderm (TE) and the inner cell mass (ICM) at E3.5. Embryos are treated with RDV from E2.5 to E3.5, and immunohistochemically examined for CDX2 and SOX2 proteins, which are markers for TE and ICM, respectively. A. Representative images of RDV-treated embryos that are stained for the nuclei (DAPI), CDX2 (green), and SOX2 (red). Scale bar =\u200950\u2009µm. B. Numbers of total nuclei, and those of CDX2-positive and SOX2-positive nuclei. Red lines indicate mean values (n\u2009=\u200919 or 20 for each treatment group). Statistically significant differences are marked with horizontal bars and p-values (t-test) between two groups. C. Distribution of SOX2-positive and CDX2-positive nuclear numbers for individual embryos. Linear trend lines representing the means of the SOX2/CDX2 ratios are superimposed for each treatment group (n\u2009=\u200919 or 20).', 'hash': '3c4653dd72c6682ac06b02aff4ecf020224f6446ed7583e715d4246b19119858'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr1_lrg.jpg', 'caption': 'A schematic diagram, depicting the superovulation and mating schedule of the mice and the embryonic stages. Hours are indicated relative to the time of the hCG injection. See Materials and methods for more details. E: embryonic day, hCG: human chorionic gonadotropin, PMSG: pregnant mare serum gonadotropin.', 'hash': '73a40ee990dca58d64d1f148b7a3da11e133a1b7710278b3332c49333d62aa0f'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr5_lrg.jpg', 'caption': 'Impact of remdesivir (RDV) and GS-441524 on the expression levels of the marker genes for the trophectoderm (TE), the epiblast (EPI), and the primitive endoderm (PrE) at E4.5. Embryos are treated from E2.5 to E4.5 with vehicle control (C), RDV at 2\u2009μM (R2), RDV at 4\u2009μM (R4), or GS-441524 at 4\u2009μM (G4). Graphs are qRT-PCR data, showing relative expression levels of each lineage marker, normalized against housekeeping gene Gapdh. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20093 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.05). No significant difference is observed for Cdx2 and Pou5f1.', 'hash': '175bc703160110bbd2bf9652468a68ba0beb02d004d45a12d5d9942123ba6818'}, {'image_id': 'gr11_lrg', 'image_file_name': 'gr11_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr11_lrg.jpg', 'caption': 'Impact of remdesivir and GS-441524 on the proliferation and viability of human embryonic stem cells. Graphs show the relative light unit of the CellTiter-Glo Luminescent Assay as a proxy for the relative number of metabolically active cells after 2 days of treatment. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20093 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01).', 'hash': '806eaa48b1013ecf9eeb30b6f68efc177e26c13a4dbae0fb1fc13011218102ba'}, {'image_id': 'gr9_lrg', 'image_file_name': 'gr9_lrg.jpg', 'image_path': '../data/media_files/PMC9122741/gr9_lrg.jpg', 'caption': 'Impact of remdesivir (RDV) on the trophectoderm (TE) and the inner cell mass (ICM) at E3.5. Embryos are treated from E2.5 to E3.5 with vehicle control (C), RDV at 4\u2009μM (R4), RDV at 8\u2009μM (R8), or GS-441524 at 8\u2009μM (G8). Graphs are qRT-PCR data, showing relative expression levels of TE and ICM markers, normalized against housekeeping gene Gapdh. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20094 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.05). No significant difference is observed for Gata3, Amotl2, and Pou5f1.', 'hash': '05cbb771a14571137eba64ee596c8ba3cc286245d192ad17d41788056a2ea547'}]
|
{'gr1_lrg': ['In the present study, to gain insights into the embryotoxic property of RDV, we examined the effects of the drug on the preimplantation development of mouse embryos. The key role of preimplantation development, which takes 4–5\xa0days after fertilization in the mouse, is to produce the blastocyst, a structure capable of implanting into the uterus [36], [43], [7]. The blastocyst consists of two distinct cell lineages: the trophectoderm (TE) and inner cell mass (ICM). Formation of these two lineages occurs after the 8-cell stage or embryonic day (E) 2.5 (\n<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>). Generally, cells situated on the surface of the embryo form the TE, whereas internally located cells give rise to the ICM. The TE creates the fluid-filled cavity inside the blastocyst, the expansion of which enables the blastocyst to hatch out of the egg coat and implant. After implantation, the TE gives rise to the trophoblast tissue of the placenta. During expansion of the blastocyst cavity, the ICM subdivides into the epiblast (EPI) and primitive endoderm (PrE). The EPI is a pluripotent precursor of the fetal body, and the PrE is an extraembryonic tissue that induces the body axis in the EPI. Thus, by E4.5, the blastocyst is produced that possesses a well-expanded cavity with three tissue types (TE, EPI, and PrE). The establishment of these tissues is controlled by the interplay of specific signaling pathways and transcription factors ). Generally, cells situated on the surface of the embryo form the TE, whereas internally located cells give rise to the ICM. The TE creates the fluid-filled cavity inside the blastocyst, the expansion of which enables the blastocyst to hatch out of the egg coat and implant. After implantation, the TE gives rise to the trophoblast tissue of the placenta. During expansion of the blastocyst cavity, the ICM subdivides into the epiblast (EPI) and primitive endoderm (PrE). The EPI is a pluripotent precursor of the fetal body, and the PrE is an extraembryonic tissue that induces the body axis in the EPI. Thus, by E4.5, the blastocyst is produced that possesses a well-expanded cavity with three tissue types (TE, EPI, and PrE). The establishment of these tissues is controlled by the interplay of specific signaling pathways and transcription factors [40], [47], [50], [53], and interference with these molecular machineries compromises further development or survival of the embryo.Fig. 1A schematic diagram, depicting the superovulation and mating schedule of the mice and the embryonic stages. Hours are indicated relative to the time of the hCG injection. See Materials and methods for more details. E: embryonic day, hCG: human chorionic gonadotropin, PMSG: pregnant mare serum gonadotropin.Fig. 1', 'To maximize the number of embryos for experiments, superovulation was performed, according to the previously described method [5]. Briefly, females were intraperitoneally injected with 5 IU pregnant mare serum gonadotropin (Millipore) and 48\u2009h later with 5 IU human chorionic gonadotropin (hCG; Millipore) (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>). Each female was placed in a cage overnight with a male, and the next day was examined for the presence of a copulation plug to verify successful mating. Superovulation is compatible with normal embryo development, and has been widely performed in various experimental studies to obtain many preimplantation embryos ). Each female was placed in a cage overnight with a male, and the next day was examined for the presence of a copulation plug to verify successful mating. Superovulation is compatible with normal embryo development, and has been widely performed in various experimental studies to obtain many preimplantation embryos [5]. For B6D2F1 mice, nearly 100% of embryos obtained from superovulated females can give rise to blastocysts in vitro, many of which are capable of implantation and full-term development after transfer to the uteri of surrogate females [2], [32]. The use of superovulation to maximize the yield of embryos per female is in line with the 3\u2009R principle (replacement, reduction, and refinement) of ethical animal research [24], [33].', 'At about 45\u2009h after the hCG injection, female mice were euthanized by cervical dislocation, and the oviducts were removed. Two-cell stage embryos, which correspond to embryonic day 1.5 (E1.5) (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>), were flushed out from the isolated oviducts with the FHM medium (Millipore). For each experiment, embryos obtained from three to four females were grouped together as a batch. Embryos were cultured in 20\u2009μL drops of KSOM with overlying mineral oil in an incubator (37\u2009°C with 5% CO), were flushed out from the isolated oviducts with the FHM medium (Millipore). For each experiment, embryos obtained from three to four females were grouped together as a batch. Embryos were cultured in 20\u2009μL drops of KSOM with overlying mineral oil in an incubator (37\u2009°C with 5% CO2 humidified air). Embryo culture was conducted in the atmospheric oxygen level (20%), which is commonly practiced and allows nearly 100% of control embryos from the B6D2F1 crossing to develop to the 8-cell stage (E2.5; 69\u2009h after the hCG injection), the early blastocyst stage (E3.5; 93\u2009h after the hCG injection), and the late blastocyst stage (E4.5; 117\u2009h after the hCG injection) with a well-expanded cavity (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>).).'], 'gr2_lrg': ['When embryos were treated with RDV (2, 4, and 8\u2009µM) from E2.5 (8-cell stage), all treatment groups had formed a blastocyst cavity by E3.5 (\n<xref rid="gr2_lrg" ref-type="fig">Fig. 2A</xref>). Their sizes and gross morphology were indistinguishable from the control embryos (0\u2009µM; vehicle only) (). Their sizes and gross morphology were indistinguishable from the control embryos (0\u2009µM; vehicle only) (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B). By E4.5, embryos exposed to 2\u2009µM RDV further expanded the cavity similarly to the control, and those exposed to 4\u2009µM RDV also expanded but less robustly than the control (B). By E4.5, embryos exposed to 2\u2009µM RDV further expanded the cavity similarly to the control, and those exposed to 4\u2009µM RDV also expanded but less robustly than the control (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B). By contrast, embryos exposed to 8\u2009µM RDV had collapsed their cavity (B). By contrast, embryos exposed to 8\u2009µM RDV had collapsed their cavity (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>A), resulting in a significantly decreased size (A), resulting in a significantly decreased size (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B). These results suggest that RDV treatment does not affect the initial blastocyst formation up to E3.5, but impairs the expansion (at 4\u2009µM) or the maintenance (at 8\u2009µM) of the cavity by E4.5.B). These results suggest that RDV treatment does not affect the initial blastocyst formation up to E3.5, but impairs the expansion (at 4\u2009µM) or the maintenance (at 8\u2009µM) of the cavity by E4.5.Fig. 2Adverse effects of remdesivir (RDV) on the expansion and maintenance of the blastocyst cavity. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5. A. Representative images of RDV-treated embryos at E2.5 (before treatment), E3.5, and E4.5. Scale bar =\u2009100\u2009µm. B. The size of RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200927 or 28 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01). No significant difference is observed among treatments at E3.5.Fig. 2'], 'gr3_lrg': ['The decrease in size upon RDV exposure implicates the possibility that the blastocysts are undergoing cell death. To test this possibility, RDV-treated E4.5 embryos were stained for live and dead cells. Embryos exposed to the lower concentrations (2 and 4\u2009μM) had some patches of dead cells (\n<xref rid="gr3_lrg" ref-type="fig">Fig. 3A</xref>), but the areas occupied by them were not significantly larger than the control (), but the areas occupied by them were not significantly larger than the control (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>B). By contrast, treatment with 8\u2009μM RDV caused a dramatic increase in areas containing dead cells (B). By contrast, treatment with 8\u2009μM RDV caused a dramatic increase in areas containing dead cells (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>A,B). This suggests that exposure to the high concentration of RDV leads to cell death, causing a collapse of the blastocyst cavity by E4.5.A,B). This suggests that exposure to the high concentration of RDV leads to cell death, causing a collapse of the blastocyst cavity by E4.5.Fig. 3Impact of remdesivir (RDV) on cell survival. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5. A. Representative images of RDV-treated embryos at E4.5 that are stained for live (green) and dead (red) cells by the LIVE/DEAD Cell Viability Assay. Binary images (see Materials and methods) are also shown for dead cells. Scale bar =\u2009100\u2009µm. B. The areas occupied by dead cells in RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200924 or 25 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01).Fig. 3'], 'gr4_lrg': ['By contrast, embryos treated with GS-441524 (4 or 8\u2009µM) from E2.5 developed into expanded blastocysts by E4.5 in a manner comparable to the control in terms of size and gross morphology (\n<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>). Thus, in spite of the similarity to RDV in chemical structure (). Thus, in spite of the similarity to RDV in chemical structure (Supplemental Fig. 1), GS-441524 did not impair the blastocyst cavity formation or maintenance at the concentrations equivalent to those tested for RDV.Fig. 4Effects of GS-441524, a major metabolite of remdesivir, on the expansion and maintenance of the blastocyst cavity. Embryos are treated with indicated concentrations of GS-441524 from E2.5 to E4.5. A. Representative images of GS-441524-treated embryos at E3.5 and E4.5. Scale bar =\u2009100\u2009µm. B. The size of GS-441524-treated embryos. No significant difference is found at E3.5 or E4.5 among treatments (mean ±\u200995% CI, n\u2009=\u200930–32\u2009for each treatment group).Fig. 4'], 'gr5_lrg': ['To evaluate the state of RDV-exposed blastocysts at the molecular level, the gene expression profiles of key cell lineage markers were examined at E4.5. The transcript levels of TE markers Cdx2 and Eomes were unchanged, while Gata3 was increased by about 20% by RDV exposure (\n<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>). By contrast, the expressions of the epiblast (EPI) markers were mostly reduced by RDV exposure. Specifically, the transcript levels of ). By contrast, the expressions of the epiblast (EPI) markers were mostly reduced by RDV exposure. Specifically, the transcript levels of Sox2 and Esrrb were progressively decreased in a concentration-dependent manner, with about 55% reduction at 4\u2009μM. Pou5f1 expression also appeared to be diminished progressively, although not statistically significant. Nanog expression was significantly decreased by RDV at 2\u2009μM, but not at 4\u2009μM. The expressions of the primitive endoderm (PrE) markers, Gata4, Pdgfra, and Sox17, were all progressively down-regulated by RDV exposure, with 75–80% reduction at 4\u2009μM. These results suggest that RDV mainly diminishes the gene expressions associated with the ICM lineage (i.e., EPI and PrE) but not the TE lineage.Fig. 5Impact of remdesivir (RDV) and GS-441524 on the expression levels of the marker genes for the trophectoderm (TE), the epiblast (EPI), and the primitive endoderm (PrE) at E4.5. Embryos are treated from E2.5 to E4.5 with vehicle control (C), RDV at 2\u2009μM (R2), RDV at 4\u2009μM (R4), or GS-441524 at 4\u2009μM (G4). Graphs are qRT-PCR data, showing relative expression levels of each lineage marker, normalized against housekeeping gene Gapdh. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20093 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.05). No significant difference is observed for Cdx2 and Pou5f1.Fig. 5', 'For comparison, the gene expression profiles were also examined for the blastocysts that were exposed to GS-441524 at 4\u2009μM. GS-441524 altered the expressions of some of the cell lineage markers, although the patterns of alterations differed from RDV (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>). Most notably, GS-441524 significantly increased the levels of the EPI markers, ). Most notably, GS-441524 significantly increased the levels of the EPI markers, Sox2, Nanog, and Esrrb, which was opposite to the effects of RDV. Thus, the impacts of RDV and GS-441524 on preimplantation embryos were different from each other at the morphological and molecular levels.'], 'gr6_lrg': ['The reduction in the EPI and PrE marker transcripts by RDV exposure was accompanied by a decrease in the cell number of these tissues. Immunofluorescence staining showed that the numbers of nuclei positive for EPI marker proteins (SOX2 and NANOG) and those positive for PrE marker proteins (SOX17 and GATA4) were significantly lower in RDV-treated E4.5 blastocysts (\n<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>). Thus, in spite of the normal appearance in the gross morphology, the formation of EPI and PrE was diminished in blastocysts treated with RDV even at 2\u2009μM.). Thus, in spite of the normal appearance in the gross morphology, the formation of EPI and PrE was diminished in blastocysts treated with RDV even at 2\u2009μM.Fig. 6Impact of remdesivir (RDV) on the epiblast (EPI) and the primitive endoderm (PrE) cells. Embryos are treated with indicated concentrations of RDV from E2.5 to E4.5, and immunohistochemically examined for SOX2 (EPI marker), NANOG (EPI marker), SOX17 (PrE marker), and GATA4 (PrE marker). A. Representative images of embryos that are stained for the nuclei (DAPI), SOX2 (green) and SOX17 (red). B. Representative images of embryos stained for the nuclei (DAPI), NANOG (green) and GATA4 (red). Scale bars =\u200950\u2009µm. C. Numbers of nuclei that are positive for the EPI marker and PrE marker per embryo. Red lines indicate mean values (n\u2009=\u200919 or 20 for each treatment group). Statistically significant differences are marked with horizontal bars and p-values (t-test) between two groups.Fig. 6'], 'gr7_lrg': ['To determine whether preimplantation development is more severely impaired when RDV exposure starts before the 8-cell stage, embryos were treated from E1.5 (2-cell stage). All treatment groups (0, 2, 4, and 8\u2009μM) progressed to the 8-cell stage by E2.5, and developed into blastocysts by E3.5 (\n<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>A). By E4.5, embryos treated with 2\u2009μM RDV further expanded the cavity in a manner comparable to the control (0\u2009μM), whereas those treated with 4\u2009μM expanded less robustly (A). By E4.5, embryos treated with 2\u2009μM RDV further expanded the cavity in a manner comparable to the control (0\u2009μM), whereas those treated with 4\u2009μM expanded less robustly (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>B). The cavity collapsed in most of the blastocysts exposed to 8\u2009μM RDV. Overall, the concentration-effect relationship was essentially indistinguishable from the RDV treatment started from E2.5 (B). The cavity collapsed in most of the blastocysts exposed to 8\u2009μM RDV. Overall, the concentration-effect relationship was essentially indistinguishable from the RDV treatment started from E2.5 (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>), indicating that earlier treatment from E1.5 did not significantly exacerbate the effects of RDV on the cavity maintenance. This suggests that embryos are impaired by RDV when exposure occurs after the 8-cell stage.), indicating that earlier treatment from E1.5 did not significantly exacerbate the effects of RDV on the cavity maintenance. This suggests that embryos are impaired by RDV when exposure occurs after the 8-cell stage.Fig. 7Impact of early exposure to remdesivir (RDV) on the expansion and maintenance of the blastocyst cavity. Embryos are treated with indicated concentrations of RDV from E1.5 to E4.5. A. Representative images of RDV-treated embryos at E2.5, E3.5, and E4.5. Scale bar =\u2009100\u2009µm. B. The size of RDV-treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200936–40\u2009for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01). No significant difference is observed among treatments at E3.5.Fig. 7'], 'gr8_lrg': ['To further examine whether the effects of RDV depend on the duration and timing of exposure, embryos were treated with RDV (8\u2009μM) at different intervals between E2.5 and E4.5, as depicted in\n<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>A. We observed that CON-RDV embryos formed expanded blastocysts that were slightly smaller than CON embryos (A. We observed that CON-RDV embryos formed expanded blastocysts that were slightly smaller than CON embryos (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>B,C). By contrast, RDV-CON embryos formed blastocysts that were much smaller in size relative to CON and CON-RDV blastocysts. However, RDV-CON embryos did not collapse the cavity, unlike RDV embryos (B,C). By contrast, RDV-CON embryos formed blastocysts that were much smaller in size relative to CON and CON-RDV blastocysts. However, RDV-CON embryos did not collapse the cavity, unlike RDV embryos (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>B,C). Thus, the blastocyst cavity expansion was more sensitively affected by the early exposure to RDV (E2.5-E3.5) than the late exposure (E3.5-E4.5), although it was most impaired with continuous exposure (E2.5-E4.5).B,C). Thus, the blastocyst cavity expansion was more sensitively affected by the early exposure to RDV (E2.5-E3.5) than the late exposure (E3.5-E4.5), although it was most impaired with continuous exposure (E2.5-E4.5).Fig. 8Duration- and timing-dependent impact of remdesivir (RDV) exposure on the blastocyst morphology and the gene expression profiles. A. A diagram depicting the RDV treatment regimen. B. Representative images of treated embryos at E4.5. Scale bar =\u2009100\u2009µm. C. The size of treated embryos. Different letters indicate statistically significant differences among treatments (mean ±\u200995% CI, n\u2009=\u200930–54\u2009for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01). D. The expression levels of the markers for the trophectoderm (TE), the epiblast (EPI), and the primitive endoderm (PrE) at E4.5. Graphs are qRT-PCR data, showing relative expression levels of each lineage marker, normalized against housekeeping gene Gapdh. C, C-R, and R-C correspond to the treatment groups CON, CON-RDV, and RDV-CON, depicted in A, respectively. The RDV group is not analyzed due to extensive cell death. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20093 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.05). No significant difference is observed for Cdx2, Pou5f1, and Nanog.Fig. 8', 'We then compared the expression profiles of cell lineage markers among the E4.5 blastocysts of CON, CON-RDV, and RDV-CON groups. In CON-RDV blastocysts, the effect of RDV was mild such that only 1 out of the 10 genes examined was altered, namely Eomes (TE; decreased) (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>D). By contrast, in RDV-CON blastocysts, the impact was much more dramatic, as expressions of 6 out of the 10 genes were altered, namely D). By contrast, in RDV-CON blastocysts, the impact was much more dramatic, as expressions of 6 out of the 10 genes were altered, namely Gata3 (TE; increased), Sox2 and Esrrb (EPI; decreased), and Gata4, Pdgfra, and Sox17 (PrE; decreased) (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>D). Notably, all of the PrE markers were reduced by greater than 80%. Altogether, these results suggest that RDV impairs the blastocyst morphology (cavity expansion) and gene expressions (the ICM lineage) most severely when exposure occurs between E2.5 and E3.5.D). Notably, all of the PrE markers were reduced by greater than 80%. Altogether, these results suggest that RDV impairs the blastocyst morphology (cavity expansion) and gene expressions (the ICM lineage) most severely when exposure occurs between E2.5 and E3.5.', 'Although exposure during E2.5 to E3.5 resulted in the most severe outcome by E4.5 (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>), RDV-exposed embryos appeared morphologically normal at E3.5 (), RDV-exposed embryos appeared morphologically normal at E3.5 (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>). To assess whether adverse effects already exist at E3.5 at the molecular levels, early lineage markers for ICM and TE were compared between control and RDV-exposed embryos. For TE markers, the expression of ). To assess whether adverse effects already exist at E3.5 at the molecular levels, early lineage markers for ICM and TE were compared between control and RDV-exposed embryos. For TE markers, the expression of Cdx2 was up-regulated by RDV (4 and 8\u2009μM), whereas Gata3 and Amotl2 were unaffected (\n<xref rid="gr9_lrg" ref-type="fig">Fig. 9</xref>). For ICM markers, the expression of ). For ICM markers, the expression of Nanog was down-regulated by RDV (8\u2009μM), while Pou5f1 and Sox2 were not significantly altered compared to the control (<xref rid="gr9_lrg" ref-type="fig">Fig. 9</xref>). By comparison, exposure to GS-441524 (8\u2009μM) led to increases in both ). By comparison, exposure to GS-441524 (8\u2009μM) led to increases in both Cdx2 and Nanog.Fig. 9Impact of remdesivir (RDV) on the trophectoderm (TE) and the inner cell mass (ICM) at E3.5. Embryos are treated from E2.5 to E3.5 with vehicle control (C), RDV at 4\u2009μM (R4), RDV at 8\u2009μM (R8), or GS-441524 at 8\u2009μM (G8). Graphs are qRT-PCR data, showing relative expression levels of TE and ICM markers, normalized against housekeeping gene Gapdh. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20094 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.05). No significant difference is observed for Gata3, Amotl2, and Pou5f1.Fig. 9'], 'gr10_lrg': ['The total number of cells per embryo was progressively reduced by RDV treatment in a concentration-dependent manner (\n<xref rid="gr10_lrg" ref-type="fig">Fig. 10</xref>A,B). The numbers of CDX2-positive (TE) and SOX2-positive (ICM) nuclei were also lower in RDV-treated blastocysts. However, when the ratio of SOX2-positive to CDX2-positive cell numbers was analyzed for individual embryos (SOX2/CDX2 ratio), the mean ratio decreased in RDV-treated blastocysts in a dose-dependent manner (A,B). The numbers of CDX2-positive (TE) and SOX2-positive (ICM) nuclei were also lower in RDV-treated blastocysts. However, when the ratio of SOX2-positive to CDX2-positive cell numbers was analyzed for individual embryos (SOX2/CDX2 ratio), the mean ratio decreased in RDV-treated blastocysts in a dose-dependent manner (<xref rid="gr10_lrg" ref-type="fig">Fig. 10</xref>C). This suggests that RDV diminishes the overall cell proliferation during E2.5 to E3.5, but more severely for the ICM than TE lineage.C). This suggests that RDV diminishes the overall cell proliferation during E2.5 to E3.5, but more severely for the ICM than TE lineage.Fig. 10Effects of remdesivir (RDV) on the trophectoderm (TE) and the inner cell mass (ICM) at E3.5. Embryos are treated with RDV from E2.5 to E3.5, and immunohistochemically examined for CDX2 and SOX2 proteins, which are markers for TE and ICM, respectively. A. Representative images of RDV-treated embryos that are stained for the nuclei (DAPI), CDX2 (green), and SOX2 (red). Scale bar =\u200950\u2009µm. B. Numbers of total nuclei, and those of CDX2-positive and SOX2-positive nuclei. Red lines indicate mean values (n\u2009=\u200919 or 20 for each treatment group). Statistically significant differences are marked with horizontal bars and p-values (t-test) between two groups. C. Distribution of SOX2-positive and CDX2-positive nuclear numbers for individual embryos. Linear trend lines representing the means of the SOX2/CDX2 ratios are superimposed for each treatment group (n\u2009=\u200919 or 20).Fig. 10'], 'gr11_lrg': ['The results above on mouse embryos raised the possibility that RDV may also impair human preimplantation development. However, testing the effects of RDV on actual human embryos is practically and ethically challenging. Here, to gain insights into the potential impact of RDV on human embryos, we used human embryonic stem cells (hESCs) as a model for the human ICM lineage. RDV exposure (0.5–8\u2009μM) significantly decreased the viability of hESCs in a concentration-dependent manner (\n<xref rid="gr11_lrg" ref-type="fig">Fig. 11</xref>). Namely, 0.5\u2009μM RDV decreased the cell number to about 70% of the control level, whereas 4 and 8\u2009μM RDV reduced it down to less than 10% (). Namely, 0.5\u2009μM RDV decreased the cell number to about 70% of the control level, whereas 4 and 8\u2009μM RDV reduced it down to less than 10% (<xref rid="gr11_lrg" ref-type="fig">Fig. 11</xref>). On the other hand, GS-441524 had no effect on cell viability at 0.5–4\u2009µM, whereas cell number decreased to 80% of the control level at 8\u2009µM (). On the other hand, GS-441524 had no effect on cell viability at 0.5–4\u2009µM, whereas cell number decreased to 80% of the control level at 8\u2009µM (<xref rid="gr11_lrg" ref-type="fig">Fig. 11</xref>). These results suggest that RDV, but not GS-441524, severely impairs the proliferation and/or survival of human cells of the ICM lineage at the concentrations found in the plasma.). These results suggest that RDV, but not GS-441524, severely impairs the proliferation and/or survival of human cells of the ICM lineage at the concentrations found in the plasma.Fig. 11Impact of remdesivir and GS-441524 on the proliferation and viability of human embryonic stem cells. Graphs show the relative light unit of the CellTiter-Glo Luminescent Assay as a proxy for the relative number of metabolically active cells after 2 days of treatment. Different letters indicate statistically significant differences among treatments (mean ±\u2009standard deviation, n\u2009=\u20093 for each treatment group, one-way ANOVA followed by t-test, p\u2009<\u20090.01).Fig. 11']}
|
Remdesivir impairs mouse preimplantation embryo development at therapeutic concentrations
|
[
"Antiviral drug",
"Blastocyst",
"COVID-19",
"Fertility",
"GS-441524",
"Preimplantation development",
"Remdesivir",
"Reproductive risk"
] |
Reprod Toxicol
|
1661065200
|
We initiated a randomized, placebo-controlled, phase 1/2 trial to evaluate the safety and immunogenicity of the S-268019-b recombinant protein vaccine, scheduled as 2 intramuscular injections given 21 days apart, in 60 randomized healthy Japanese adults. We evaluated 2 regimens of the S-910823 antigen (5 μg [n = 24] and 10 μg [n = 24]) with an oil-in-water emulsion formulation and compared against placebo (n = 12). Reactogenicity was mild in most participants. No serious adverse events were noted. For both regimens, vaccination resulted in robust IgG and neutralizing antibody production at days 36 and 50 and predominant T-helper 1-mediated immune reaction, as evident through antigen-specific polyfunctional CD4+ T-cell responses with IFN-γ, IL-2, and IL-4 production on spike protein peptides stimulation. Based on the interim analysis, the S-268019-b vaccine is safe, produces neutralizing antibodies titer comparable with that in convalescent serum from COVID-19-recovered patients. However, further evaluation of the vaccine in a large clinical trial is warranted.
|
[
"Adult",
"Antibodies, Neutralizing",
"Antibodies, Viral",
"COVID-19",
"COVID-19 Vaccines",
"Double-Blind Method",
"Humans",
"Immunization, Passive",
"Immunogenicity, Vaccine",
"Japan",
"SARS-CoV-2",
"Vaccines, Synthetic",
"COVID-19 Serotherapy"
] |
other
|
PMC9122741
| null |
18
|
[
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"{'Citation': 'WMA Declaration of Helsinki – Ethical principles for medical research involving human subjects. Available from: https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/ Last accessed: October 9, 2021.'}",
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"{'Citation': 'Maghodia A.B., Geisler C., Jarvis D.L. Characterization of an Sf-rhabdovirus-negative S. frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system. Protein Expr Purif. 2016;122:45–55. doi: 10.1016/j.pep.2016.02.014.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.pep.2016.02.014'}, {'@IdType': 'pmc', '#text': 'PMC4842140'}, {'@IdType': 'pubmed', '#text': '26923062'}]}}",
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] |
Reprod Toxicol. 2022 Aug 21; 111:135-147
|
NO-CC CODE
|
|
Four different constructs of SARS-CoV-2 spike protein. (A) Schematic of four different constructs of SARS-CoV-2 spike protein. S, full-length spike protein; IgkS, spike protein in which the signal peptide was substituted by Igk; Stc, spike protein with C-terminal 19 amino acids deletion; IgkStc, the spike protein with signal peptide replacement by Igk and C-terminal 19 amino acids deletion. (B) Immunofluorescence analysis of the four different constructs of SARS-CoV-2 spike protein expression at 48 h post transfection of HEK293 cells by confocal microscopy. Control, cells transfected with the empty vector pCMV.
|
gr1_lrg
|
2
|
0e15316568a7000b115996628dcec87d2e117c048039b1631f2fb24ae505e9cb
|
gr1_lrg.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
716,
515
] |
[{'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC8233049/gr4_lrg.jpg', 'caption': 'Detection of neutralizing ability of recombinant ACE2 and human antibodies against SARS2pp infection. (A) SARS2pp showed a good linear relationship between MOI 2-0.0002. (B) ACE2-Fc was superior to ACE2-his for SARS2pp infection inhibition (MOI 0.3). VSVpp containing the spike glycoprotein of the vesicular stomatitis virus (VSV-G) as the control pseudotype particles. (C) and (D) Two SARS-CoV-2 neutralizing antibodies (REGN10933 and REGN10987) re-evaluated against SARS2pp and its South Africa variant SARS2pp(B.1.351) (MOI 0.3). Data were the results from three replicates.', 'hash': 'f3d839897571bc0884cb61e07c11b0faa71b942f72d5d6c463b56e831fb6ffea'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC8233049/gr5_lrg.jpg', 'caption': 'Different cell lines showed different susceptibility to SARSpp and SARS2pp (MOI 2). (A) Fluorescence detection of SARSpp and SARS2pp infectivity to kidney, liver, and small intestine-derived cell lines. (B) Bioluminescence detection of SARSpp and SARS2pp infectivity to kidney, liver, and small intestine-derived cell lines. The luciferase activity above three times of signals obtained from the unsusceptible BHK21 cells was regarded as positive (the dashed line). Data were the results from three replicates.', 'hash': '963bc2468db849d2e4d4751c1b8213bd183ff528cd8d884d8afef1f4487e3ef0'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC8233049/gr1_lrg.jpg', 'caption': 'Four different constructs of SARS-CoV-2 spike protein. (A) Schematic of four different constructs of SARS-CoV-2 spike protein. S, full-length spike protein; IgkS, spike protein in which the signal peptide was substituted by Igk; Stc, spike protein with C-terminal 19 amino acids deletion; IgkStc, the spike protein with signal peptide replacement by Igk and C-terminal 19 amino acids deletion. (B) Immunofluorescence analysis of the four different constructs of SARS-CoV-2 spike protein expression at 48 h post transfection of HEK293 cells by confocal microscopy. Control, cells transfected with the empty vector pCMV.', 'hash': '0e15316568a7000b115996628dcec87d2e117c048039b1631f2fb24ae505e9cb'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC8233049/gr3_lrg.jpg', 'caption': 'Three factors to increase SARS2pp titer. (A) and (B) Fluorescence and bioluminescence detection of SARS2pp infectivity (293/hACE2 cells) packaged by SARS-CoV-2 spike protein expressed by different promoters (CAG and CMV). (C) and (D) Two optimized spike protein sequences with SARS2pp production improved by approximately 2.5 times. (E) and (F) 1 % BSA addition to the medium enhanced SARS2pp production about 2-3 times. Control, non-transduced cells. Data were the results from three replicates.', 'hash': '33042d85832692c6da3fd212257355f698f7858f4add3e1d5b5450868d61b098'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC8233049/gr2_lrg.jpg', 'caption': 'Dual-reporter SARS2pp production and infectivity. (A) Specification of the HIV dual-reporter plasmid for SARS2pp infection indication. eGFP-T2A-luc2, dual-reporter genes eGFP and luc2 connected by the T2A peptide. (B) and (C) Fluorescence and bioluminescence detection of SARS2pp infectivity (293/hACE2 cells) packaged by different constructs of SARS-CoV-2 spike proteins. The luciferase activities were normalized against "S" (set as 1). Control, non-transduced cells. Data were the results from three replicates.', 'hash': '59576d12cc0ed44580ff60162e117a63abd7632cea74df02091b7ae6ed671215'}]
|
{'gr1_lrg': ['For successful SARS2pp production, the following four different constructs of the SARS-CoV-2 spike protein plasmids were designed and synthesized: 1) full-length spike protein (S), 2) spike protein (IgkS) with the signal peptide substituted by the leader sequence from mouse immunoglobulin κ light chain Igk, 3) spike protein (Stc) with a C-terminal 19 amino acids deletion, and 4) spike protein (IgkStc) with signal peptide substitution and C-terminal 19 amino acids deletion (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>\nA). After transfection into HEK293 cells, the expression of these four different constructs of spike protein was detected by immunofluorescence analysis. All these four constructs were expressed on the cell membrane and showed cell fusion (\nA). After transfection into HEK293 cells, the expression of these four different constructs of spike protein was detected by immunofluorescence analysis. All these four constructs were expressed on the cell membrane and showed cell fusion (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B).B).Fig. 1Four different constructs of SARS-CoV-2 spike protein. (A) Schematic of four different constructs of SARS-CoV-2 spike protein. S, full-length spike protein; IgkS, spike protein in which the signal peptide was substituted by Igk; Stc, spike protein with C-terminal 19 amino acids deletion; IgkStc, the spike protein with signal peptide replacement by Igk and C-terminal 19 amino acids deletion. (B) Immunofluorescence analysis of the four different constructs of SARS-CoV-2 spike protein expression at 48 h post transfection of HEK293 cells by confocal microscopy. Control, cells transfected with the empty vector pCMV.Fig. 1'], 'gr2_lrg': ['A dual-reporter vector bearing eGFP and Luc2 was designed for simplified observation and analysis (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\nA). SARS2pps packaged with the four constructs of SARS-CoV-2 spike protein were produced in HEK293 T cells, and its infectivity was determined in 293/hACE2 cells. SARS2pps packaged with S and \nA). SARS2pps packaged with the four constructs of SARS-CoV-2 spike protein were produced in HEK293 T cells, and its infectivity was determined in 293/hACE2 cells. SARS2pps packaged with S and IgkS showed lower eGFP expression and luciferase activity with that packaged with Stc and IgkStc (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B, C), suggesting that the spike C-terminal 19 amino acids mainly restrained the SARS2pp production. Compared with that packaged with Stc, SARS2pp packaged by B, C), suggesting that the spike C-terminal 19 amino acids mainly restrained the SARS2pp production. Compared with that packaged with Stc, SARS2pp packaged by IgkStc showed approximately twofold increase in eGFP expression and luciferase activity (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B, C), indicating that the signal peptide replacement by B, C), indicating that the signal peptide replacement by Igk could further facilitate SARS2pp production.Fig. 2Dual-reporter SARS2pp production and infectivity. (A) Specification of the HIV dual-reporter plasmid for SARS2pp infection indication. eGFP-T2A-luc2, dual-reporter genes eGFP and luc2 connected by the T2A peptide. (B) and (C) Fluorescence and bioluminescence detection of SARS2pp infectivity (293/hACE2 cells) packaged by different constructs of SARS-CoV-2 spike proteins. The luciferase activities were normalized against "S" (set as 1). Control, non-transduced cells. Data were the results from three replicates.Fig. 2'], 'gr3_lrg': ['Several factors including the promoters for spike protein expression, different spike optimized sequences, and the production conditions were optimized to obtain high SARS2pp titers. First, SARS2pp titer was increased up to 10 times when the CMV promoter other than CAG was used for spike protein expression (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\nA). Second, the SARS2pp titer was improved by approximately 2–3 times through comparing different spike optimized sequences (\nA). Second, the SARS2pp titer was improved by approximately 2–3 times through comparing different spike optimized sequences (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>B). Finally, 1 % BSA addition to the medium benefitted SARS2pp production and improved the titer by 2–3 times (B). Finally, 1 % BSA addition to the medium benefitted SARS2pp production and improved the titer by 2–3 times (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C). These three optimized conditions were adopted in the subsequent studies.C). These three optimized conditions were adopted in the subsequent studies.Fig. 3Three factors to increase SARS2pp titer. (A) and (B) Fluorescence and bioluminescence detection of SARS2pp infectivity (293/hACE2 cells) packaged by SARS-CoV-2 spike protein expressed by different promoters (CAG and CMV). (C) and (D) Two optimized spike protein sequences with SARS2pp production improved by approximately 2.5 times. (E) and (F) 1 % BSA addition to the medium enhanced SARS2pp production about 2-3 times. Control, non-transduced cells. Data were the results from three replicates.Fig. 3'], 'gr4_lrg': ['Infection efficiency was firstly detected at different MOIs in 293/hACE2 cells to examine the applicability of this SARS2pp system, and a good linear relationship was observed between MOI 2-0.0002 (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\nA). Inhibition assay was then performed to test the activity of recombinant ACE2-Fc and ACE2-his proteins (fusion proteins consisting of the extracellular domain (Met 1-Ser 740) of human ACE2 linked to the Fc region of human IgG1 or his tag at the C-terminus) against SARS2pp infection. The results showed that SARS2pp but not VSVpp was inhibited specifically by both recombinant proteins, and the action of ACE2-Fc was superior to that of ACE2-his (\nA). Inhibition assay was then performed to test the activity of recombinant ACE2-Fc and ACE2-his proteins (fusion proteins consisting of the extracellular domain (Met 1-Ser 740) of human ACE2 linked to the Fc region of human IgG1 or his tag at the C-terminus) against SARS2pp infection. The results showed that SARS2pp but not VSVpp was inhibited specifically by both recombinant proteins, and the action of ACE2-Fc was superior to that of ACE2-his (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>B). Two human SARS-CoV-2 neutralizing antibodies (REGN10933 and REGN10987) (B). Two human SARS-CoV-2 neutralizing antibodies (REGN10933 and REGN10987) (Baum et al., 2020) were also re-evaluated against SARS2pp and the Beta variant SARS2pp(B.1.351). REGN10987 maintained about 25 % of its neutralization activity against SARS2pp(B.1.351), but REGN10933 had lost its neutralization activity against SARS2pp(B.1.351) at least 1.5 log units relative to that of SARS2pp. When REGN10933 and REGN10987 were combined into a cocktail (1:1), these two antibodies remained effective against SARS2pp and SARS2pp(B.1.351) (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>C, D).C, D).Fig. 4Detection of neutralizing ability of recombinant ACE2 and human antibodies against SARS2pp infection. (A) SARS2pp showed a good linear relationship between MOI 2-0.0002. (B) ACE2-Fc was superior to ACE2-his for SARS2pp infection inhibition (MOI 0.3). VSVpp containing the spike glycoprotein of the vesicular stomatitis virus (VSV-G) as the control pseudotype particles. (C) and (D) Two SARS-CoV-2 neutralizing antibodies (REGN10933 and REGN10987) re-evaluated against SARS2pp and its South Africa variant SARS2pp(B.1.351) (MOI 0.3). Data were the results from three replicates.Fig. 4'], 'gr5_lrg': ['A panel of SARS2pp susceptible cell lines derived from kidney, liver, and small intestine were selected to compare their susceptibility to SARSpp. Baby Syrian hamster kidney cell line BHK21 served as the unsusceptible control. In accordance with SARS-CoV and SARS-CoV-2 causing multi-organ damage, all the tested cell lines showed susceptibility to SARSpp and SARS2pp but to a different degree, except BHK21. For the kidney-derived cell lines 293 T and Vero-E6, SARSpp showed higher transduction levels than SARS2pp. For liver- and small intestine-derived HuH7, HuH7.5.1, and Caco-2 cell lines, SARS2pp was more infectious than SARSpp (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>\n).\n).Fig. 5Different cell lines showed different susceptibility to SARSpp and SARS2pp (MOI 2). (A) Fluorescence detection of SARSpp and SARS2pp infectivity to kidney, liver, and small intestine-derived cell lines. (B) Bioluminescence detection of SARSpp and SARS2pp infectivity to kidney, liver, and small intestine-derived cell lines. The luciferase activity above three times of signals obtained from the unsusceptible BHK21 cells was regarded as positive (the dashed line). Data were the results from three replicates.Fig. 5']}
|
An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research
| null |
J Virol Methods
|
1632553200
|
The presence of IgG and IgM antibodies in the venous blood of 76 patients with confirmed COVID-19 infection was determined by ELISA using Russian test systems. Different levels of IgM antibodies to N-protein and receptor binding domain of the Spike protein (RBD) were revealed. The dynamics of IgG antibodies to the whole virion antigen and recombinant antigens showed high values on weeks 4-5 of the disease. The level of IgG antibodies to Nprotein remained low throughout the observation period. The characteristic dynamics of IgG measured using test systems with sorbed whole virion or recombinant spike proteins reflects the duration of the disease.
|
[
"Antibodies, Viral",
"Antigens, Viral",
"COVID-19",
"Coronavirus Nucleocapsid Proteins",
"Enzyme-Linked Immunosorbent Assay",
"Humans",
"Immunity, Humoral",
"Immunoglobulin G",
"Immunoglobulin M",
"SARS-CoV-2",
"Spike Glycoprotein, Coronavirus",
"Time Factors",
"Virion"
] |
other
|
PMC8233049
| null |
7
|
[
"{'Citation': 'Amanat F, Krammer F. SARS-CoV-2 vaccines: status report. Immunity. 2020;52(4):583–589. doi: 10.1016/j.immuni.2020.03.007.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.immuni.2020.03.007'}, {'@IdType': 'pmc', '#text': 'PMC7136867'}, {'@IdType': 'pubmed', '#text': '32259480'}]}}",
"{'Citation': 'Bundschuh C, Egger M, Wiesinger K, Gabriel C, Clodi M, Mueller T, Dieplinger B. Evaluation of the EDI enzyme linked immunosorbent assays for the detection of SARS-CoV-2 IgM and IgG antibodies in human plasma. Clin. Chim. Acta. 2020;509:79–82. doi: 10.1016/j.cca.2020.05.047.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1016/j.cca.2020.05.047'}, {'@IdType': 'pmc', '#text': 'PMC7278646'}, {'@IdType': 'pubmed', '#text': '32526218'}]}}",
"{'Citation': 'Salvatori G, Luberto L, Maffei M, Aurisicchio L, Roscilli G, Palombo F, Marra E. SARS-CoV-2 Spike protein: an optimal immunological target for vaccines. J. Transl. Med. 2020;18(1):222. doi: 10.1186/s12967-020-02392-y.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1186/s12967-020-02392-y'}, {'@IdType': 'pmc', '#text': 'PMC7268185'}, {'@IdType': 'pubmed', '#text': '32493510'}]}}",
"{'Citation': 'Tai W, He L, Zhang X, Pu J, Voronin D, Jiang S, Zhou Y, Du L. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell. Mol. Immunol. 2020;17(6):613–620. doi: 10.1038/s41423-020-0400-4.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1038/s41423-020-0400-4'}, {'@IdType': 'pmc', '#text': 'PMC7091888'}, {'@IdType': 'pubmed', '#text': '32203189'}]}}",
"{'Citation': 'Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the SARSCoV-2 Spike glycoprotein. Cell. 2020;181(2):281-292.e6. doi: 10.1016/j.cell.2020.02.058', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC7102599'}, {'@IdType': 'pubmed', '#text': '32155444'}]}}",
"{'Citation': 'Xiang F, Wang X, He X, Peng Z, Yang B, Zhang J, Zhou Q, Ye H, Ma Y, Li H, Wei X, Cai P, Ma WL. Antibody detection and dynamic characteristics in Patients with COVID-19. Clin. Infect. Dis. 2020;71(8):1930–1934. doi: 10.1093/cid/ciaa461.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1093/cid/ciaa461'}, {'@IdType': 'pmc', '#text': 'PMC7188146'}, {'@IdType': 'pubmed', '#text': '32306047'}]}}",
"{'Citation': 'Zhao J, Yuan Q, Wang H, Liu W, Liao X, Su Y, Wang X, Yuan J, Li T, Li J, Qian S, Hong C, Wang F, Liu Y, Wang Z, He Q, Li Z, He B, Zhang T, Fu Y, Ge S, Liu L, Zhang J, Xia N, Zhang Z. Antibody response to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin. Infect. Dis. 2020;71(16):2027–2034. doi: 10.1093/cid/ciaa344.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'doi', '#text': '10.1093/cid/ciaa344'}, {'@IdType': 'pmc', '#text': 'PMC7184337'}, {'@IdType': 'pubmed', '#text': '32221519'}]}}"
] |
J Virol Methods. 2021 Sep 25; 295:114221
|
NO-CC CODE
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GR24 promotes D53 protein degradationa, Confocal scanning images showing that AEBSF, Pepstatin A and Leupetin are not effective in blocking D53-GFP fusion protein degradation in transgenic seedlings treated with 5 μM GR24. Scale bars, 100 μm. b, Degradation of D53-GFP fusion protein, but not D3-GFP and D14-GFP fusion proteins expressed in rice protoplasts, in the presence of 5 μM GR24. Pre-treatment with 40 μM MG132 for one hour before addition of GR24 effectively blocks D53-GFP degradation. c. D53-GFP is degraded in the d53 mutant protoplasts in the presence of GR24, but not in d3 or d14 protoplasts. For b and c, each figure represents at least fifty cells observed. Scale bars, 10 μm.
|
nihms541100f13
|
2
|
ee91799a23c20732a71e0dd467613f6e2e93aaf77b85c08d7ec9e95487f76b9d
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nihms541100f13.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
649,
950
] |
[{'image_id': 'nihms541100f12', 'image_file_name': 'nihms541100f12.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f12.jpg', 'caption': 'Mapping of the D14-binding domain of D53a, Schematic structure of the D53 protein. Numbers indicate amino-acid (aa) residues. b, Y2H analysis showing interaction between full-length and various domain deletion variants of D53 with D14 in the presence or absence of 5 μM GR24. –LT, control medium (SD–Leu/–Trp); –LTHA, selective medium (SD–Leu/–Trp/–His/–Ade).', 'hash': '936e34d073e9fd19b0aff60d7efe454f79d985abd84576dff9c0fedb8c97eae5'}, {'image_id': 'nihms541100f5', 'image_file_name': 'nihms541100f5.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f5.jpg', 'caption': 'A schematic model depicting SLs promote D14-SCFD3-mediated degradation of D53.', 'hash': 'af2d18c039c4623d3804cc903778db1744c4e714061962d9a8fe0ecc892f99ba'}, {'image_id': 'nihms541100f2', 'image_file_name': 'nihms541100f2.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f2.jpg', 'caption': 'Map-based cloning and characterization of D53a,\nD53 was fine-mapped on chromosome 11. The numbers of recombinants are shown in brackets. b, Molecular lesions in d53 mutant. c, Phenotypic comparison of proActin::D53-GFP and proActin::d53-GFP transgenic plants. Vector, proActin::GFP control. Scale bar, 10 cm. d, e, Tiller number (d) and relative expression of D53\n(e) of transgenic plants in c. f,\nD53 expression in various organs, including young panicles (YP), young roots (YR), shoots (S), leaf blades (LB), leaf sheaths (LS), culms (C) and nodes (N). g, GR24 treatment induces D53 expression. h, Relative expression levels of D53 in two wild-type varieties Norin 8 (N) and Shiokari (S), and six rice d mutants. Each value in d–h represents the mean ± s.d. (d and f–h, n = 3 replicates; e, n = 20 plants). The Student’s t-test analysis indicates a significant difference (compared with control, *P<0.05, **P<0.01).', 'hash': '13933adfe2f98e66d2ddcb8dd6d9c8e97d18598d96b75c23b73f05ae2c07e5dd'}, {'image_id': 'nihms541100f14', 'image_file_name': 'nihms541100f14.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f14.jpg', 'caption': 'D53-RNAi transgenic lines in d3 and d14 backgroundsa, Comparison of plant height, diameter of the 3rd internode and tiller number between WT, d53, d14, d3 and their double mutants. Values are mean ± s.d. (n=10). b, c, Real-time RT-PCR assay (b) and Western blot analysis (c) showing that the endogenous level of D53 mRNAs and proteins are down-regulated in three representative D53-RNAi lines in d53, d14 and d3 mutant backgrounds, compared to WT control. Data are means ± s.d. (n=3). The double asterisks represent the significant difference determined by the t-test at P<0.01. Anti-HSP82 was used as a loading control. d, Tiller number of representative D53-RNAi transgenic lines in d3 and d14 mutant backgrounds at the heading stage. Each value represents the mean ± s.d. of six plants. L2, L6 and L11 represent three independent lines in d3 background, and L1, L4, L6, L9 and L10 represent five independent lines in d14 background. Akumuro (S) and Norin 8 (N) are the wild-type varieties correspond to d3 and d14 mutants, respectively. d3Vec. and d14Vec. transgenic lines were used as the controls. The single and double asterisks represent significance difference compared with vector control determined by the t-test at P<0.05 and P<0.01, respectively.', 'hash': 'ad0c34b7e149f4c03e6716c53ccfbb3094d10ee8a4bb18a82ae8a346a3f362e9'}, {'image_id': 'nihms541100f13', 'image_file_name': 'nihms541100f13.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f13.jpg', 'caption': 'GR24 promotes D53 protein degradationa, Confocal scanning images showing that AEBSF, Pepstatin A and Leupetin are not effective in blocking D53-GFP fusion protein degradation in transgenic seedlings treated with 5 μM GR24. Scale bars, 100 μm. b, Degradation of D53-GFP fusion protein, but not D3-GFP and D14-GFP fusion proteins expressed in rice protoplasts, in the presence of 5 μM GR24. Pre-treatment with 40 μM MG132 for one hour before addition of GR24 effectively blocks D53-GFP degradation. c. D53-GFP is degraded in the d53 mutant protoplasts in the presence of GR24, but not in d3 or d14 protoplasts. For b and c, each figure represents at least fifty cells observed. Scale bars, 10 μm.', 'hash': 'ee91799a23c20732a71e0dd467613f6e2e93aaf77b85c08d7ec9e95487f76b9d'}, {'image_id': 'nihms541100f3', 'image_file_name': 'nihms541100f3.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f3.jpg', 'caption': 'GR24 promotes the D53-D14 and D14-D3 interactiona, Y2H showing that D53 and d53 interact with D14 in the presence of GR24. Yeast transformants were spotted on the control medium: –LT and selective medium: –LTHA + X-α-gal. b, BiFC analysis of D53 and D14. The positions of nuclei are indicated by DAPI staining. Scale bar, 10 μm. c,\nIn vitro pull-down assay of recombinant MBP-D3 or MBP using resins containing GST-D14. Asterisks indicate the full-length MBP-D3 protein. d, Pull-down assay showing co-IP of D14 from the d3 mutant plant extracts, using GST-D3-OSK1 as the bait. ‘Input’ shows that roughly equal amount of total plant proteins was used.', 'hash': '42c0fb6e9bd51c8dfafb1ac7eacf4f9f5063de9de18ec1f644c1eac459c427be'}, {'image_id': 'nihms541100f4', 'image_file_name': 'nihms541100f4.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f4.jpg', 'caption': 'GR24 promotes D14- and D3-dependent proteasomal degradation of D53a, b, Western blot analysis showing that GR24 promotes D53 protein degradation in wild-type (a), but not in d mutants (b). 10 μg of total protein were applied in each lane. c, Confocal scanning images showing different degradation patterns of D53-GFP and d53-GFP fusion proteins in wild-type, d3 or d14 backgrounds. d, Relative luciferase activity of D53-LUC or d53-LUC in wild-type, d14, d3 or d53 protoplasts. Values are means ± s.d. of three independent experiments. The double asterisks represent significance difference compared with control (LUC) determined by the Student’s t-test at P<0.01. ns, not significant. e, f, Phenotype of d14 d53\n(e) and d3 d53\n(f) double mutants. g, Phenotypes of D53-RNAi transgenic plants in d3 and d14 backgrounds. Scale bars, 100 μm in c and 20 cm in e–g.', 'hash': 'cd7b5fba9e20a57181764f5e8f3ba57eb42aa3f4e21c54222e3fb69ff71bca7c'}, {'image_id': 'nihms541100f9', 'image_file_name': 'nihms541100f9.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f9.jpg', 'caption': 'Phylogenetic analysis of D53 proteinUsing the D53 protein sequence as the query in tblastn searches, homologs were identified from different organisms with a permissive cutoff E value of 1E-3. The sequences chosen from representative genome were aligned and used to generate the neighbor-joining phylogenetic tree with 1,000 bootstrap replicates. The clade names were given based on known sequences in each clade, which is supported by a bootstrap value >85.', 'hash': 'bc84687939fb722f6a5c6145e21fe59388dc78052e0df61107575dd6f4841167'}, {'image_id': 'nihms541100f7', 'image_file_name': 'nihms541100f7.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f7.jpg', 'caption': 'd53 mutation behaves in a semi-dominant mannerComparison of WT, heterozygous (F1) and homozygous d53 plants at the heading stage (a), flag leaf (b), cross section of the first internode (c), panicle (d), plant height (e), tiller number (f) and diameter of the third internode (g). Scale bars, 20 cm in a, 5 cm in b and d and 500 μm in c. For e–g, each value represents the mean ± s.d. (n=25). h, Segregation of F2 progeny from a self-pollinated F1 plant (d53 × Norin 8).', 'hash': '44d74702369633470e8007f53df502e98bf735cc567219a3c970a39719bfe841'}, {'image_id': 'nihms541100f10', 'image_file_name': 'nihms541100f10.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f10.jpg', 'caption': 'Multiple sequence alignment of the deduced amino acid sequence of D53 with its homologsD53 protein is predicted to contain an N-terminal domain, a D1 ATPase domain, an M domain, and a D2 ATPase domain (http://toolkit.tuebingen.mpg.de/hhpred). The beginning and ending sites of each domain are indicated above the sequences. The predicted Walker A (P-loop) and Walker B motifs are shown in red boxes in the D1 domain and green boxes in the D2 domain, respectively. Note that the deletion of five amino acids in the D2 domain of d53 protein overlaps with the GYVG loop in ClpC. The conserved putative EAR motif in D53 and ClpP-binding loop in ClpC are also shown. The sequences used for alignment are D53 (Oryza sativa, LOC_Os11g01330), D53-like (Oryza sativa, LOC_Os12g01360), SMXL6 (Arabidopsis, At1g07200), SMXL7 (Arabidopsis, At2g29970), and ClpC (Bacillus subtilis, GI: 16077154).', 'hash': 'a1a2cdb7cc2662440bf68a4300a28c8ab67139dae49fa0be595f8975f3b1e548'}, {'image_id': 'nihms541100f1', 'image_file_name': 'nihms541100f1.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f1.jpg', 'caption': 'Phenotype of d53 mutanta, b, Phenotype of wild type (WT) and d53 mutant at four-week-old seedling stage (a) or heading stage (b). White arrows indicate the first tillers in d53, which is usually absent in WT, and red arrows show the second tillers. c, Comparison of tillering kinetics at different developmental stages. d, Real-time RT-PCR assay showing altered expression of D10 and FC1 in d mutants. e, Responses of rice seedlings to GR24 treatment. Red and white arrowheads indicate the first and second tillers, respectively. f, LC/MS-MS measurement of epi-5DS levels in root exudates. gfw, per gram fresh weight. Scale bars, 5 cm in a, 30 cm in b and 2 cm in e. Values are means ± s.d. (c, n = 30 plants; d and f, n = 3 replicates). The Student’s t-test analysis indicates a significant difference (compared with WT, *P<0.05, **P<0.01).', 'hash': 'f53df76a3f189a9b454663ede3243dea241bc35024010142bb22feea470662af'}, {'image_id': 'nihms541100f6', 'image_file_name': 'nihms541100f6.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f6.jpg', 'caption': 'Phenotypes of d53 mutanta, Comparison of crown root growth in wild type (WT) and d53 mutant. DAG, day after germination. Each value represents the mean ± s.d. of 25 seedlings. b, Root phenotype of seven-week-old WT and d53 at the tillering stage. Red dots indicate the main culms. c, Comparison of different types of tillers between WT and d53 at the heading stage. Pt, primary tillers; St, secondary tillers; Tt, tertiary tillers; Qt, quaternary tillers. Each value represents the mean ± s.d. of 20 seedlings. d, Morphology comparison of tiller buds at the second node between WT and d53. White arrows and arrowheads indicate the tiller buds and the second nodes, respectively. e, Transverse sections of the first internode of WT and d53. f, Number of vascular bundles (VB) calculated from transverse sections of the first internode of WT and d53. SVB, small vascular bundle; LVB, large vascular bundle. Data are means ± s.d. (n=10). g, Longitudinal sections of the first internode of WT and d53. h, Comparison of parenchyma (PC) cell length in first internode and root between WT and d53. Data are means ± s.d. (n=10). Differences with respect to the WT that were found to be significant in a t-test are indicated with asterisks (*P<0.05; ** P<0.01; ns, not significant). Scale bars, 10 cm in b, 2 cm in d and 100 μm in e and g.', 'hash': 'fb2ed0b3360704f2f4cda6a6835b9310b9b0ca5bceb5445c2fda257a2b0fce0d'}, {'image_id': 'nihms541100f8', 'image_file_name': 'nihms541100f8.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f8.jpg', 'caption': 'd53 is insensitive to GR24 treatment and confers enhanced tillering promoting activitya, Response of five-week-old WT, d53, d14 and d27 to the application of 1 μM GR24. b, Tiller bud length of two-week-old WT, d53, d14 and d27 seedling treated with (+) or without (−) 1 μM GR24. Data are means + s.d. (n=10). c, Numbers of tillers showing outgrowth (>2 mm) for five-week-old WT, d53, d14 and d27 plants treated with (+) or without (−) 1 μM GR24. Data are means ± s.d. (n=10). Asterisks in b and c denote significant differences between treated and untreated samples within the same genotype (two tailed Mann-Whitney U test, P<0.01; ns, not significant). d,\nD53 RNAi transgenic plants exhibit reduced tillering in the d53 mutant background. Vector, d53 transformed with the pCUbi1390-ΔFAD2 control. e, Tiller number of RNAi transgenic lines in d at the tillering stage. Each value represents the mean ± s.d. of six plants (T1 generation). L4, L10 and L11 represent three independent lines. The t-test analysis indicated a significant difference (compared with vector control, **P<0.01). Scale bars, 20 cm in a and 10 cm in d.', 'hash': 'd9a93441d48a89e5d77fafda18775255c379a55ada601a8af3d755ea584ee904'}, {'image_id': 'nihms541100f11', 'image_file_name': 'nihms541100f11.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f11.jpg', 'caption': 'Histochemical staining of the pD53::GUS reporter gene and subcellular localization of D53 proteina–h, Histochemical staining of young root (a), shoot (b), leaf (c), leaf sheath (d), panicle (e), transverse section of the leaf sheath (f), stem (g) and node (h). Scale bars, 1 mm in a, b, c, d, f and h; 1 cm in e and 100 μm in g. i–l, Subcellular localization of D53-GFP fusion protein in rice protoplast cells. A nuclear marker protein, OsMADS3, fused with mCherry, was used as a positive control. Scale bars, 5 μm. m–p, Confocal scanning images showing nuclear localization of the D53-GFP fusion protein in transgenic root cells. Scale bars, 100 μm.', 'hash': '2e9fd820df0162de61bad18af37fd862f0ccb8b1521b2ad478fbc039ee5a4909'}]
|
{'nihms541100f1': ['Previous studies have identified several rice mutants defective in SL biosynthesis or signaling10–14. Because of their highly branched and dwarf phenotype, these mutants were termed “d mutants”, such as d3, d10, d14 (also known as d88 or htd2), d17 (htd1), and d27. The rice d53 mutant31 also displayed reduced height and increased tillering, as well as thinner stem and shorter crown root, compared to the wild-type strain (<xref rid="nihms541100f1" ref-type="fig">Fig. 1a, b</xref> and and <xref rid="nihms541100f6" ref-type="fig">Extended Data Fig. 1a, b</xref>). Kinetic analysis showed that at the heading stage, the total tiller number of ). Kinetic analysis showed that at the heading stage, the total tiller number of d53 was about three times that of the wild type, resulting from an increase in both higher-order and high nodes tillers (<xref rid="nihms541100f1" ref-type="fig">Fig. 1c</xref> and and <xref rid="nihms541100f6" ref-type="fig">Extended Data Fig. 1c, d</xref>). Histological analysis revealed that the sizes of vascular bundles and parenchyma cells in internodes were largely comparable between ). Histological analysis revealed that the sizes of vascular bundles and parenchyma cells in internodes were largely comparable between d53 and wild-type plants, implying that the shortening and thinning of d53 stem were mainly caused by a reduction in cell number (<xref rid="nihms541100f6" ref-type="fig">Extended Data Fig. 1e–h</xref>). The phenotypes of F). The phenotypes of F1 heterozygous plants were intermediate between the homozygous parental plants (<xref rid="nihms541100f7" ref-type="fig">Extended Data Fig. 2a–g</xref>). Genetic analyses of an F). Genetic analyses of an F2 population derived from a cross of d53 and the wild-type parent (Norin 8) showed that the normal, intermediate and dwarf plants segregated as 1:2:1 (33:58:28, χ2=0.09, P>0.05), indicating that the d53 mutation behaved in a semi-dominant manner (<xref rid="nihms541100f7" ref-type="fig">Extended Data Fig. 2h</xref>).).', 'The phenotypic similarity between d53 and the previously reported rice d mutants prompted us to examine whether d53 is defective in SL-mediated inhibition of axillary bud outgrowth. Quantitative real-time RT-PCR analysis showed that expression of D10 (CCD8), was similarly up-regulated in d53 (<xref rid="nihms541100f1" ref-type="fig">Fig. 1d</xref>) as in other ) as in other d mutants due to feedback regulation in the SL pathway11. In addition, expression of an inhibitor of axillary bud outgrowth, FINE CULM1 (FC1)32, which is orthologous to the maize TEOSINTE BRANCHED1 (TB1)33 and the Arabidopsis BRANCHED1 (BRC1)34 was also similarly down-regulated in the d53, d14 and d27 mutants (<xref rid="nihms541100f1" ref-type="fig">Fig. 1d</xref>), suggesting that ), suggesting that D53 is likely involved in SL biosynthesis or signaling. Moreover, exogenous application of a SL analogue, GR24, effectively inhibited the outgrowth axillary buds of d27, but not d14 or d53 (<xref rid="nihms541100f1" ref-type="fig">Fig 1e</xref> and and <xref rid="nihms541100f8" ref-type="fig">Extended Data Fig. 3a–c</xref>). Further, measurement of SLs produced in the root exudates showed that ). Further, measurement of SLs produced in the root exudates showed that d53 accumulated markedly higher levels of 2′-epi-5-deoxystrigol, a native SL of rice, than the wild-type cultivar Norin 8 (<xref rid="nihms541100f1" ref-type="fig">Fig. 1f</xref>). These results indicate that ). These results indicate that d53 is a SL-insensitive mutant.'], 'nihms541100f2': ['D53 was previously mapped to the terminal region of the short arm of rice chromosome 11 (ref. 35). To decipher the molecular defect in d53, we isolated D53 by a map-based cloning approach. Using an F2 population of ~ 12,000 plants generated from the cross between Ketan Nangka and the mutant, we further delimited the D53 locus to a 34-kb DNA region on the BAC clone OSJNBa0032J07, which contains three putative genes (<xref rid="nihms541100f2" ref-type="fig">Fig. 2a</xref>). Sequence analysis revealed a single-nucleotide substitution and 15 nucleotides deletion in the third exon of LOC_Os11g01330 in ). Sequence analysis revealed a single-nucleotide substitution and 15 nucleotides deletion in the third exon of LOC_Os11g01330 in d53, which resulted in an amino acid substitution (R812T) and deletion of five amino acids (G813KTGI817) (<xref rid="nihms541100f2" ref-type="fig">Fig. 2b</xref>). To verify that this mutation caused the tillering dwarf phenotype, we generated transgenic plants expressing the wild type or mutant ). To verify that this mutation caused the tillering dwarf phenotype, we generated transgenic plants expressing the wild type or mutant D53 gene under the control of rice Actin1 promoter, in a wild-type background. Strikingly, all transgenic plants expressing the mutant d53 gene showed a more exaggerated tillering phenotype than those expressing the wild-type D53 gene. The severity of tillering phenotype in these transgenic plants was correlated with the expression level of the transgene. Notably, overexpression of the wild-type D53 gene also caused a moderate increase in tillering, compared to the vector control plants (<xref rid="nihms541100f2" ref-type="fig">Fig. 2c–e</xref>). These observations suggested that the D53 protein acts as a repressor in the SL-mediated branching-inhibition pathway and that the dominant tillering phenotype of the ). These observations suggested that the D53 protein acts as a repressor in the SL-mediated branching-inhibition pathway and that the dominant tillering phenotype of the d53 mutant was most likely caused by a gain-of-function mutation in d53. To further confirm this, we generated D53 knockdown transgenic plants using a RNA interference (RNAi) approach. As expected, reducing D53 expression in d53 background drastically reduced the tiller number (<xref rid="nihms541100f8" ref-type="fig">Extended Data Fig. 3d, e</xref>). Taken together, these data support the proposition that ). Taken together, these data support the proposition that d53 mutation enhanced D53 activity in repressing SL signaling.', 'Real-time PCR analysis revealed that D53 was widely expressed in the examined rice tissues (<xref rid="nihms541100f2" ref-type="fig">Fig. 2f</xref>). ). D53 promoter driven GUS (β-glucuronidase) reporter gene (pD53::GUS) assay showed that GUS staining was observed in vasculature in roots, shoots, leaves, leaf sheaths, nodes, internodes and young panicles, preferentially in the parenchyma cells surrounding the xylem (<xref rid="nihms541100f11" ref-type="fig">Extended Data Fig. 6a–h</xref>). Moreover, ). Moreover, D53 expression was up-regulated by GR24 treatment in wild-type plants, but down-regulated in six d mutants, suggesting that expression of D53 is regulated by SLs signaling (<xref rid="nihms541100f2" ref-type="fig">Fig. 2g, h</xref>). The D53-GFP (green fluorescent protein) fusion protein is exclusively localized to the nucleus in rice protoplasts and the ). The D53-GFP (green fluorescent protein) fusion protein is exclusively localized to the nucleus in rice protoplasts and the pActin::D53-GFP transgenic root cells (<xref rid="nihms541100f11" ref-type="fig">Extended Data Fig. 6i–p</xref>).).'], 'nihms541100f9': ['D53 is predicted to encode a protein of 1131 amino acids. A BLAST search identified a closely related homolog of D53 (designated D53-like, LOC_Os12g01360) with 96.6% amino acid sequence identity in the rice genome. In addition, D53 homologs were found in other monocots and dicots, but not in lower plants, animals or microbes, indicating that D53-like proteins are specific in higher plants (<xref rid="nihms541100f9" ref-type="fig">Extended Data Fig. 4</xref>). Sequence analysis by the HHpred structure prediction server revealed that D53 shares a similar secondary structure composition, despite low primary sequence homology, to proteins of the class I Clp ATPases family, which are characterized by an N-terminal domain, a D1 ATPase domain, an M domain, and a D2 ATPase domain). Sequence analysis by the HHpred structure prediction server revealed that D53 shares a similar secondary structure composition, despite low primary sequence homology, to proteins of the class I Clp ATPases family, which are characterized by an N-terminal domain, a D1 ATPase domain, an M domain, and a D2 ATPase domain36. Notably, the D2 domain of D53 contains a highly conserved linear sequence, FDLNL, which closely matches the ETHYLENE RESPONSE FACTOR-associated amphiphilic repression (EAR) motif (<xref rid="nihms541100f10" ref-type="fig">Extended Data Fig. 5</xref>), which is known to interact with the TOPLESS family of proteins and involved in transcriptional repression), which is known to interact with the TOPLESS family of proteins and involved in transcriptional repression37.'], 'nihms541100f3': ['Previous studies have identified the F-box protein D3 and the α/β hydrolase D14 as two key components of SL signaling in rice10,12, of which D14 and its orthologues in Arabidopsis (AtD14) and Petunia (DAD2) have been proposed to directly participate in SL perception19,27,28. Yeast two-hybrid assay (Y2H) showed that both D53 and d53 could physically interact with D14 in the presence of GR24 (<xref rid="nihms541100f3" ref-type="fig">Fig. 3a</xref>). Domain deletion analysis indicated that the D1 domain of D53 was essential for the GR24-dependent D53-D14 interaction. Interestingly, its binding activity was inhibited by the M and D2 domains, although their negative effect can be overcome by the N domain (). Domain deletion analysis indicated that the D1 domain of D53 was essential for the GR24-dependent D53-D14 interaction. Interestingly, its binding activity was inhibited by the M and D2 domains, although their negative effect can be overcome by the N domain (<xref rid="nihms541100f12" ref-type="fig">Extended Data Fig. 7</xref>). We verified the D53-D14 interaction in ). We verified the D53-D14 interaction in N. benthamiana leaf cell nucleus both in the presence or absence of exogenously applied GR24 using a bimolecular fluorescence complementation (BiFC) assay (<xref rid="nihms541100f3" ref-type="fig">Fig. 3b</xref>). The observed interaction between D53 and D14 in the absence of exogenously applied GR24 might be due to the effect of endogenous SLs present in the tobacco leaf cells. Consistent with the previously reported GR24-depedent interaction between DAD2 and PhMAX2A (an ortholog of D3 in Petunia) in yeast). The observed interaction between D53 and D14 in the absence of exogenously applied GR24 might be due to the effect of endogenous SLs present in the tobacco leaf cells. Consistent with the previously reported GR24-depedent interaction between DAD2 and PhMAX2A (an ortholog of D3 in Petunia) in yeast19, our in vitro pull-down assay also revealed a direct physical interaction between D14 and D3 in a GR24-dependent manner (<xref rid="nihms541100f3" ref-type="fig">Fig. 3c</xref>). Furthermore, using recombinant GST-D3-OSK1 fusion protein as the bait, our ). Furthermore, using recombinant GST-D3-OSK1 fusion protein as the bait, our in vitro pull-down assay showed that D14 could be more efficiently co-immunoprecipitated (co-IP) from d3 plant extracts in the presence of exogenously applied GR24 (<xref rid="nihms541100f3" ref-type="fig">Fig. 3d</xref>). Together, these results suggest that SLs may act to promote complex formation among D14, D3 and D53, linking D53 to the hormone-perception components of the SL signaling pathway.). Together, these results suggest that SLs may act to promote complex formation among D14, D3 and D53, linking D53 to the hormone-perception components of the SL signaling pathway.', 'It has been speculated that perception of SLs triggers the degradation of putative repressors by the SCFMAX2 ubiquitin ligase complex to suppress shoot branching21,29,30. In this study, we established that D53 acts as a repressor of SL signaling in rice. Consistent with the previous observation of GR24-dependent interaction between DAD2 and PhMAX2 (ref. 19), we found that GR24 also promotes the interaction between D14 with D53 and D3 (<xref rid="nihms541100f3" ref-type="fig">Fig. 3</xref>). Further, we showed that D53 is targeted for degradation by the proteasome in a D14- and D3-dependent manner (). Further, we showed that D53 is targeted for degradation by the proteasome in a D14- and D3-dependent manner (<xref rid="nihms541100f4" ref-type="fig">Fig. 4a–d</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8</xref>). Together, these data collectively support the notion that SL perception by D14 acts to promote ubiquitination of D53 by the D14-SCF). Together, these data collectively support the notion that SL perception by D14 acts to promote ubiquitination of D53 by the D14-SCFD3 ubiquitin ligase, and subsequent degradation of D53 by the proteasome, leading to the propagation of SL signal and downstream physiological responses (<xref rid="nihms541100f5" ref-type="fig">Fig. 5</xref>). Our findings revealed a remarkable similarity between the hormonal perception and signaling mechanism of SL and several other classes of plant hormones, including auxin, jasmonate and gibberellin). Our findings revealed a remarkable similarity between the hormonal perception and signaling mechanism of SL and several other classes of plant hormones, including auxin, jasmonate and gibberellin25,38–40.'], 'nihms541100f4': ['To investigate how SL regulates D53, we performed a set of additional experiments. Both western blot analysis and fluorescence microscopy examination showed that GR24 treatment induced rapid degradation of the D53 protein in wild-type cells, but not in d3 and d14 mutant cells (<xref rid="nihms541100f4" ref-type="fig">Fig. 4a–c</xref>). We further showed that D53 was degraded by the proteasome, as a proteasome inhibitor, MG132, but not other protease inhibitors, effectively blocked GR24-induced D53-GFP degradation (). We further showed that D53 was degraded by the proteasome, as a proteasome inhibitor, MG132, but not other protease inhibitors, effectively blocked GR24-induced D53-GFP degradation (<xref rid="nihms541100f4" ref-type="fig">Fig. 4c, d</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8a, b</xref>). Notably, unlike the wild-type D53-GFP fusion protein, the mutant d53-GFP fusion protein appeared to be stable in the presence of GR24 (). Notably, unlike the wild-type D53-GFP fusion protein, the mutant d53-GFP fusion protein appeared to be stable in the presence of GR24 (<xref rid="nihms541100f4" ref-type="fig">Fig. 4c</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8b</xref>). Interestingly, we noted that D53-GFP and D53-LUC were still degraded in the ). Interestingly, we noted that D53-GFP and D53-LUC were still degraded in the d53 mutant cells, but not in d3 or d14 mutant cells (<xref rid="nihms541100f4" ref-type="fig">Fig. 4d</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8c</xref>), indicating that the D53 degradation pathway was still operational in the ), indicating that the D53 degradation pathway was still operational in the d53 mutant. Together, these results suggest that SL triggers proteasome-mediated degradation of D53 in a D14- and D3-dependent manner. Importantly, the insensitivity of d53 protein to SL-triggered turnover is consistent with the observed dominant gain-of-function mutant phenotype of d53.'], 'nihms541100f14': ['To provide genetic support for the functional relationship between D53, D3 and D14, we generated d3 d53 and d14 d53 double mutants. The d3 mutant had more tillers and it was shorter than the d14 and d53 single mutants (<xref rid="nihms541100f14" ref-type="fig">Extended Data Fig. 9a</xref>). The ). The d14 d53 double mutants exhibited a dwarf tillering phenotype resembling the d14 and d53 parental plants, whereas the d3 d53 double mutant exhibited a dwarf tillering phenotype resembling d3 (<xref rid="nihms541100f4" ref-type="fig">Fig. 4e, f</xref> and and <xref rid="nihms541100f14" ref-type="fig">Extended Data Fig. 9a</xref>). The lack of obvious additive effects among these mutants suggests that ). The lack of obvious additive effects among these mutants suggests that D3, D14 and D53 act in the same signaling pathway. To further test their epistasis relationship, we knocked down D53 gene expression in the d3 and d14 backgrounds. As shown in <xref rid="nihms541100f4" ref-type="fig">Fig. 4g</xref> and and <xref rid="nihms541100f14" ref-type="fig">Extended Data Fig. 9b–d</xref>, the mutant phenotype of , the mutant phenotype of d3 and d14 was restored to nearly wild type levels, demonstrating that D53 acts downstream of D3 and D14, and that accumulation of D53 protein is responsible for blocking SL signaling and conferring the dwarf tillering phenotype in these mutants.']}
|
D3 D14-SCF-dependent degradation of D53 regulates strigolactone signaling
| null |
Nature
|
1387440000
|
Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.
|
[
"Amino Acid Sequence",
"Cloning, Molecular",
"Gene Expression Regulation, Plant",
"Lactones",
"Molecular Sequence Data",
"Mutation",
"Oryza",
"Phenotype",
"Plant Growth Regulators",
"Plant Proteins",
"Proteasome Endopeptidase Complex",
"Protein Binding",
"Proteolysis",
"SKP Cullin F-Box Protein Ligases",
"Signal Transduction"
] |
other
|
PMC4096652
| null |
50
|
[
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Nature. 2013 Dec 19; 504(7480):406-410
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NO-CC CODE
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GR24 promotes the D53-D14 and D14-D3 interactiona, Y2H showing that D53 and d53 interact with D14 in the presence of GR24. Yeast transformants were spotted on the control medium: –LT and selective medium: –LTHA + X-α-gal. b, BiFC analysis of D53 and D14. The positions of nuclei are indicated by DAPI staining. Scale bar, 10 μm. c,
In vitro pull-down assay of recombinant MBP-D3 or MBP using resins containing GST-D14. Asterisks indicate the full-length MBP-D3 protein. d, Pull-down assay showing co-IP of D14 from the d3 mutant plant extracts, using GST-D3-OSK1 as the bait. ‘Input’ shows that roughly equal amount of total plant proteins was used.
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nihms541100f3
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42c0fb6e9bd51c8dfafb1ac7eacf4f9f5063de9de18ec1f644c1eac459c427be
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
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[{'image_id': 'nihms541100f12', 'image_file_name': 'nihms541100f12.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f12.jpg', 'caption': 'Mapping of the D14-binding domain of D53a, Schematic structure of the D53 protein. Numbers indicate amino-acid (aa) residues. b, Y2H analysis showing interaction between full-length and various domain deletion variants of D53 with D14 in the presence or absence of 5 μM GR24. –LT, control medium (SD–Leu/–Trp); –LTHA, selective medium (SD–Leu/–Trp/–His/–Ade).', 'hash': '936e34d073e9fd19b0aff60d7efe454f79d985abd84576dff9c0fedb8c97eae5'}, {'image_id': 'nihms541100f5', 'image_file_name': 'nihms541100f5.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f5.jpg', 'caption': 'A schematic model depicting SLs promote D14-SCFD3-mediated degradation of D53.', 'hash': 'af2d18c039c4623d3804cc903778db1744c4e714061962d9a8fe0ecc892f99ba'}, {'image_id': 'nihms541100f2', 'image_file_name': 'nihms541100f2.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f2.jpg', 'caption': 'Map-based cloning and characterization of D53a,\nD53 was fine-mapped on chromosome 11. The numbers of recombinants are shown in brackets. b, Molecular lesions in d53 mutant. c, Phenotypic comparison of proActin::D53-GFP and proActin::d53-GFP transgenic plants. Vector, proActin::GFP control. Scale bar, 10 cm. d, e, Tiller number (d) and relative expression of D53\n(e) of transgenic plants in c. f,\nD53 expression in various organs, including young panicles (YP), young roots (YR), shoots (S), leaf blades (LB), leaf sheaths (LS), culms (C) and nodes (N). g, GR24 treatment induces D53 expression. h, Relative expression levels of D53 in two wild-type varieties Norin 8 (N) and Shiokari (S), and six rice d mutants. Each value in d–h represents the mean ± s.d. (d and f–h, n = 3 replicates; e, n = 20 plants). The Student’s t-test analysis indicates a significant difference (compared with control, *P<0.05, **P<0.01).', 'hash': '13933adfe2f98e66d2ddcb8dd6d9c8e97d18598d96b75c23b73f05ae2c07e5dd'}, {'image_id': 'nihms541100f14', 'image_file_name': 'nihms541100f14.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f14.jpg', 'caption': 'D53-RNAi transgenic lines in d3 and d14 backgroundsa, Comparison of plant height, diameter of the 3rd internode and tiller number between WT, d53, d14, d3 and their double mutants. Values are mean ± s.d. (n=10). b, c, Real-time RT-PCR assay (b) and Western blot analysis (c) showing that the endogenous level of D53 mRNAs and proteins are down-regulated in three representative D53-RNAi lines in d53, d14 and d3 mutant backgrounds, compared to WT control. Data are means ± s.d. (n=3). The double asterisks represent the significant difference determined by the t-test at P<0.01. Anti-HSP82 was used as a loading control. d, Tiller number of representative D53-RNAi transgenic lines in d3 and d14 mutant backgrounds at the heading stage. Each value represents the mean ± s.d. of six plants. L2, L6 and L11 represent three independent lines in d3 background, and L1, L4, L6, L9 and L10 represent five independent lines in d14 background. Akumuro (S) and Norin 8 (N) are the wild-type varieties correspond to d3 and d14 mutants, respectively. d3Vec. and d14Vec. transgenic lines were used as the controls. The single and double asterisks represent significance difference compared with vector control determined by the t-test at P<0.05 and P<0.01, respectively.', 'hash': 'ad0c34b7e149f4c03e6716c53ccfbb3094d10ee8a4bb18a82ae8a346a3f362e9'}, {'image_id': 'nihms541100f13', 'image_file_name': 'nihms541100f13.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f13.jpg', 'caption': 'GR24 promotes D53 protein degradationa, Confocal scanning images showing that AEBSF, Pepstatin A and Leupetin are not effective in blocking D53-GFP fusion protein degradation in transgenic seedlings treated with 5 μM GR24. Scale bars, 100 μm. b, Degradation of D53-GFP fusion protein, but not D3-GFP and D14-GFP fusion proteins expressed in rice protoplasts, in the presence of 5 μM GR24. Pre-treatment with 40 μM MG132 for one hour before addition of GR24 effectively blocks D53-GFP degradation. c. D53-GFP is degraded in the d53 mutant protoplasts in the presence of GR24, but not in d3 or d14 protoplasts. For b and c, each figure represents at least fifty cells observed. Scale bars, 10 μm.', 'hash': 'ee91799a23c20732a71e0dd467613f6e2e93aaf77b85c08d7ec9e95487f76b9d'}, {'image_id': 'nihms541100f3', 'image_file_name': 'nihms541100f3.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f3.jpg', 'caption': 'GR24 promotes the D53-D14 and D14-D3 interactiona, Y2H showing that D53 and d53 interact with D14 in the presence of GR24. Yeast transformants were spotted on the control medium: –LT and selective medium: –LTHA + X-α-gal. b, BiFC analysis of D53 and D14. The positions of nuclei are indicated by DAPI staining. Scale bar, 10 μm. c,\nIn vitro pull-down assay of recombinant MBP-D3 or MBP using resins containing GST-D14. Asterisks indicate the full-length MBP-D3 protein. d, Pull-down assay showing co-IP of D14 from the d3 mutant plant extracts, using GST-D3-OSK1 as the bait. ‘Input’ shows that roughly equal amount of total plant proteins was used.', 'hash': '42c0fb6e9bd51c8dfafb1ac7eacf4f9f5063de9de18ec1f644c1eac459c427be'}, {'image_id': 'nihms541100f4', 'image_file_name': 'nihms541100f4.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f4.jpg', 'caption': 'GR24 promotes D14- and D3-dependent proteasomal degradation of D53a, b, Western blot analysis showing that GR24 promotes D53 protein degradation in wild-type (a), but not in d mutants (b). 10 μg of total protein were applied in each lane. c, Confocal scanning images showing different degradation patterns of D53-GFP and d53-GFP fusion proteins in wild-type, d3 or d14 backgrounds. d, Relative luciferase activity of D53-LUC or d53-LUC in wild-type, d14, d3 or d53 protoplasts. Values are means ± s.d. of three independent experiments. The double asterisks represent significance difference compared with control (LUC) determined by the Student’s t-test at P<0.01. ns, not significant. e, f, Phenotype of d14 d53\n(e) and d3 d53\n(f) double mutants. g, Phenotypes of D53-RNAi transgenic plants in d3 and d14 backgrounds. Scale bars, 100 μm in c and 20 cm in e–g.', 'hash': 'cd7b5fba9e20a57181764f5e8f3ba57eb42aa3f4e21c54222e3fb69ff71bca7c'}, {'image_id': 'nihms541100f9', 'image_file_name': 'nihms541100f9.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f9.jpg', 'caption': 'Phylogenetic analysis of D53 proteinUsing the D53 protein sequence as the query in tblastn searches, homologs were identified from different organisms with a permissive cutoff E value of 1E-3. The sequences chosen from representative genome were aligned and used to generate the neighbor-joining phylogenetic tree with 1,000 bootstrap replicates. The clade names were given based on known sequences in each clade, which is supported by a bootstrap value >85.', 'hash': 'bc84687939fb722f6a5c6145e21fe59388dc78052e0df61107575dd6f4841167'}, {'image_id': 'nihms541100f7', 'image_file_name': 'nihms541100f7.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f7.jpg', 'caption': 'd53 mutation behaves in a semi-dominant mannerComparison of WT, heterozygous (F1) and homozygous d53 plants at the heading stage (a), flag leaf (b), cross section of the first internode (c), panicle (d), plant height (e), tiller number (f) and diameter of the third internode (g). Scale bars, 20 cm in a, 5 cm in b and d and 500 μm in c. For e–g, each value represents the mean ± s.d. (n=25). h, Segregation of F2 progeny from a self-pollinated F1 plant (d53 × Norin 8).', 'hash': '44d74702369633470e8007f53df502e98bf735cc567219a3c970a39719bfe841'}, {'image_id': 'nihms541100f10', 'image_file_name': 'nihms541100f10.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f10.jpg', 'caption': 'Multiple sequence alignment of the deduced amino acid sequence of D53 with its homologsD53 protein is predicted to contain an N-terminal domain, a D1 ATPase domain, an M domain, and a D2 ATPase domain (http://toolkit.tuebingen.mpg.de/hhpred). The beginning and ending sites of each domain are indicated above the sequences. The predicted Walker A (P-loop) and Walker B motifs are shown in red boxes in the D1 domain and green boxes in the D2 domain, respectively. Note that the deletion of five amino acids in the D2 domain of d53 protein overlaps with the GYVG loop in ClpC. The conserved putative EAR motif in D53 and ClpP-binding loop in ClpC are also shown. The sequences used for alignment are D53 (Oryza sativa, LOC_Os11g01330), D53-like (Oryza sativa, LOC_Os12g01360), SMXL6 (Arabidopsis, At1g07200), SMXL7 (Arabidopsis, At2g29970), and ClpC (Bacillus subtilis, GI: 16077154).', 'hash': 'a1a2cdb7cc2662440bf68a4300a28c8ab67139dae49fa0be595f8975f3b1e548'}, {'image_id': 'nihms541100f1', 'image_file_name': 'nihms541100f1.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f1.jpg', 'caption': 'Phenotype of d53 mutanta, b, Phenotype of wild type (WT) and d53 mutant at four-week-old seedling stage (a) or heading stage (b). White arrows indicate the first tillers in d53, which is usually absent in WT, and red arrows show the second tillers. c, Comparison of tillering kinetics at different developmental stages. d, Real-time RT-PCR assay showing altered expression of D10 and FC1 in d mutants. e, Responses of rice seedlings to GR24 treatment. Red and white arrowheads indicate the first and second tillers, respectively. f, LC/MS-MS measurement of epi-5DS levels in root exudates. gfw, per gram fresh weight. Scale bars, 5 cm in a, 30 cm in b and 2 cm in e. Values are means ± s.d. (c, n = 30 plants; d and f, n = 3 replicates). The Student’s t-test analysis indicates a significant difference (compared with WT, *P<0.05, **P<0.01).', 'hash': 'f53df76a3f189a9b454663ede3243dea241bc35024010142bb22feea470662af'}, {'image_id': 'nihms541100f6', 'image_file_name': 'nihms541100f6.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f6.jpg', 'caption': 'Phenotypes of d53 mutanta, Comparison of crown root growth in wild type (WT) and d53 mutant. DAG, day after germination. Each value represents the mean ± s.d. of 25 seedlings. b, Root phenotype of seven-week-old WT and d53 at the tillering stage. Red dots indicate the main culms. c, Comparison of different types of tillers between WT and d53 at the heading stage. Pt, primary tillers; St, secondary tillers; Tt, tertiary tillers; Qt, quaternary tillers. Each value represents the mean ± s.d. of 20 seedlings. d, Morphology comparison of tiller buds at the second node between WT and d53. White arrows and arrowheads indicate the tiller buds and the second nodes, respectively. e, Transverse sections of the first internode of WT and d53. f, Number of vascular bundles (VB) calculated from transverse sections of the first internode of WT and d53. SVB, small vascular bundle; LVB, large vascular bundle. Data are means ± s.d. (n=10). g, Longitudinal sections of the first internode of WT and d53. h, Comparison of parenchyma (PC) cell length in first internode and root between WT and d53. Data are means ± s.d. (n=10). Differences with respect to the WT that were found to be significant in a t-test are indicated with asterisks (*P<0.05; ** P<0.01; ns, not significant). Scale bars, 10 cm in b, 2 cm in d and 100 μm in e and g.', 'hash': 'fb2ed0b3360704f2f4cda6a6835b9310b9b0ca5bceb5445c2fda257a2b0fce0d'}, {'image_id': 'nihms541100f8', 'image_file_name': 'nihms541100f8.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f8.jpg', 'caption': 'd53 is insensitive to GR24 treatment and confers enhanced tillering promoting activitya, Response of five-week-old WT, d53, d14 and d27 to the application of 1 μM GR24. b, Tiller bud length of two-week-old WT, d53, d14 and d27 seedling treated with (+) or without (−) 1 μM GR24. Data are means + s.d. (n=10). c, Numbers of tillers showing outgrowth (>2 mm) for five-week-old WT, d53, d14 and d27 plants treated with (+) or without (−) 1 μM GR24. Data are means ± s.d. (n=10). Asterisks in b and c denote significant differences between treated and untreated samples within the same genotype (two tailed Mann-Whitney U test, P<0.01; ns, not significant). d,\nD53 RNAi transgenic plants exhibit reduced tillering in the d53 mutant background. Vector, d53 transformed with the pCUbi1390-ΔFAD2 control. e, Tiller number of RNAi transgenic lines in d at the tillering stage. Each value represents the mean ± s.d. of six plants (T1 generation). L4, L10 and L11 represent three independent lines. The t-test analysis indicated a significant difference (compared with vector control, **P<0.01). Scale bars, 20 cm in a and 10 cm in d.', 'hash': 'd9a93441d48a89e5d77fafda18775255c379a55ada601a8af3d755ea584ee904'}, {'image_id': 'nihms541100f11', 'image_file_name': 'nihms541100f11.jpg', 'image_path': '../data/media_files/PMC4096652/nihms541100f11.jpg', 'caption': 'Histochemical staining of the pD53::GUS reporter gene and subcellular localization of D53 proteina–h, Histochemical staining of young root (a), shoot (b), leaf (c), leaf sheath (d), panicle (e), transverse section of the leaf sheath (f), stem (g) and node (h). Scale bars, 1 mm in a, b, c, d, f and h; 1 cm in e and 100 μm in g. i–l, Subcellular localization of D53-GFP fusion protein in rice protoplast cells. A nuclear marker protein, OsMADS3, fused with mCherry, was used as a positive control. Scale bars, 5 μm. m–p, Confocal scanning images showing nuclear localization of the D53-GFP fusion protein in transgenic root cells. Scale bars, 100 μm.', 'hash': '2e9fd820df0162de61bad18af37fd862f0ccb8b1521b2ad478fbc039ee5a4909'}]
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{'nihms541100f1': ['Previous studies have identified several rice mutants defective in SL biosynthesis or signaling10–14. Because of their highly branched and dwarf phenotype, these mutants were termed “d mutants”, such as d3, d10, d14 (also known as d88 or htd2), d17 (htd1), and d27. The rice d53 mutant31 also displayed reduced height and increased tillering, as well as thinner stem and shorter crown root, compared to the wild-type strain (<xref rid="nihms541100f1" ref-type="fig">Fig. 1a, b</xref> and and <xref rid="nihms541100f6" ref-type="fig">Extended Data Fig. 1a, b</xref>). Kinetic analysis showed that at the heading stage, the total tiller number of ). Kinetic analysis showed that at the heading stage, the total tiller number of d53 was about three times that of the wild type, resulting from an increase in both higher-order and high nodes tillers (<xref rid="nihms541100f1" ref-type="fig">Fig. 1c</xref> and and <xref rid="nihms541100f6" ref-type="fig">Extended Data Fig. 1c, d</xref>). Histological analysis revealed that the sizes of vascular bundles and parenchyma cells in internodes were largely comparable between ). Histological analysis revealed that the sizes of vascular bundles and parenchyma cells in internodes were largely comparable between d53 and wild-type plants, implying that the shortening and thinning of d53 stem were mainly caused by a reduction in cell number (<xref rid="nihms541100f6" ref-type="fig">Extended Data Fig. 1e–h</xref>). The phenotypes of F). The phenotypes of F1 heterozygous plants were intermediate between the homozygous parental plants (<xref rid="nihms541100f7" ref-type="fig">Extended Data Fig. 2a–g</xref>). Genetic analyses of an F). Genetic analyses of an F2 population derived from a cross of d53 and the wild-type parent (Norin 8) showed that the normal, intermediate and dwarf plants segregated as 1:2:1 (33:58:28, χ2=0.09, P>0.05), indicating that the d53 mutation behaved in a semi-dominant manner (<xref rid="nihms541100f7" ref-type="fig">Extended Data Fig. 2h</xref>).).', 'The phenotypic similarity between d53 and the previously reported rice d mutants prompted us to examine whether d53 is defective in SL-mediated inhibition of axillary bud outgrowth. Quantitative real-time RT-PCR analysis showed that expression of D10 (CCD8), was similarly up-regulated in d53 (<xref rid="nihms541100f1" ref-type="fig">Fig. 1d</xref>) as in other ) as in other d mutants due to feedback regulation in the SL pathway11. In addition, expression of an inhibitor of axillary bud outgrowth, FINE CULM1 (FC1)32, which is orthologous to the maize TEOSINTE BRANCHED1 (TB1)33 and the Arabidopsis BRANCHED1 (BRC1)34 was also similarly down-regulated in the d53, d14 and d27 mutants (<xref rid="nihms541100f1" ref-type="fig">Fig. 1d</xref>), suggesting that ), suggesting that D53 is likely involved in SL biosynthesis or signaling. Moreover, exogenous application of a SL analogue, GR24, effectively inhibited the outgrowth axillary buds of d27, but not d14 or d53 (<xref rid="nihms541100f1" ref-type="fig">Fig 1e</xref> and and <xref rid="nihms541100f8" ref-type="fig">Extended Data Fig. 3a–c</xref>). Further, measurement of SLs produced in the root exudates showed that ). Further, measurement of SLs produced in the root exudates showed that d53 accumulated markedly higher levels of 2′-epi-5-deoxystrigol, a native SL of rice, than the wild-type cultivar Norin 8 (<xref rid="nihms541100f1" ref-type="fig">Fig. 1f</xref>). These results indicate that ). These results indicate that d53 is a SL-insensitive mutant.'], 'nihms541100f2': ['D53 was previously mapped to the terminal region of the short arm of rice chromosome 11 (ref. 35). To decipher the molecular defect in d53, we isolated D53 by a map-based cloning approach. Using an F2 population of ~ 12,000 plants generated from the cross between Ketan Nangka and the mutant, we further delimited the D53 locus to a 34-kb DNA region on the BAC clone OSJNBa0032J07, which contains three putative genes (<xref rid="nihms541100f2" ref-type="fig">Fig. 2a</xref>). Sequence analysis revealed a single-nucleotide substitution and 15 nucleotides deletion in the third exon of LOC_Os11g01330 in ). Sequence analysis revealed a single-nucleotide substitution and 15 nucleotides deletion in the third exon of LOC_Os11g01330 in d53, which resulted in an amino acid substitution (R812T) and deletion of five amino acids (G813KTGI817) (<xref rid="nihms541100f2" ref-type="fig">Fig. 2b</xref>). To verify that this mutation caused the tillering dwarf phenotype, we generated transgenic plants expressing the wild type or mutant ). To verify that this mutation caused the tillering dwarf phenotype, we generated transgenic plants expressing the wild type or mutant D53 gene under the control of rice Actin1 promoter, in a wild-type background. Strikingly, all transgenic plants expressing the mutant d53 gene showed a more exaggerated tillering phenotype than those expressing the wild-type D53 gene. The severity of tillering phenotype in these transgenic plants was correlated with the expression level of the transgene. Notably, overexpression of the wild-type D53 gene also caused a moderate increase in tillering, compared to the vector control plants (<xref rid="nihms541100f2" ref-type="fig">Fig. 2c–e</xref>). These observations suggested that the D53 protein acts as a repressor in the SL-mediated branching-inhibition pathway and that the dominant tillering phenotype of the ). These observations suggested that the D53 protein acts as a repressor in the SL-mediated branching-inhibition pathway and that the dominant tillering phenotype of the d53 mutant was most likely caused by a gain-of-function mutation in d53. To further confirm this, we generated D53 knockdown transgenic plants using a RNA interference (RNAi) approach. As expected, reducing D53 expression in d53 background drastically reduced the tiller number (<xref rid="nihms541100f8" ref-type="fig">Extended Data Fig. 3d, e</xref>). Taken together, these data support the proposition that ). Taken together, these data support the proposition that d53 mutation enhanced D53 activity in repressing SL signaling.', 'Real-time PCR analysis revealed that D53 was widely expressed in the examined rice tissues (<xref rid="nihms541100f2" ref-type="fig">Fig. 2f</xref>). ). D53 promoter driven GUS (β-glucuronidase) reporter gene (pD53::GUS) assay showed that GUS staining was observed in vasculature in roots, shoots, leaves, leaf sheaths, nodes, internodes and young panicles, preferentially in the parenchyma cells surrounding the xylem (<xref rid="nihms541100f11" ref-type="fig">Extended Data Fig. 6a–h</xref>). Moreover, ). Moreover, D53 expression was up-regulated by GR24 treatment in wild-type plants, but down-regulated in six d mutants, suggesting that expression of D53 is regulated by SLs signaling (<xref rid="nihms541100f2" ref-type="fig">Fig. 2g, h</xref>). The D53-GFP (green fluorescent protein) fusion protein is exclusively localized to the nucleus in rice protoplasts and the ). The D53-GFP (green fluorescent protein) fusion protein is exclusively localized to the nucleus in rice protoplasts and the pActin::D53-GFP transgenic root cells (<xref rid="nihms541100f11" ref-type="fig">Extended Data Fig. 6i–p</xref>).).'], 'nihms541100f9': ['D53 is predicted to encode a protein of 1131 amino acids. A BLAST search identified a closely related homolog of D53 (designated D53-like, LOC_Os12g01360) with 96.6% amino acid sequence identity in the rice genome. In addition, D53 homologs were found in other monocots and dicots, but not in lower plants, animals or microbes, indicating that D53-like proteins are specific in higher plants (<xref rid="nihms541100f9" ref-type="fig">Extended Data Fig. 4</xref>). Sequence analysis by the HHpred structure prediction server revealed that D53 shares a similar secondary structure composition, despite low primary sequence homology, to proteins of the class I Clp ATPases family, which are characterized by an N-terminal domain, a D1 ATPase domain, an M domain, and a D2 ATPase domain). Sequence analysis by the HHpred structure prediction server revealed that D53 shares a similar secondary structure composition, despite low primary sequence homology, to proteins of the class I Clp ATPases family, which are characterized by an N-terminal domain, a D1 ATPase domain, an M domain, and a D2 ATPase domain36. Notably, the D2 domain of D53 contains a highly conserved linear sequence, FDLNL, which closely matches the ETHYLENE RESPONSE FACTOR-associated amphiphilic repression (EAR) motif (<xref rid="nihms541100f10" ref-type="fig">Extended Data Fig. 5</xref>), which is known to interact with the TOPLESS family of proteins and involved in transcriptional repression), which is known to interact with the TOPLESS family of proteins and involved in transcriptional repression37.'], 'nihms541100f3': ['Previous studies have identified the F-box protein D3 and the α/β hydrolase D14 as two key components of SL signaling in rice10,12, of which D14 and its orthologues in Arabidopsis (AtD14) and Petunia (DAD2) have been proposed to directly participate in SL perception19,27,28. Yeast two-hybrid assay (Y2H) showed that both D53 and d53 could physically interact with D14 in the presence of GR24 (<xref rid="nihms541100f3" ref-type="fig">Fig. 3a</xref>). Domain deletion analysis indicated that the D1 domain of D53 was essential for the GR24-dependent D53-D14 interaction. Interestingly, its binding activity was inhibited by the M and D2 domains, although their negative effect can be overcome by the N domain (). Domain deletion analysis indicated that the D1 domain of D53 was essential for the GR24-dependent D53-D14 interaction. Interestingly, its binding activity was inhibited by the M and D2 domains, although their negative effect can be overcome by the N domain (<xref rid="nihms541100f12" ref-type="fig">Extended Data Fig. 7</xref>). We verified the D53-D14 interaction in ). We verified the D53-D14 interaction in N. benthamiana leaf cell nucleus both in the presence or absence of exogenously applied GR24 using a bimolecular fluorescence complementation (BiFC) assay (<xref rid="nihms541100f3" ref-type="fig">Fig. 3b</xref>). The observed interaction between D53 and D14 in the absence of exogenously applied GR24 might be due to the effect of endogenous SLs present in the tobacco leaf cells. Consistent with the previously reported GR24-depedent interaction between DAD2 and PhMAX2A (an ortholog of D3 in Petunia) in yeast). The observed interaction between D53 and D14 in the absence of exogenously applied GR24 might be due to the effect of endogenous SLs present in the tobacco leaf cells. Consistent with the previously reported GR24-depedent interaction between DAD2 and PhMAX2A (an ortholog of D3 in Petunia) in yeast19, our in vitro pull-down assay also revealed a direct physical interaction between D14 and D3 in a GR24-dependent manner (<xref rid="nihms541100f3" ref-type="fig">Fig. 3c</xref>). Furthermore, using recombinant GST-D3-OSK1 fusion protein as the bait, our ). Furthermore, using recombinant GST-D3-OSK1 fusion protein as the bait, our in vitro pull-down assay showed that D14 could be more efficiently co-immunoprecipitated (co-IP) from d3 plant extracts in the presence of exogenously applied GR24 (<xref rid="nihms541100f3" ref-type="fig">Fig. 3d</xref>). Together, these results suggest that SLs may act to promote complex formation among D14, D3 and D53, linking D53 to the hormone-perception components of the SL signaling pathway.). Together, these results suggest that SLs may act to promote complex formation among D14, D3 and D53, linking D53 to the hormone-perception components of the SL signaling pathway.', 'It has been speculated that perception of SLs triggers the degradation of putative repressors by the SCFMAX2 ubiquitin ligase complex to suppress shoot branching21,29,30. In this study, we established that D53 acts as a repressor of SL signaling in rice. Consistent with the previous observation of GR24-dependent interaction between DAD2 and PhMAX2 (ref. 19), we found that GR24 also promotes the interaction between D14 with D53 and D3 (<xref rid="nihms541100f3" ref-type="fig">Fig. 3</xref>). Further, we showed that D53 is targeted for degradation by the proteasome in a D14- and D3-dependent manner (). Further, we showed that D53 is targeted for degradation by the proteasome in a D14- and D3-dependent manner (<xref rid="nihms541100f4" ref-type="fig">Fig. 4a–d</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8</xref>). Together, these data collectively support the notion that SL perception by D14 acts to promote ubiquitination of D53 by the D14-SCF). Together, these data collectively support the notion that SL perception by D14 acts to promote ubiquitination of D53 by the D14-SCFD3 ubiquitin ligase, and subsequent degradation of D53 by the proteasome, leading to the propagation of SL signal and downstream physiological responses (<xref rid="nihms541100f5" ref-type="fig">Fig. 5</xref>). Our findings revealed a remarkable similarity between the hormonal perception and signaling mechanism of SL and several other classes of plant hormones, including auxin, jasmonate and gibberellin). Our findings revealed a remarkable similarity between the hormonal perception and signaling mechanism of SL and several other classes of plant hormones, including auxin, jasmonate and gibberellin25,38–40.'], 'nihms541100f4': ['To investigate how SL regulates D53, we performed a set of additional experiments. Both western blot analysis and fluorescence microscopy examination showed that GR24 treatment induced rapid degradation of the D53 protein in wild-type cells, but not in d3 and d14 mutant cells (<xref rid="nihms541100f4" ref-type="fig">Fig. 4a–c</xref>). We further showed that D53 was degraded by the proteasome, as a proteasome inhibitor, MG132, but not other protease inhibitors, effectively blocked GR24-induced D53-GFP degradation (). We further showed that D53 was degraded by the proteasome, as a proteasome inhibitor, MG132, but not other protease inhibitors, effectively blocked GR24-induced D53-GFP degradation (<xref rid="nihms541100f4" ref-type="fig">Fig. 4c, d</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8a, b</xref>). Notably, unlike the wild-type D53-GFP fusion protein, the mutant d53-GFP fusion protein appeared to be stable in the presence of GR24 (). Notably, unlike the wild-type D53-GFP fusion protein, the mutant d53-GFP fusion protein appeared to be stable in the presence of GR24 (<xref rid="nihms541100f4" ref-type="fig">Fig. 4c</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8b</xref>). Interestingly, we noted that D53-GFP and D53-LUC were still degraded in the ). Interestingly, we noted that D53-GFP and D53-LUC were still degraded in the d53 mutant cells, but not in d3 or d14 mutant cells (<xref rid="nihms541100f4" ref-type="fig">Fig. 4d</xref> and and <xref rid="nihms541100f13" ref-type="fig">Extended Data Fig. 8c</xref>), indicating that the D53 degradation pathway was still operational in the ), indicating that the D53 degradation pathway was still operational in the d53 mutant. Together, these results suggest that SL triggers proteasome-mediated degradation of D53 in a D14- and D3-dependent manner. Importantly, the insensitivity of d53 protein to SL-triggered turnover is consistent with the observed dominant gain-of-function mutant phenotype of d53.'], 'nihms541100f14': ['To provide genetic support for the functional relationship between D53, D3 and D14, we generated d3 d53 and d14 d53 double mutants. The d3 mutant had more tillers and it was shorter than the d14 and d53 single mutants (<xref rid="nihms541100f14" ref-type="fig">Extended Data Fig. 9a</xref>). The ). The d14 d53 double mutants exhibited a dwarf tillering phenotype resembling the d14 and d53 parental plants, whereas the d3 d53 double mutant exhibited a dwarf tillering phenotype resembling d3 (<xref rid="nihms541100f4" ref-type="fig">Fig. 4e, f</xref> and and <xref rid="nihms541100f14" ref-type="fig">Extended Data Fig. 9a</xref>). The lack of obvious additive effects among these mutants suggests that ). The lack of obvious additive effects among these mutants suggests that D3, D14 and D53 act in the same signaling pathway. To further test their epistasis relationship, we knocked down D53 gene expression in the d3 and d14 backgrounds. As shown in <xref rid="nihms541100f4" ref-type="fig">Fig. 4g</xref> and and <xref rid="nihms541100f14" ref-type="fig">Extended Data Fig. 9b–d</xref>, the mutant phenotype of , the mutant phenotype of d3 and d14 was restored to nearly wild type levels, demonstrating that D53 acts downstream of D3 and D14, and that accumulation of D53 protein is responsible for blocking SL signaling and conferring the dwarf tillering phenotype in these mutants.']}
|
D3 D14-SCF-dependent degradation of D53 regulates strigolactone signaling
| null |
Nature
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1387440000
|
Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.
|
[
"Amino Acid Sequence",
"Cloning, Molecular",
"Gene Expression Regulation, Plant",
"Lactones",
"Molecular Sequence Data",
"Mutation",
"Oryza",
"Phenotype",
"Plant Growth Regulators",
"Plant Proteins",
"Proteasome Endopeptidase Complex",
"Protein Binding",
"Proteolysis",
"SKP Cullin F-Box Protein Ligases",
"Signal Transduction"
] |
other
|
PMC4096652
| null |
50
|
[
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] |
Nature. 2013 Dec 19; 504(7480):406-410
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NO-CC CODE
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ADAM8 multimerises and interacts with integrin β1 in tumour cells(A)Western blot of integrin β1 subunit (“Int β1”) in Panc1_ctrl (ctrl) and Panc1_A8 (A8) cells (~115 kD). Beta-Tubulin (“β-tub”) was used as loading control. (B) In Panc1 cells transiently expressing empty vector (EV) or ADAM8-Bipro (A8BiPro), anti-BiPro or unrelated IgG was used for immunoprecipitation (IP). Blots were probed with polyclonal β1 integrin antibody. (C) Re-probing of blot (B) using polyclonal anti-ADAM8 antibody. (D) FRET/FLIM analysis of ADAM8-ADAM8 interactions. Panc1 cells were co-transfected with ADAM8-GFP and ADAM8-mCherry (“A8-mCh”) constructs; upper panel, fluorescence lifetime in the absence (Control IgG), lower panel in the presence of anti-β1 antibody (anti-β1), determined in nanoseconds (ns). (E) FRET efficiency (in %) for ADAM8-ADAM8 interaction in anti-β1 antibody treated Panc1 cells (n=10). As statistical test, ANOVA was used; **, p<0.001. (F-H) Localisation of ADAM8 protein in MDA-MB-231 control (“shCtrl”, F) or (H) in MDA-MB-231 cells with a stable β1 integrin knockdown. Staining of ADAM8 in red and β1 integrin in green. Boxed areas in F, a lamellipod structure zoomed in. In contrast to boxed area in F, the one in G shows diffuse staining of ADAM8 mainly in vesicles, the entire cell is flattened and cell area increases significantly. Scale bars (F and G), 10 μm. (H) Localisation of ADAM8 in MDA-MB-231 cells treated with peptide BK-1362 (1µM) or with BK-1361 (1µM). Similar to G, cells flatten and localisation of ADAM8 in lamellipodia is changed. (I) Quantification of cell areas of BK-1362 and BK-1361 treated cells, given as mean ±S.D. from 50 cell areas. **, p<0.001 (Student’s t-test). (J) Quantification of ADAM8 in the cell membrane as average integrated density (AID)/pixel (sum of density 5µm in from cell periphery, divided by total pixels). ANOVA was used as statistical test. *, p<0.01, **, p<0.001 (Student’s t-test). (K) Interaction of ADAM8-GFP and ADAM8-mCh in MDA-MB-231 control (“shCtrl”) and MDA-MB-231 with a stable β1 integrin knockdown (“shβ1”). In the right panel, antibody 12G10 was used to detect activated β1 integrin. (L) Quantification of FRET efficiency between ADAM8-GFP and ADAM8-mCh in MDA-MB-231 control (“shCtrl”) and MDA-MB-231 with stable β1 integrin knockdown (“shβ1”). ANOVA was used; *, p<0.01 and **, p<0.001.(Student’s t-test).
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emss-61632-f004
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2
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a78b7e88a53f915550696011da4a899d32c8554d3cb62b993f80dc2f5625fa1c
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emss-61632-f004.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
800,
1109
] |
[{'image_id': 'emss-61632-f007', 'image_file_name': 'emss-61632-f007.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f007.jpg', 'caption': 'Application of peptide BK-1361 in PDAC mice(A) Kaplan-Meier analysis of KPC/saline, KPC/BK-1362 (control peptide) and KPC/BK-1361 mice (n=10 in each group). Median survival times (MST) are given in the diagram; Statistical analysis was carried out using a Log rank test with p<0.001. (B) Concentration of soluble ADAM8 in normal mouse pancreas (WT) and in pancreata of saline treated and BK-1361 treated KPC mice, as determined by ELISA (n=5) in ng/mg from pancreas. ANOVA was used as statistical test. **, p<0.01, **, p<0.001. (C) Frequency of metastases in liver and lung of KPC/control and KPC/BK-1361 mice. (D) IHC of mice at week 12. Representative PAS staining in KPC/saline vs. KPC/BK-1361 pancreas; scale bar, 200 µm. (E) Whisker plot with quantification of invasive PDAC in saline and BK-1361 treated KPC mice. Tukey-type analysis was used for statistics; **, p<0.001 (Student’s t-test). (F), Whisker plot of normal acinar area in KPC/saline and KPC/BK-1361 treated pancreata (n=5), Tukey-type analysis, *p<0.05 (Student’s t-test). (G,H) PAS staining, ADAM8 and pERK1/2 IHC in KPC/control (G) and KPC/BK-1361 (H) mice. Arrowheads indicate focal acinar cell staining, arrow indicates nuclear staining, and asterisks mark identical positions. Note loss of ductal architecture in control vs. BK-1361 treated mice with positive staining for ADAM8 and pERK1/2. Quantification of IHC for KPC/saline and KPC/BK-1361 pancreas reveals less staining for pERK1/2 in BK-1361 vs. saline treated pancreata. Tukey-type statistical analysis was performed. **< p<0.001 (Student’s t-test). Scale bar in D, 100 µm.', 'hash': '53410f80a2434b27141a7812ae51ae8e5b3a2d225f4a5b4eed30fd039e46f8e6'}, {'image_id': 'emss-61632-f001', 'image_file_name': 'emss-61632-f001.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f001.jpg', 'caption': 'Extracellular ADAM8 processing and inhibition of ADAM8 activity(A) ADAM8 processing: (1) autocatalytic prodomain (red) removal; (2) for active ADAM8; after membrane transport, ADAM8 cleaves membrane proteins (3a); alternatively, removal of a soluble MP (blue) domain (3b) leads to ECM cleavage and formation of remnant ADAM8. A putative interaction site (magenta) located in the disintegrin domain (DI, green). (B) Homology modelling of ADAM8 disintegrin/cysteine-rich (DC) domain based on homology to pdb file 2ao741 within the integrin-binding loop region of ADAM15. The amino acid motif “KD” is potentially accessible to peptidomimetics such as BK-1361 (see table 1). (C) FRET analysis of ADAM8 monomers in the presence of BK-1362 (control peptide “CP”) and BK-1361 (500 nM, respectively) in Panc1 cells. Fluorescence lifetime (in nanoseconds “ns”) and FRET efficiency are calculated from >12 cells in 3 independent experiments. ANOVA was used as statistical test; data are presented as mean ± SEM; p-value *, p<0.05 (Student’s t-test). (D) left panel, complex of pro-ADAM8 (100 ng/lane) at 0 and 24 hours analysed by native gel electrophoresis and immunoblotting. With CP, pro-ADAM8 forms complexes (dimers and trimers, arrowheads 2 and 3); with BK-1361, only monomers of pro-ADMA8 are detected (arrowhead 1); right panel, activation of recombinant pro-ADAM8 in vitro in the presence of CP and BK1361. Bar graph: Autocatalytic activation of pro-ADAM8 ± BK-1361 (200 nM) in vitro. Pro-ADAM8 (100 ng) incubated for indicated times; Fluorescence activity using CD23 fluorogenic peptide monitored over 5 days in triplicates. “FU”, fluorescence units. (E) Effect of BK-1361 and control peptides (see table 1) on ADAM8 MP domain removal in ADAM8-BiPro transfected COS7 cells after 12 hours. Anti-BiPro antibody detects pro (triangle), mature (circle), and remnant (diamond) ADAM8 in cell lysates (WCL); soluble ADAM8 activity from processed ADAM8 is detected in cell supernatants (SN) by CD 23 peptide cleavage. (F) Test of BK-1361 for membrane-bound CD23 in WCL and soluble CD 23 (sCD23) in SNs. Cells were incubated with CP or BK-1361 (500 nM) for 12h. (G) Dose-dependent inhibition of CD23 shedding determined by ELISA (n=5) with mean values ± SD; IC50 value for BK-1361: 182 ± 23 nM, whereas CP shows no significant inhibition of CD23 shedding.', 'hash': '14005d063aa5c402a31af1254fa74df2e32fed8f9c401518627cfb82005fc508'}, {'image_id': 'emss-61632-f006', 'image_file_name': 'emss-61632-f006.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f006.jpg', 'caption': 'Effect of ADAM8 inhibition in an orthotopic pancreatic cancer model(A) Pancreas morphology after orthotopic injection of Panc1_A8, Panc1_ctrl, or Panc1_A8 cells followed by BK-1361 injection (10 µg/g daily, n=12 per group). (B) Tumour load [in g] 12 days after implantation (n=12); Tukey’s linear contrast test was used. **, 0.001, ***, p<0.0001 (Student’s t-test) (C) Representative ADAM8 IHC/Periodic Acid Schiffs (PAS) staining of pancreas tumours from mice injected with Panc1_A8, Panc1_ctrl compared to implanted Panc1_A8/BK-1361 treated. Significant invasion of Panc1_A8 cells (arrows, upper panel left); Panc1_ctrl and Panc1_A8/BK-1361 injected tumours show less invasion from implantation site. Lower panel, ADAM8 staining; infiltration and loss of ductal architecture, increase in ADAM8 in Panc1_ctrl implanted tumours, and delineated between implantation site and surrounding pancreas tissue in Panc1_A8/BK-1361 treated mice. Lower panel, left, enlarged image shows invasion of ADAM8-positive tumour cells; enlarged images (boxed in upper panel); box1: upregulation of ADAM8 in cell mass of Panc1_ctrl cells; box 2: enlarged view of the border between implantation site and surrounding ductal tissue; box 3: signs of intratumoural necrosis in tumours treated with BK-1361 (bar, 100 µm). (D) pERK1/2 staining of tumours. Only for Panc1_A8 (top), tumour cells stain positive for pERK1/2 (bar, 100 µm). (E) Morphology of spleen and diaphragm from mice injected with either Panc1_A8, Panc1_ctrl, or Panc1_A8/BK-1361. In PAS stains (lower panel), infiltration of tumour cells in the diaphragm is only seen in Panc1_A8. Scale bar, 100 µm. (F) Quantification of infiltration areas in peritoneum, diaphragm and spleen. Values are in % of total tissue area ±SEM (n=12). (G) ADAM8+ micrometastases in livers from Panc1_A8 injected mice; in contrast, metastases were mostly absent in livers from Panc1_ctrl and Panc1_A8/BK-1361 treated mice (bar, 200 µm); relative frequency of liver metastases in mice injected with Panc1_A8, Panc1_ctrl, and Panc1_A8/BK-1361, respectively (n=12). (H) Pancreas histology (PAS, ADAM8 IHC) after orthotopic implantation of AsPC-1_shCtrl and AsPC-1_shA8/2 cells. Scale bar in I, left upper panel, 100 µm. ADAM8+ cells in pancreas injected with AsPC-1_shCtrl cells, weaker staining with AsPC-1_shA8 cells. Infiltration areas in pancreata from 6 mice/group were quantified. Mean values ±SEM. **, p<0.01 (Student’s t-test).', 'hash': '763c34d87b1eb793b57ef88acb362fd24b6bf3309dba6f33680108bfb83eaf17'}, {'image_id': 'emss-61632-f005', 'image_file_name': 'emss-61632-f005.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f005.jpg', 'caption': 'ADAM8 intracellular kinase signalling(A) Antibody detection of FAK phosphorylation (Y397) in Panc1_A8 and Panc1_ctrl cells 24h after plating cells on plastic. Total FAK antibody was used to control equal loading and anti-ADAM8 confirms ADAM8 expression. A beta-Tubulin antibody was used as loading control. Note a 2.8-fold increase in pFAK in Panc1_A8 cells compared to Panc1_ctrl cells. (B) Western Blot of relative phosphorylation of ERK1/2, in Panc1_ctrl and Panc1_A8 cells as determined by antibodies directed against phosphorylated forms of respective kinases. Lower panel, ERK1/2 phosphorylation in Panc1_ctrl and Panc1_A8 cells in the presence of 500 nM BK-1361. (C) Relative invasiveness of Panc1_ctrl and Panc1_A8 cells (in duplicates) in the presence of ERK1/2 inhibitor U0126 (10 µM) after 18h incubation, indicating a strong correlation between ERK1/2 phosphorylation and invasion in Panc1 cells. ANOVA statistical test was used; *, p<0.01; **, p<0.001 (Student’s t-test). (D) Correlation of ADAM8 and pERK1/2 levels in AsPC-1 control (“shCtrl”) and knockdown (“shA8”) cells. In clone AsPC-1_shA8/2, levels of pERK1/2 are reduced by 3-fold. (E) Relative kinase phosphorylation of ERK and AKT signalling pathways after 18 h incubation with (“+”) or without (“−“) U0126 (10 µM) in protein lysates of AsPC-1_shCtrl and AsPC-1_shA8 cells. Kinases and their respective phosphorylation sites are indicated on the right. (F) Membrane-bound ADAM8 with cytoplasmic domain is required for FAK and ERK1/2 activation. Panc1 cells were transiently transfected with full-length ADAM8 construct (“A8 wt”), ADAM8 lacking the cytoplasmic domain (“A8ΔCD”), or with a construct encoding the soluble ectodomain (“A8ecto”) of ADAM8, respectively. To control for cellular ADAM8 activity, the amount of soluble CD23 was determined in supernatants by using an HA-tag antibody. The presence of ADAM8 protein variants, relative levels of pFAK and pERK1/2 were detected by WB, using beta–Tubulin as loading control. Levels of FAK and ERK phosphorylation were quantified from 3 independent experiments, given as mean values ± S.E.M.', 'hash': '3e11970020ea0f91379e3930cb5bd04cb87d7fe6249a47fd6d9ec70faa946eff'}, {'image_id': 'emss-61632-f002', 'image_file_name': 'emss-61632-f002.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f002.jpg', 'caption': 'Effect of ADAM8 expression in Panc1 cells(A) Western blot of ADAM8 in PDAC cell lines Panc1, Capan-1, AsPC-1 (B) ADAM8 immunofluorescence (anti-ADAM8, green) in non-permeabilised Panc1_ctrl and Panc1_A8 cells. ADAM8 membrane staining in Panc1_ctrl (Upper panel), and Panc1_A8 (lower panel) cells. Insert (lower panel), ADAM8 localisation in membrane protrusions. Scale bar, 50 µm; (C) Western Blot of whole cell lysates (WCL) and supernatants (SN) from Panc1_ctrl and Panc1_A8 cells; proform (arrowhead), mature (circle) and remnant form (diamond) of ADAM8, respectively. In Panc1_A8 supernatant, a ~30 kDa soluble ADAM8 MP fragment is detectable (filled arrowhead). (D) ADAM8 activities in supernatants of Panc1_ctrl and Panc1_A8 cells were measured as fluorescence units (“FU”, n=5) by cleavage of CD23 substrate; ***, p-value < 0.0001 (Student’s t-test). (E), Scratch assay of Panc1_ctrl vs. Panc1_A8 cells treated either with BB-94 (100 nM “+”, 1µM “++”) or with BK-1361 (200 “+”, 500 nM “++”). Cell counts after t=0h and 12h (3 independent experiments in triplicates, n=9); ANOVA was used as statistical test; p-values *, <0.01, **, p<0.001 (Student’s t-test). (F) Invasion of Panc1_ctrl and Panc1_A8 cells in collagen I (Coll I), collagen IV (Coll IV), fibronectin (FN) and Matrigel (MG) analysed in Boyden chambers (18 h). Data presented as mean ± SD from 5 independent experiments. ANOVA test was used. *, p<0.01, **, p<0.001, ***, p<0.0001 (Student’s t-test). (G) PrAMA inference analysis of supernatants (SN) and membranes (Mem) from Panc1_ctrl and Panc1_A8 cells using 5 fluorogenic peptides to detect MMP activities. Activities of MMP-2, MMP-14, and ADAM8 are shown as relative changes to Panc1_ctrl. (H) Western blot and gelatine zymography of Panc1_ctrl and Panc1_A8 supernatants for MMP-2, MMP-2 activity, and MMP-14. (I) Invasion of Panc1_A8 cells treated with BK-1361 (10, 100, 1000 nM) or 1000 nM control peptides for 6 hours, respectively (see table 1). Invasion was determined after 18 hours. ANOVA test was used. *, p<0.005, **, p<0.001, ***, p<0.0001. (J) PrAMA from Panc1_A8 cells treated with vehicle, BK-1361, BB-94, and U0126 (1µM, respectively) for 24 hours. Values are given relative to Panc1_A8 treated with vehicle only. ANOVA was used as statistical test; p-values *, p<0.1, **, p<0.01, ***, p<0.001 (Student’s t-test).', 'hash': '3633cac218a283d902a5a9da416589683c65db39271f165c703daba91890813c'}, {'image_id': 'emss-61632-f003', 'image_file_name': 'emss-61632-f003.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f003.jpg', 'caption': 'ADAM8 inhibition reduces MMP2 and 14 activity and affects invasiveness.(A) Stable ADAM8 knockdown (shA8) of AsPC-1 cell clones were generated and characterised for ADAM8 protein (B) and mRNA (C) expression. Values are given as mean values from 3 independent experiments performed in triplicates ± SEM. Using these cell clones, (D) scratch and (E) matrigel invasion assays were performed. In both assays, an ADAM8 gene dosage dependent effect was observed. (F) Matrigel invasion was determined after addition of BK-1361 (0-1000 nM) or control peptides (see table 1). For D-F, values are given as mean values ± SEM. ANOVA was used as statistical test. P-values *, p<0.1, **, p<0.01, ***, p<0.005 (Student’s t-test). (G) PrAMA inference analysis of two AsPC-1 ADAM8 knockdown clones and of AsPC-1_shCtrl cells treated with BK-1361, relative to AsPC-1_shCtrl cells. Note that both, genetic and pharmacological inhibition of ADAM8 show similar effects, i.e. reduction of MMP-2 and MMP-14 activity with the exception of MMP-14 in AsPC-1 cells.', 'hash': '3a328ba9ba9e4287bfb9e119e3999bf96b6bb03dae5a98c96acad24f4c15b6e3'}, {'image_id': 'emss-61632-f004', 'image_file_name': 'emss-61632-f004.jpg', 'image_path': '../data/media_files/PMC5014123/emss-61632-f004.jpg', 'caption': 'ADAM8 multimerises and interacts with integrin β1 in tumour cells(A)Western blot of integrin β1 subunit (“Int β1”) in Panc1_ctrl (ctrl) and Panc1_A8 (A8) cells (~115 kD). Beta-Tubulin (“β-tub”) was used as loading control. (B) In Panc1 cells transiently expressing empty vector (EV) or ADAM8-Bipro (A8BiPro), anti-BiPro or unrelated IgG was used for immunoprecipitation (IP). Blots were probed with polyclonal β1 integrin antibody. (C) Re-probing of blot (B) using polyclonal anti-ADAM8 antibody. (D) FRET/FLIM analysis of ADAM8-ADAM8 interactions. Panc1 cells were co-transfected with ADAM8-GFP and ADAM8-mCherry (“A8-mCh”) constructs; upper panel, fluorescence lifetime in the absence (Control IgG), lower panel in the presence of anti-β1 antibody (anti-β1), determined in nanoseconds (ns). (E) FRET efficiency (in %) for ADAM8-ADAM8 interaction in anti-β1 antibody treated Panc1 cells (n=10). As statistical test, ANOVA was used; **, p<0.001. (F-H) Localisation of ADAM8 protein in MDA-MB-231 control (“shCtrl”, F) or (H) in MDA-MB-231 cells with a stable β1 integrin knockdown. Staining of ADAM8 in red and β1 integrin in green. Boxed areas in F, a lamellipod structure zoomed in. In contrast to boxed area in F, the one in G shows diffuse staining of ADAM8 mainly in vesicles, the entire cell is flattened and cell area increases significantly. Scale bars (F and G), 10 μm. (H) Localisation of ADAM8 in MDA-MB-231 cells treated with peptide BK-1362 (1µM) or with BK-1361 (1µM). Similar to G, cells flatten and localisation of ADAM8 in lamellipodia is changed. (I) Quantification of cell areas of BK-1362 and BK-1361 treated cells, given as mean ±S.D. from 50 cell areas. **, p<0.001 (Student’s t-test). (J) Quantification of ADAM8 in the cell membrane as average integrated density (AID)/pixel (sum of density 5µm in from cell periphery, divided by total pixels). ANOVA was used as statistical test. *, p<0.01, **, p<0.001 (Student’s t-test). (K) Interaction of ADAM8-GFP and ADAM8-mCh in MDA-MB-231 control (“shCtrl”) and MDA-MB-231 with a stable β1 integrin knockdown (“shβ1”). In the right panel, antibody 12G10 was used to detect activated β1 integrin. (L) Quantification of FRET efficiency between ADAM8-GFP and ADAM8-mCh in MDA-MB-231 control (“shCtrl”) and MDA-MB-231 with stable β1 integrin knockdown (“shβ1”). ANOVA was used; *, p<0.01 and **, p<0.001.(Student’s t-test).', 'hash': 'a78b7e88a53f915550696011da4a899d32c8554d3cb62b993f80dc2f5625fa1c'}]
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{'emss-61632-f001': ['Cellular activation of ADAM8 occurs in two steps. The first is intracellular prodomain removal in vesicles while the second is metalloprotease (MP) domain removal from membrane-bound activated ADAM8 (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1A</xref>). Autocatalysis implies that ADAM8 multimerises (). Autocatalysis implies that ADAM8 multimerises (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1A</xref>) and that the ADAM8 disintegrin/cysteine-rich (DC) domain is critical for multimerisation as demonstrated previously by using an antibody directed against the DC domain) and that the ADAM8 disintegrin/cysteine-rich (DC) domain is critical for multimerisation as demonstrated previously by using an antibody directed against the DC domain35. To define the regions in the DC domain involved in ADAM8-ADAM8 interactions, homology modelling of the ADAM8 DC domain was performed based on the ADAM10 sequence derived from Janes et al.40 (Supplementary Fig. 1 and 2). We hypothesised that an extended loop structure exposing the RGD-like positions of the “KDX” motif in the integrin binding loop (IBL) in human and mouse ADAM8 might be responsible for the observed interactions (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1B</xref>).).', 'In mouse and human ADAM8, the amino acid residues K and D are exposed towards the outer aspect of the disintegrin (DI) domain thereby forming a potential contact interface (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1B</xref>, labelled in magenta). To generate a peptidomimetic compound, a series of cyclic peptides (6 aa) mimicking the motif “RLSKDK” of mouse ADAM8 in the IBL were generated. Amino acids R, L, or S were inserted in the peptide as D-amino acids to alter the conformational constraint of the KDK motif and to generate a potentially more stable peptide for , labelled in magenta). To generate a peptidomimetic compound, a series of cyclic peptides (6 aa) mimicking the motif “RLSKDK” of mouse ADAM8 in the IBL were generated. Amino acids R, L, or S were inserted in the peptide as D-amino acids to alter the conformational constraint of the KDK motif and to generate a potentially more stable peptide for in vivo work. The cyclic peptide sequence RLsKDK with “s” as D-serine named BK-1361 was most effective in blocking ADAM8-dependent cell adhesion in mouse and human cells with similar efficiencies (Supplementary Fig. 3) and ADAM8-ADAM8 interactions as shown by reduced FRET/FLIM efficiency. In contrast, a control peptide (CP, RLsADK; <xref ref-type="fig" rid="emss-61632-f001">Fig. 1C</xref>) had no effect.) had no effect.', 'Multimerisation of ADAM8 was investigated by native gel electrophoresis (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1 D</xref>). Under native conditions, 100 ng of pro-ADAM8 associates in dimeric (~120 kD) and trimeric (~180 kD) complexes of pro-ADAM8, while in the presence of BK-1361, only monomers (~60 kD) were detected (). Under native conditions, 100 ng of pro-ADAM8 associates in dimeric (~120 kD) and trimeric (~180 kD) complexes of pro-ADAM8, while in the presence of BK-1361, only monomers (~60 kD) were detected (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1D</xref>, left panel). Detection of dimers and trimers suggests that dimers associate by disintegrin domain (homophilic) interactions, whereas trimers could be formed by a different mode of interaction. At higher concentrations of recombinant ADAM8, we detected even larger complexes as a result of greater order multimerisation (, left panel). Detection of dimers and trimers suggests that dimers associate by disintegrin domain (homophilic) interactions, whereas trimers could be formed by a different mode of interaction. At higher concentrations of recombinant ADAM8, we detected even larger complexes as a result of greater order multimerisation (Supplementary Figure 3E) in agreement with more than one interaction mode that results in ADAM8 complex formation. These interactions can be blocked by BK-1361 and we further analysed if prevention of complex formation affects ADAM8 activity in vitro.', 'Activation of pro-ADAM8 was detected over a time course of 120 h (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1D</xref>, right panel) but blocked by BK-1361 (, right panel) but blocked by BK-1361 (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1D</xref>, bar graph) with an IC, bar graph) with an IC50 of 120 ± 19 nM. In a cell-based assay, BK-1361 and other peptides (see table 1) were tested for their ability to inhibit ADAM8-dependent extracellular resulting in an active soluble MP domain (<xref ref-type="fig" rid="emss-61632-f001">Figure 1 A</xref>, , <xref ref-type="fig" rid="emss-61632-f003">3b</xref>). In cell lysates, presence of the remnant form indicates cellular processing of ADAM8 as seen with no or inactive control peptides (). In cell lysates, presence of the remnant form indicates cellular processing of ADAM8 as seen with no or inactive control peptides (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1 E</xref>, upper panel). In cell supernatants, processing results in detectable activity of released ADAM8 MP ‥ BK-1361, but not other BK peptide variants, decreased this activity (, upper panel). In cell supernatants, processing results in detectable activity of released ADAM8 MP ‥ BK-1361, but not other BK peptide variants, decreased this activity (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1 E</xref>, lower panel). BK-1361 blocks mouse and human ADAM8 , lower panel). BK-1361 blocks mouse and human ADAM8 in vitro with similar efficiencies, a prerequisite for testing BK-1361 in orthotopic PDAC models using human donor cells in mouse hosts.', 'We next tested BK-1361 and variants for their ability to inhibit shedding of CD23, a known substrate of ADAM830 in cell-based shedding assays (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1F</xref> and and table 1). Co-transfection of ADAM8 with a tagged CD23 construct in COS7 cells resulted in significant shedding of CD23 as soluble 21 kD fragment (sCD23); sCD23 was detectable in supernatants when ADAM8 was co-expressed. In the presence of 500 nM BK-1361, sCD23 was undetectable, demonstrating that BK-1361 inhibits the in vivo shedding activity of ADAM8. ELISA assays were performed to determine [sCD23] vs. [BK-1361] with an IC50 of 182 ± 23 nM for BK-1361; BK-1362 had no significant effect (<xref ref-type="fig" rid="emss-61632-f001">Fig. 1G</xref>). We conclude that BK-1361 affects ADAM8-ADAM8 interactions thereby inhibiting cellular shedding and autocatalytic activation of ADAM8 ). We conclude that BK-1361 affects ADAM8-ADAM8 interactions thereby inhibiting cellular shedding and autocatalytic activation of ADAM8 in vitro and in a cell-based assay in a specific manner, as neither catalytic activities of ADAM 9, 10, 12, 17 nor MMP-2, -9, and -14 were inhibited in concentrations of up to 10 µM (Table 2).'], 'emss-61632-f002': ['As shown earlier, ADAM8 expression is correlated with invasiveness in vitro in various cell lines15,20,21. However, no functional in vivo data on ADAM8 in pancreatic malignancies are yet available. To establish cell lines for analysis of PDAC in vivo, we selected Panc1 (very low ADAM8) and AsPC-1 cells (high endogenous levels of ADAM8) as determined by Western Blot15 (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2A</xref>). Panc1 cell lines with a moderate over-expression of ADAM8 (Panc1_A8; NM_001109.4; ). Panc1 cell lines with a moderate over-expression of ADAM8 (Panc1_A8; NM_001109.4; <xref ref-type="fig" rid="emss-61632-f002">Fig. 2B</xref>) and AsPC-1 cell lines with ) and AsPC-1 cell lines with ADAM8 shRNA knockdown constructs were generated. Microarray analysis of Panc1_ctrl vs. Panc1_A8 cells revealed that Panc1_A8 cells showed no off-target effects of the ADAM8 knockdown, as expression levels of >95% of genes were unchanged including genes encoding MMPs, other ADAMs, ADAMTS, and TIMP1-4 levels (Supplementary Fig. 4) Cellular localisation of ADAM8, analysed by CLSM, confirmed low ADAM8 expression in Panc1_ctrl cells compared to enhanced ADAM8 expression in Panc1_A8 cells. In Panc1_A8 cells, ADAM8 is localised in the cell membrane and membrane extrusions (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2B</xref>). In supernatants of different Panc1_A8 cell clones characterised (n=30), ADAM8 catalytic activity correlated with the ). In supernatants of different Panc1_A8 cell clones characterised (n=30), ADAM8 catalytic activity correlated with the ADAM8 dosage, determined by western blotting (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2C</xref>) and CD23 fluorescence assay () and CD23 fluorescence assay (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2D</xref>). Migration and invasion behaviour of Panc1_ctrl cells was compared to Panc1_A8 cells ). Migration and invasion behaviour of Panc1_ctrl cells was compared to Panc1_A8 cells in vitro (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2E and F</xref>). In wound-healing (“scratch”) assays, migration rates of Panc1_A8 cells were significantly increased (8 ± 3.8-fold) compared to Panc1_ctrl cells. Panc1_A8 cell migration is inhibited by BB-94, a metalloprotease inhibitor. In addition, application of BK-1361 reduced migration rates of Panc1_A8 cells significantly, but not to the same level as BB-94, suggesting that ADAM8 modulates other metalloprotease activities accounting for migration above control levels. In accordance, invasion into different ECM substrates collagen I, collagenIV, fibronectin, and Matrigel was enhanced by ADAM8 (). In wound-healing (“scratch”) assays, migration rates of Panc1_A8 cells were significantly increased (8 ± 3.8-fold) compared to Panc1_ctrl cells. Panc1_A8 cell migration is inhibited by BB-94, a metalloprotease inhibitor. In addition, application of BK-1361 reduced migration rates of Panc1_A8 cells significantly, but not to the same level as BB-94, suggesting that ADAM8 modulates other metalloprotease activities accounting for migration above control levels. In accordance, invasion into different ECM substrates collagen I, collagenIV, fibronectin, and Matrigel was enhanced by ADAM8 (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2F</xref>).).', 'A proteolytic activity matrix assay (PrAMA)41 was used for simultaneous detection of multiple activities in Panc1_ctrl and Panc1_A8 cells that could account for the observed invasiveness. Briefly, PrAMA is based on the knowledge of individual FRET-substrate MMP/ADAM cleavage signatures using purified enzymes41. Panels of FRET-substrate cleavage measurements can be used to infer a dynamic, quantitative and specific profile of MMP/ADAM proteolytic activities from complex enzyme mixtures such a supernatants and solubilised membranes (see M&M and Supplementary Fig. 5). PrAMA inference revealed increased activities of MMP-2 in supernatants and MMP-14 (MT1-MMP) in cell membranes of Panc1_A8 cells compared to Panc1_ctrl cells (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2G</xref> and and Supplementary Fig. 5). Enhanced ADAM8 activities were detected in supernatants and membranes of Panc1_A8 cells. Gelatine zymography and Western blot for MMP-2 and MMP-14 confirmed increased activity of MMP-2 and higher membrane concentration of MT1-MMP (<xref ref-type="fig" rid="emss-61632-f002">Fig 2H</xref>). However, elevated MMP activities in Panc1_A8 cells are not due to transcriptional activation of ). However, elevated MMP activities in Panc1_A8 cells are not due to transcriptional activation of MMP-2 and MMP-14 (Supplementary Fig. 4).', 'We further investigated whether BK-1361 is able to affect ADAM8-dependent invasiveness and MMP secretion of Panc1_A8 cells (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2I and J</xref>). BK-1361 and peptide variants were tested for their ability to block invasiveness of Panc1_A8 cells (). BK-1361 and peptide variants were tested for their ability to block invasiveness of Panc1_A8 cells (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2I</xref>). A dose-dependent effect of BK-1361 on invasion of Panc1_A8 cells was observed. From control peptides, only BK-1364 had a slight effect on invasion. In parallel, we performed PrAMA assays in Panc1_A8 cells to evaluate MMP-2, MMP-14, and ADAM8 activities in the presence of BK-1361, BB-94, and the ERK1/2 inhibitor U0126, respectively (). A dose-dependent effect of BK-1361 on invasion of Panc1_A8 cells was observed. From control peptides, only BK-1364 had a slight effect on invasion. In parallel, we performed PrAMA assays in Panc1_A8 cells to evaluate MMP-2, MMP-14, and ADAM8 activities in the presence of BK-1361, BB-94, and the ERK1/2 inhibitor U0126, respectively (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2J</xref>). BK-1361 and BB-94 reduced activities of MMP-2, MMP-14, and ADAM8. ERK inhibition had a slight effect on MMP-2 activity and a greater effect on MMP-14 activity whilst ADAM8 activity was not affected. These findings argue for an ERK1/2- mediated effect on MMP-14, and, to a lesser extent, on MMP-2 activation. ADAM8 as regulator of ERK1/2 activation is not directly affected by U0126.). BK-1361 and BB-94 reduced activities of MMP-2, MMP-14, and ADAM8. ERK inhibition had a slight effect on MMP-2 activity and a greater effect on MMP-14 activity whilst ADAM8 activity was not affected. These findings argue for an ERK1/2- mediated effect on MMP-14, and, to a lesser extent, on MMP-2 activation. ADAM8 as regulator of ERK1/2 activation is not directly affected by U0126.', 'Our data suggest an effect of pharmacological ADAM8 inhibition on invasiveness of PDAC cells. To determine the effect of a genetic ADAM8 knockdown on cellular invasiveness, we selected AsPC-1 cells with high endogenous ADAM8 levels (<xref ref-type="fig" rid="emss-61632-f002">Fig. 2A</xref>) and generated AsPC_1 cell clones carrying a stable knockdown of ) and generated AsPC_1 cell clones carrying a stable knockdown of ADAM8 (sh_A8). Three representative cell clones from different sh_A8 constructs were analysed for ADAM8 expression (<xref ref-type="fig" rid="emss-61632-f003">Fig. 3A-C</xref>), cell migration and invasion (), cell migration and invasion (<xref ref-type="fig" rid="emss-61632-f003">Fig. 3 D,E</xref>, , Supplementary Movie 1). Knockdown of ADAM8 in AsPC-1 cells caused a significant drop in cell migration depending on the gene dosage of ADAM8. Invasion of AsPC-1 cells was similarly affected by ADAM8 dosage (<xref ref-type="fig" rid="emss-61632-f003">Fig.3E</xref>). BK-1361 treatment of wild-type AsPC-1 cells was similar to the genetic knockdown of ). BK-1361 treatment of wild-type AsPC-1 cells was similar to the genetic knockdown of ADAM8 with 87 ± 3.5% inhibition (<xref ref-type="fig" rid="emss-61632-f003">Fig. 3F</xref>). PrAMA assays were performed with AsPC-1_shCtrl ± BK-1361, and AsPC-1_shA8 cell clones 1 and 2 (). PrAMA assays were performed with AsPC-1_shCtrl ± BK-1361, and AsPC-1_shA8 cell clones 1 and 2 (<xref ref-type="fig" rid="emss-61632-f003">Fig. 3G</xref>). Reduction of MMP-2 and MMP-14 activities were observed in AsPC-1_shA8 clones. In BK-1361 treated AsPC-1_shCtrl cells, MMP-2 was similarly affected, however, the effect on MMP-14 was less pronounced (). Reduction of MMP-2 and MMP-14 activities were observed in AsPC-1_shA8 clones. In BK-1361 treated AsPC-1_shCtrl cells, MMP-2 was similarly affected, however, the effect on MMP-14 was less pronounced (<xref ref-type="fig" rid="emss-61632-f003">Fig. 3G</xref>).).'], 'emss-61632-f004': ['The membrane localisation of ADAM8 in Panc1_A8 cells suggests that ADAM8 is complexed with cellular integrins thereby enhancing cell migration and invasiveness. To investigate this, co-immunoprecipitation (co-IP) experiments were performed in Panc1 cells expressing either control or a tagged ADAM8 construct (ADAM8-BiPro).,As a result, β1 integrin, present in comparable amounts in Panc1_ctrl and Panc1_A8 cells, was co-immunoprecipitated with ADAM8 (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4 A,B,C</xref>). To analyse cellular ADAM8-β1 integrin and ADAM8-ADAM8 interactions, FRET/FLIM analyses were performed to detect FRET in cell lines expressing fusion proteins ADAM8-GFP and ADAM8-mCherry, respectively. ADAM8 multimerisation was detected in Panc1 cells (). To analyse cellular ADAM8-β1 integrin and ADAM8-ADAM8 interactions, FRET/FLIM analyses were performed to detect FRET in cell lines expressing fusion proteins ADAM8-GFP and ADAM8-mCherry, respectively. ADAM8 multimerisation was detected in Panc1 cells (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4D&E</xref>), indicated by FRET efficiency of 7.5 ± 0.93%. In cells, complex formation and membrane localisation of ADAM8 ), indicated by FRET efficiency of 7.5 ± 0.93%. In cells, complex formation and membrane localisation of ADAM8 in vivo involves β1 integrin, since treatment of Panc1 cells with a β1 integrin-blocking antibody (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4D and E</xref>) resulted in a significant drop to 3.1 ± 0.8% FRET efficiency. Moreover, MDA-MB-231 breast cancer cells lacking β1 integrin () resulted in a significant drop to 3.1 ± 0.8% FRET efficiency. Moreover, MDA-MB-231 breast cancer cells lacking β1 integrin (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4F and G</xref>) show a significant change in cellular morphology whilst ADAM8 localisation in lamellipod structures is lost. Interestingly, administration of BK-1361 causes a similar change in cell morphology () show a significant change in cellular morphology whilst ADAM8 localisation in lamellipod structures is lost. Interestingly, administration of BK-1361 causes a similar change in cell morphology (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4 H and I</xref>). We conclude that β1 integrin knockdown or specific ADAM8 inhibition have similar effects on ADAM8 membrane localisation (). We conclude that β1 integrin knockdown or specific ADAM8 inhibition have similar effects on ADAM8 membrane localisation (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4J</xref>). In addition, areas of positive ADAM8-FRET in MDA-MB-231 cells were analysed for β1 integrin (). In addition, areas of positive ADAM8-FRET in MDA-MB-231 cells were analysed for β1 integrin (<xref ref-type="fig" rid="emss-61632-f004">Fig. 4 K and L</xref>). In most areas, activated β1 integrin was detected by antibody 12G10, suggesting that ADAM8 interaction and β1 integrin activation are correlated.). In most areas, activated β1 integrin was detected by antibody 12G10, suggesting that ADAM8 interaction and β1 integrin activation are correlated.', 'Equal amounts of protein were loaded onto 10% reducing polyacrylamide gels. The proteins were transferred on nitrocellulose, unspecific binding sites were blocked (5% skimmed milk, 0.1% Tween-20 in PBS) and specific antibodies were used to detect the proteins of interest. An enhanced chemiluminescent (ECL) substrate (Thermo Scientific) for horseradish peroxidase (HRP) enzyme and a chemiluminescence reader (Intas, Goettingen, Germany) were used for visualization. Images in <xref ref-type="fig" rid="emss-61632-f004">Figure 4</xref> and and <xref ref-type="fig" rid="emss-61632-f005">5</xref> have been cropped for presentation. Full size images are presented in have been cropped for presentation. Full size images are presented in supplementary Figures 11 and 12.'], 'emss-61632-f005': ['To analyse if ADAM8 interactions cause altered intracellular signalling, a MAP kinase array was used to screen for kinase phosphorylation in Panc1_ctrl and Panc1_A8 cells (Supplementary Fig. 6). As potential downstream effectors of the observed ADAM8-β1 integrin interaction, we investigated phosphorylation of Focal adhesion kinase (FAK), ERK1/2, Akt, and p38γin Panc1_A8 vs. Panc1_ctrl cells. FAK was described as β1 integrin interacting protein42. In Western Blots, increased phosphorylation of FAK at residue Tyr397 correlates with ADAM8 expression levels in Panc1 cells (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5A</xref>). In addition to pFAK, we detected increased phosphorylation of ERK1/2 (p44/p42) in Panc1_A8 cells by western blotting using corresponding phospho-specific antibodies (3.4-fold ±0.2 Panc1_A8 vs. Panc1_ctrl, ). In addition to pFAK, we detected increased phosphorylation of ERK1/2 (p44/p42) in Panc1_A8 cells by western blotting using corresponding phospho-specific antibodies (3.4-fold ±0.2 Panc1_A8 vs. Panc1_ctrl, <xref ref-type="fig" rid="emss-61632-f005">Fig. 5B</xref>). The observed increase in pERK1/2 was reduced in Panc1_A8 cells treated with BK-1361 (). The observed increase in pERK1/2 was reduced in Panc1_A8 cells treated with BK-1361 (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5B</xref>). To correlate ERK1/2 phosphorylation with the observed invasiveness of Panc1 cells, matrigel invasion assays were performed in the presence of U0126 (). To correlate ERK1/2 phosphorylation with the observed invasiveness of Panc1 cells, matrigel invasion assays were performed in the presence of U0126 (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5C</xref>). U0126 blocked ERK1/2 phosphorylation in Panc1_ctrl and Panc1_A8 cells and resulted in decreased invasion of Panc1_A8 cells. In addition, AsPC-1_shCtrl and three AsPC-1_shA8 cell clones with different ADAM8 levels were analysed for pERK1/2 levels (). U0126 blocked ERK1/2 phosphorylation in Panc1_ctrl and Panc1_A8 cells and resulted in decreased invasion of Panc1_A8 cells. In addition, AsPC-1_shCtrl and three AsPC-1_shA8 cell clones with different ADAM8 levels were analysed for pERK1/2 levels (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5 D</xref>). In cell clone AsPC-1_shA8/2, pERK1/2 levels were reduced by 2.9 ± 0.3 fold suggesting that ADAM8 expression levels are correlated with pERK1/2. In AsPC-1 cells, ADAM8 levels affect MEK1/2, p-Akt and c-Raf activation (). In cell clone AsPC-1_shA8/2, pERK1/2 levels were reduced by 2.9 ± 0.3 fold suggesting that ADAM8 expression levels are correlated with pERK1/2. In AsPC-1 cells, ADAM8 levels affect MEK1/2, p-Akt and c-Raf activation (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5E</xref>). In addition, a β1-integrin antibody that blocks activation was able to reduce pERK1/2 levels in Panc1_A8 cells, demonstrating that β1-integrin is required for ADAM8-dependent ERK1/2 activation (). In addition, a β1-integrin antibody that blocks activation was able to reduce pERK1/2 levels in Panc1_A8 cells, demonstrating that β1-integrin is required for ADAM8-dependent ERK1/2 activation (Supplementary Fig. 7).', 'To investigate whether the observed FAK and ERK1/2 activation depends on membrane bound ADAM8, Panc1 cells were transfected with wild-type ADAM8 (“A8 wt”), an ADAM8 construct lacking the cytoplasmic domain (“A8ΔCD”), or a soluble ADAM8 (“A8ecto”) construct (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5F</xref>). First, we confirmed that all constructs are catalytically active, as all three ADAM8 proteins shed CD23 (“sCD23”) from the cell membrane. Interestingly, neither ∆CD nor the ectodomain of ADAM8 were able to activate FAK and ERK1/2 (). First, we confirmed that all constructs are catalytically active, as all three ADAM8 proteins shed CD23 (“sCD23”) from the cell membrane. Interestingly, neither ∆CD nor the ectodomain of ADAM8 were able to activate FAK and ERK1/2 (<xref ref-type="fig" rid="emss-61632-f005">Fig. 5F</xref>), suggesting that intracellular signalling mediated by ADAM8 requires membrane localisation of ADAM8 and the presence of the cytoplasmic domain. As potential substrates for ERK activation i.e. the EGFR ligand family such as HB-EGF, EGF, or amphiregulin, were screened (), suggesting that intracellular signalling mediated by ADAM8 requires membrane localisation of ADAM8 and the presence of the cytoplasmic domain. As potential substrates for ERK activation i.e. the EGFR ligand family such as HB-EGF, EGF, or amphiregulin, were screened (Supplementary Fig. 8). We have not identified significant EGFR ligand release, so that ADAM8-β1 integrin interactions might act independent from EGFR signalling.'], 'emss-61632-f006': ['Initially, orthotopic injections of Panc1 cells into mouse pancreas were performed (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6A-G</xref>) in three cohorts (n=12 each); cohort 1 received Panc1_A8 cells, cohort 2 Panc1_ctrl cells, and cohort 3 Panc1_A8 cells followed by daily i.p. injection of 10 µg/g BK-1361. Mice were monitored for 12 days, by which time most of the mice injected with Panc1_A8 cells were moribund and reached endpoint criteria. In contrast, mice injected with Panc1_ctrl cells or Panc1_A8/BK-1361 treatment showed improved clinical parameters. At endpoint, pancreatic tumours formed from Panc1_ctrl cells were significantly smaller than from Panc1_A8 cells () in three cohorts (n=12 each); cohort 1 received Panc1_A8 cells, cohort 2 Panc1_ctrl cells, and cohort 3 Panc1_A8 cells followed by daily i.p. injection of 10 µg/g BK-1361. Mice were monitored for 12 days, by which time most of the mice injected with Panc1_A8 cells were moribund and reached endpoint criteria. In contrast, mice injected with Panc1_ctrl cells or Panc1_A8/BK-1361 treatment showed improved clinical parameters. At endpoint, pancreatic tumours formed from Panc1_ctrl cells were significantly smaller than from Panc1_A8 cells (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6A, B</xref>). Moreover tumours obtained from mice that received Panc1_A8 cells and daily injections of BK-1361 were significantly smaller. These data indicate that inhibition of ADAM8 reduced tumour load to almost the value of Panc1_ctrl derived tumours (0.42 grams for Panc1_ctrl vs. 0.62 for Panc1_A8/BK-1361). By histology, a significant invasion of Panc1_A8 cells into the pancreatic tissue was detected, whereas in tumours derived from Panc1_A8/BK-1361 cells, tumour masses embedded in matrigel were primarily localised to the implantation site even after 12 days, as the boundaries of pancreas and implanted tumour mass were still distinct (). Moreover tumours obtained from mice that received Panc1_A8 cells and daily injections of BK-1361 were significantly smaller. These data indicate that inhibition of ADAM8 reduced tumour load to almost the value of Panc1_ctrl derived tumours (0.42 grams for Panc1_ctrl vs. 0.62 for Panc1_A8/BK-1361). By histology, a significant invasion of Panc1_A8 cells into the pancreatic tissue was detected, whereas in tumours derived from Panc1_A8/BK-1361 cells, tumour masses embedded in matrigel were primarily localised to the implantation site even after 12 days, as the boundaries of pancreas and implanted tumour mass were still distinct (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6C</xref>). In addition, there were signs of necrosis inside the implanted tumour treated with BK-1361 (). In addition, there were signs of necrosis inside the implanted tumour treated with BK-1361 (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6C</xref>), inferring that non-invasive Panc1 cells undergo necrotic changes. Moreover, ADAM8 levels in Panc1_ctrl cells located in the tumour were increased under hypoxic conditions. Co-staining for pERK1/2 was observed in infiltrative ADAM8-positive tumour cells (), inferring that non-invasive Panc1 cells undergo necrotic changes. Moreover, ADAM8 levels in Panc1_ctrl cells located in the tumour were increased under hypoxic conditions. Co-staining for pERK1/2 was observed in infiltrative ADAM8-positive tumour cells (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6D</xref>, upper panel)., upper panel).', 'Metastasis and infiltration is the major cause for the observed morbidity in PDAC5, 6. Since ADAM8 was discussed in the context of infiltration and metastasis26,34, we investigated orthotopic mice for infiltration of close structures such as peritoneum, diaphragm and spleen and liver metastasis. From mice injected with Panc1_A8 cells, we found significant infiltrates in adjacent organs (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6E&F</xref>). Macroscopic inspection and hematotoxylin/eosin (HE) stain of tissue sections revealed higher invasion into spleen and diaphragm of Panc1_A8 injected mice (). Macroscopic inspection and hematotoxylin/eosin (HE) stain of tissue sections revealed higher invasion into spleen and diaphragm of Panc1_A8 injected mice (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6E</xref>). Analysis of infiltration areas showed enhanced invasive behaviour of Panc1_A8 cells vs. Panc1_ctrl and Panc1_A8/BK-1361 cells (). Analysis of infiltration areas showed enhanced invasive behaviour of Panc1_A8 cells vs. Panc1_ctrl and Panc1_A8/BK-1361 cells (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6F</xref>) in peritoneum, diaphragm and spleen.) in peritoneum, diaphragm and spleen.', 'ADAM8 staining of liver sections revealed occurrence of micrometastases with higher frequencies in Panc1_A8 implanted mice compared to Panc1_ctrl and Panc1_A8/BK-1361 (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6G</xref>). Metastases frequencies were markedly different between Panc1_A8, Panc1_ctrl, and Panc1_A8/BK-1361. Furthermore, the implantation of AsPC-1_shCtrl and AsPC-1_shA8 cells was analysed (). Metastases frequencies were markedly different between Panc1_A8, Panc1_ctrl, and Panc1_A8/BK-1361. Furthermore, the implantation of AsPC-1_shCtrl and AsPC-1_shA8 cells was analysed (<xref ref-type="fig" rid="emss-61632-f006">Fig. 6H</xref>). AsPC-1_shCtrl cells caused large streams of tumour cells invading the pancreatic tissue with an infiltration area of 21 ± 2.8 %. In contrast, AsPC-1_shA8 cells were located close to the injection site and showed less invasive behaviour with infiltration areas of 3.4 ± 1.2% (p<0.01). Thus, data derived from genetic ). AsPC-1_shCtrl cells caused large streams of tumour cells invading the pancreatic tissue with an infiltration area of 21 ± 2.8 %. In contrast, AsPC-1_shA8 cells were located close to the injection site and showed less invasive behaviour with infiltration areas of 3.4 ± 1.2% (p<0.01). Thus, data derived from genetic ADAM8 knockdown support the results obtained with ADAM8 inhibition using BK-1361.'], 'emss-61632-f007': ['The therapeutic effect of ADAM8 inhibition in vivo was analysed in mice with genotype KrasLSL-G12D, Trp53R172H/+, PdxCre/+(KPC) 2, a genetically engineered PDAC mouse model. Injections of BK-1361 were started around the onset of Pancreatic Intraepithelial Neoplasias (PanINs). KPC control groups received injections of either saline (as in a clinical setting) or control peptide (BK-1362). Control groups showed progression to PDAC with a median survival of 15.5 weeks for saline and 16 weeks for BK-1362. In contrast, BK-1361 treated KPC mice have extended median survival times of 24.2 weeks (<xref ref-type="fig" rid="emss-61632-f007">Fig. 7A</xref>). The ). The in vivo efficacy of BK-1361 was demonstrated by determining soluble ADAM8 levels (<xref ref-type="fig" rid="emss-61632-f007">Fig. 7B</xref>). Lower frequencies of metastases in liver and lung were observed in BK-1361 treated KPC mice (). Lower frequencies of metastases in liver and lung were observed in BK-1361 treated KPC mice (<xref ref-type="fig" rid="emss-61632-f007">Fig. 7C</xref>). Pancreas morphology in BK-1361 treated mice showed reduced infiltration areas in the pancreas compared to control mice (). Pancreas morphology in BK-1361 treated mice showed reduced infiltration areas in the pancreas compared to control mice (<xref ref-type="fig" rid="emss-61632-f007">Fig. 7D and E</xref>), while the areas of intact acinar structures are increased (0.74% for BK-1361 treated vs. 0.18% for saline treated mice, ), while the areas of intact acinar structures are increased (0.74% for BK-1361 treated vs. 0.18% for saline treated mice, <xref ref-type="fig" rid="emss-61632-f007">Fig. 7F</xref>). Tumour progression was associated with increased staining for ADAM8 and pERK1/2 in control KPC mice. In BK-1361 treated KPC mice, ADAM8 and pERK1/2 staining is restricted to acinar structures, suggesting despite occurrence of neoplasias, tumour infiltration was reduced while the acinar architecture was more conserved (). Tumour progression was associated with increased staining for ADAM8 and pERK1/2 in control KPC mice. In BK-1361 treated KPC mice, ADAM8 and pERK1/2 staining is restricted to acinar structures, suggesting despite occurrence of neoplasias, tumour infiltration was reduced while the acinar architecture was more conserved (<xref ref-type="fig" rid="emss-61632-f007">Fig. 7G</xref>). Staining intensities of pERK1/2 and ADAM8 is reduced in BK-1361 treated KPC mice (). Staining intensities of pERK1/2 and ADAM8 is reduced in BK-1361 treated KPC mice (<xref ref-type="fig" rid="emss-61632-f007">Fig. 7H</xref>), suggesting that ), suggesting that in vivo, ADAM8 inhibition leads to reduced activation of pERK1/2.']}
|
ADAM8 as a drug target in Pancreatic Cancer
| null |
Nat Commun
|
1422432000
|
[{'@Label': 'BACKGROUND/OBJECTIVES', '#text': 'Prader-Willi syndrome (PWS) is a type of human genetic obesity that may give us information regarding the physiology of non-syndromic obesity. The objective of this study was to investigate the functional correlates of hunger and satiety in individuals with PWS in comparison with healthy controls with obesity, hypothesizing that we would see significant differences in activation in the left dorsolateral prefrontal cortex (DLPFC) based on prior findings.'}, {'@Label': 'SUBJECTS/METHODS', '#text': 'This study compared the central effects of food consumption in nine individuals with PWS (7 men, 2 women; body fat 35.3±10.0%) and seven controls (7 men; body fat 28.8±7.6%), matched for percentage body fat. H2(15)O-PET (positron emission tomography) scans were performed before and after consumption of a standardized liquid meal to obtain quantitative measures of regional cerebral blood flow (rCBF), a marker of neuronal activity.'}, {'@Label': 'RESULTS', '#text': 'Compared with obese controls, PWS showed altered (P<0.05 family-wise error cluster-level corrected; voxelwise P<0.001) rCBF before and after meal consumption in multiple brain regions. There was a significant differential rCBF response within the left DLPFC after meal ingestion with decreases in DLPFC rCBF in PWS; in controls, DLPFC rCBF tended to remain unchanged. In more liberal analyses (P<0.05 family-wise error cluster-level corrected; voxelwise P<0.005), rCBF of the right orbitofrontal cortex (OFC) increased in PWS and decreased in controls. In PWS, ΔrCBF of the right OFC was associated with changes in appetite ratings.'}, {'@Label': 'CONCLUSIONS', '#text': 'The pathophysiology of eating behavior in PWS is characterized by a paradoxical meal-induced deactivation of the left DLPFC and activation in the right OFC, brain regions implicated in the central regulation of eating behavior.'}]
|
[
"Adult",
"Brain Mapping",
"Cerebrovascular Circulation",
"Feeding Behavior",
"Female",
"Functional Neuroimaging",
"Humans",
"Magnetic Resonance Imaging",
"Male",
"Meals",
"Postprandial Period",
"Prader-Willi Syndrome",
"Prefrontal Cortex",
"Reward",
"Satiation",
"Satiety Response"
] |
other
|
PMC5014123
| null |
41
|
[
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Nat Commun. 2015 Jan 28; 6:6175
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PINK1 directly stimulates mitophagy upon mitochondrial damageRepresentative data of mt-mKeima-expressing a, WT, PINK1 KO or c, PINK1 KO rescued with PINK1-WT cells treated with OA then analyzed by FACS. b, d, Average percent mitophagy for two replicates of a and c, respectively. e, Representative images of WT HeLa cells expressing mCherry-OPTN and treated with OA as indicated were immunostained for Tom20 (n=3). f, Quantification of mCherry-OPTN translocation from cells in e. Data displayed as mean ± s.d. from 3 independent experiments and using one-way ANOVA tests (***P<0.001, ns, not significant).
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multiple panels: images & plots
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[{'image_id': 'nihms-707551-f0009', 'image_file_name': 'nihms-707551-f0009.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0009.jpg', 'caption': 'GABARAPs do not translocate to damaged mitochondria and early stages of autophagosome biogenesis mediated by WIPI1 and DFCP1 are inhibited in autophagy receptor deficient cell linesRepresentative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCherry-Parkin (mCh-Parkin) and either (a) GFP-tagged GABARAP, GABARAPL1 or GABARAPL2, (b) GFP-WIPI1 or (c) GFP-DFCP1 immunostained for Tom20 (n=3 for each condition, see Figure 4b, c for quantification of b and c). d, mCh-Parkin cell lines as indicated were subjected to either Phos-Tag SDS-PAGE or standard SDS-PAGE followed by immunoblotting. Arrows indicate the position of phosphorylated Beclin species. e, Representative images of untreated WT, N/O (NDP52/OPTN) DKO and pentaKO cell lines expressing mCh-Parkin and GFP-ULK1 were immunostained for Tom20 and GFP (n=3). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.', 'hash': 'c0da7b10102537e159bfc84f03a2fcaa4b7b027ed9641f17de28c625b8e7d322'}, {'image_id': 'nihms-707551-f0007', 'image_file_name': 'nihms-707551-f0007.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0007.jpg', 'caption': 'OPTN and NDP52 preferentially bind phospho-mimetic ubiquitina, HeLa cells expressing mCherry-Parkin (Parkin) and HA-ubiquitin (HA-UB) WT, S65D or S65A were treated with CCCP. HA-UB was co-immunoprecipitated and the bound fraction was analyzed by immunoblotting. Quantification of the total bound fraction of OPTN, NDP52 and p62 are shown. b, HA-ubiquitin transfected into HeLa cells with mCherry-Parkin were treated with CCCP. HA-ubiquitin was immunoprecipitated. The bound fraction was treated with the deubiquitinase USP2 and washed to remove all unbound protein following deubiquitination. Quantification of the total bound fraction of OPTN, NDP52 and p62 are shown in the right panel. c,d, Strep-tagged ubiquitin (Strep-UB) was incubated with either WT or kinase-dead (KD) PINK1 in an in vitro phosphorylation reaction, immunoblotted with an anti-phosphoS65 ubiquitin antibody (c) and was then incubated with cytosol harvested from untreated, WT HeLa cells. The ubiquitin was then pulled down using Strep-Tactin beads and (d) analyzed by immunoblotting. e, Quantification of bound OPTN and p62 normalized to total ubiquitin. Data displayed in a, b and e as mean ± s.d. from 3 independent experiments and use one-way ANOVA tests. (***P<0.001, **P<0.005, *P<0.05). †, non-specific band. a.u., arbitrary units.', 'hash': '3c7d27b24c9b501aa58d36d0788b2491dd2574d2c4311a285ba849c5ad2e0335'}, {'image_id': 'nihms-707551-f0011', 'image_file_name': 'nihms-707551-f0011.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0011.jpg', 'caption': 'Identifying autophagy receptors required for PINK1/Parkin mitophagya, WT, OPTN KO, NDP52 KO, N/O (NDP52/OPTN) DKO, N/O/Tx (NDP52/OPTN/TAX1BP1) TKO, and pentaKO (NDP52/OPTN/TAX1BP1/NBR1/p62) HeLa cells were confirmed by immunoblotting. b, Cells as indicated with or without mCherry-Parkin (mCh-Parkin) were analyzed by immunoblotting and c, CoxII levels quantified. d, Representative images of mCh-Parkin expressing WT, pentaKO and ATG5 KO cells immunostained to label mitochondrial DNA (green) and e, quantified for mitophagy (24 h OA). >75 cells were counted per sample. f, Lysates from pentaKOs expressing mCh-Parkin and GFP-tagged autophagy receptors were immunoblotted and g, CoxII levels were quantified. Quantification in c, e and g are mean ± s.d. from 3 independent experiments and use one-way ANOVA (***P<0.001). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.', 'hash': '1c97e7cd1f88093fda7d65485f0632e790015d64dfb99b3f62e721780fe31d67'}, {'image_id': 'nihms-707551-f0006', 'image_file_name': 'nihms-707551-f0006.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0006.jpg', 'caption': 'PINK1 directly stimulates mitophagy upon mitochondrial damageRepresentative data of mt-mKeima-expressing a, WT, PINK1 KO or c, PINK1 KO rescued with PINK1-WT cells treated with OA then analyzed by FACS. b, d, Average percent mitophagy for two replicates of a and c, respectively. e, Representative images of WT HeLa cells expressing mCherry-OPTN and treated with OA as indicated were immunostained for Tom20 (n=3). f, Quantification of mCherry-OPTN translocation from cells in e. Data displayed as mean ± s.d. from 3 independent experiments and using one-way ANOVA tests (***P<0.001, ns, not significant).', 'hash': '2d93761138dbedcad549000ca97643ef1f7bc5e0e0aba8fa74598983d4cad6b4'}, {'image_id': 'nihms-707551-f0001', 'image_file_name': 'nihms-707551-f0001.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0001.jpg', 'caption': 'Analysis of knockout cell lines and characterization of autophagy receptor translocation to damaged mitochondriaa, ATG5 KO cell line confirmed by immunoblotting. b, Representative images of mitochondrial DNA nucleoids in HeLa cells immunostained with an α-DNA antibody (green) confirming colocalization with the mitochondrial marker Tom20 (red) (n=3). c, Mitochondrial fractions from mCherry-Parkin (mCh-Parkin) expressing pentaKO and WT cells were assessed by immunoblotting. d, mCh-Parkin expressing WT, pentaKO and ATG5 KOs were treated with OA or OA and MG132. Cell lysates were assessed by immunoblotting. e, Expression levels of GFP-tagged OPTN, NDP52, p62, NBR1 and TAX1BP1 re-expressed in pentaKOs by immunoblotting. f, Representative images of mCh-Parkin expressing pentaKOs from e immunostained for Tom20 (n=3). g, Expression of GFP-Tollip in mCh-Parkin pentaKOs. h, pentaKOs mCh-Parkin and with or without GFP-Tollip expression were immunoblotted. i, Representative images of mCh-Parkin pentaKOs expressing GFP-Tollip immunostained for Tom20 (n=3). Scale bars, 10 μm.', 'hash': 'aa315f38af7790da94ba7fd955b738bc92bcdf9b978cd674f5cbf902ab478145'}, {'image_id': 'nihms-707551-f0008', 'image_file_name': 'nihms-707551-f0008.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0008.jpg', 'caption': 'Analysis of LC3 family members and their translocation to damaged mitochondria in autophagy receptor KO cell linesa, Representative images of WT, pentaKO and ATG5 KO HeLa cells expressing mCherry-Parkin (mCh-Parkin) and GFP-LC3B were immunostained for Tom20 (n=3). b, Cell lysates from mCh-Parkin expressing WT, pentaKO and ATG5 KO cells were immunoblotted. c, Representative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCh-Parkin and either GFP-tagged LC3A, LC3B or LC3C were immunostained for Tom20 (n=3, see Figure 4a for quantification). d, Representative images of WT and N/O/Tx (NDP52/OPTN/TAX1BP1) TKO cells expressing mCh-Parkin and GFP-LC3C were immunostained for Tom20 (n=3) and e, quantified for GFP-LC3C translocation to mitochondria. Quantification in e is displayed as mean ± s.d. from 3 independent experiments and use one-way ANOVA tests (***P<0.001). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.', 'hash': 'd9672583653421af2a357b6dae1f79014bd95b4ae3119088499317d57dd62609'}, {'image_id': 'nihms-707551-f0010', 'image_file_name': 'nihms-707551-f0010.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0010.jpg', 'caption': 'OPTN and NDP52 rescue DFCP1 and ULK1 recruitment deficit in pentaKOsa, Representative images of pentaKOs expressing mCherry-Parkin (mCh-Parkin), GFP-DFCP1 and the indicated FLAG/HA-tagged autophagy receptors immunostained for HA (n=2). Right-hand panels display co-localization of FLAG/HA-tagged constructs and GFP-DFCP1 by fluorescence intensity line measurement. b, Representative images of pentaKOs expressing mCherry-Parkin and GFP-ULK1 were rescued with FLAG/HA-OPTN, FLAG/HA-NDP52, and FLAG/HA-p62, and immunostained for HA and GFP. Arrows indicate HA-tagged receptor puncta (n=2). Right panels display colocalization of HA and GFP by fluorescence intensity line measurement. c, d, Representative images of pentaKOs stably expressing FRB-Fis1 and transiently expressing PINK1Δ110-YFP-2xFKBP and vector or myc-tagged receptors, were (c) untreated or (d) treated with rapalog and imaged live (n=3, see Figure 4h, i for quantification of c, d). OA, Oligomycin and Antimycin A. Scale bars, 10 μm. e, Old and new models of PINK1/Parkin mitophagy. The old model is dominated by Parkin ubiquitination of mitochondrial proteins. Here PINK1 plays a small initiator role whose main function is to bring Parkin to the mitochondria. The new model depicts Parkin-dependent and independent pathways leading to robust and low-level mitophagy, respectively. Based on our data, PINK1 is central to mitophagy both before and after Parkin recruitment by phosphorylating UB to recruit both Parkin and autophagy receptors mitochondria, to induce clearance. In the absence of Parkin (right panel), this occurs at a low level due to the relatively low basal UB on mitochondria. When Parkin is present it serves to amplify the PINK1 generated UB-PO4 signal, allowing for robust and rapid mitophagy induction.', 'hash': '0a4861f8210ec8727906f91bdc8211c3ff6df89aee17106a8a5993949c9d5b57'}, {'image_id': 'nihms-707551-f0014', 'image_file_name': 'nihms-707551-f0014.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0014.jpg', 'caption': 'Characterization of autophagy receptor function during mitophagymCherry-Parkin (mCh-Parkin) expressing WT, N/O (NDP52/OPTN) DKO and pentaKOs were quantified for a, GFP-LC3A, LC3B and LC3C translocation to mitochondria, b, GFP-WIPI1 or c, GFP-DFCP1 structures per cell (>100 cells counted for each sample) or d, were immunoblotted using phospho-specific anti-S757 and S317 ULK1 antibodies. e, mCherry-Parkin WT, N/O DKO and pentaKOs stably expressing GFP-ULK1 were quantified for GFP-ULK1 puncta per cell (left graph) and the percentage of those puncta on mitochondria (right graph). f, Representative data of e, cells were immunostained for Tom20 and GFP. g, pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCherry-ULK1 (mCh-ULK1) and myc-tagged receptors, were treated with rapalog then imaged live. h, Quantification of mitochondrial ULK1 puncta in g. i, Quantification of mitochondrial ULK1 puncta in pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCh-ULK1 and myc-OPTN mutants, treated with rapalog then imaged live. Quantification in a, b, c, e, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA. (***P<0.001, **P<0.005, *P<0.05, ns, not significant). For live cell quantification >75 cells counted in a blinded manner. Quantification in h and i were performed after removal of outliers, see Online Methods for details. OA, Oligomycin and Antimycin A. Scale bars, 10 μm. a.u., arbitrary units. See Extended Data Figs. 8c, 9b, c and 10d for representative images of a, b, c, and i respectively. See Extended Data Fig. 9e untreated samples of f and Extended Data Fig. 10c for untreated images of h and i.', 'hash': 'ebf477ffa9c8c53dd259a2a9853b166e9287607b6263c55d8a995935e31910c1'}, {'image_id': 'nihms-707551-f0013', 'image_file_name': 'nihms-707551-f0013.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0013.jpg', 'caption': 'PINK1 recruits Optineurin and NDP52 independent of Parkin to promote mitophagya, Representative images of HeLa cells expressing FRB-Fis1, WT (PINK1-WT) or kinase-dead (PINK1-KD) PINK1Δ110-YFP-2xFKBP and mCherry-OPTN (mCh-OPTN) or mCherry-NDP52 (mCh-NDP52) treated with rapalog. b, Quantification of receptor translocation in cells from a and Extended Data Fig. 4c–e. >100 cells were counted per sample. c, Cells were treated with rapalog and analyzed by FACS for lysosomal positive mt-mKeima. Representative data for WT or KD PINK1Δ110-YFP-2xFKBP in pentaKOs without or with FLAG/HA-OPTN expression. Quantification in b is displayed as mean ± s.d. from 3 independent experiments and uses one-way ANOVA tests (***P<0.001, *P<0.05, ns, not significant). For images of untreated cells from a, see Extended Data Fig. 4b. Scale bars, 10 μm.', 'hash': 'ca00cd897f2cc7a94131342e4195f3347d041e22a212276e8ef0f5ca3e4b0974'}, {'image_id': 'nihms-707551-f0002', 'image_file_name': 'nihms-707551-f0002.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0002.jpg', 'caption': 'OPTN, NDP52 and TAX1BP1 triple knockout analysis and disease-associated mutationsa, KO cell lines with or without mCherry-Parkin (mCh-Parkin) expression were immunoblotted and b, CoxII levels were quantified. c, A panel of human tissue lysates was immunoblotted. d, Expression of WT or mutant GFP-OPTN in mCh-Parkin pentaKOs. e, Quantification of cells in f. >100 cells per condition. f, Representative images of mCh-Parkin pentaKOs expressing GFP-OPTN mutants immunostained for Tom20 (n=3). g, pentaKOs expressing mCh-Parkin were rescued with WT or mutant GFP-OPTN, analyzed by immunoblotting. See Fig. 2e for quantification of CoxII. h, Expression of WT or mutant GFP-NDP52 in mCh-Parkin pentaKOs. i, Quantification of cells in j. >100 cells per condition. j, Representative images of mCh-Parkin pentaKOs expressing WT or mutant GFP-NDP52 were immunostained for Tom20 (n=3). k, pentaKOs expressing mCh-Parkin rescued with WT or mutant GFP-NDP52 were analyzed by immunoblotting. See Fig. 2f for quantification of CoxII. Quantification in b and i are displayed as mean ± s.d. from 3 independent experiments using one-way ANOVA tests (***P<0.001, ns, not significant) and in e as mean from 2 independent experiments. Scale bars, 10 μm.', 'hash': '90566506574ed8f22523f8cce64afddd69a7d137fac189685823a4b948c2d177'}, {'image_id': 'nihms-707551-f0005', 'image_file_name': 'nihms-707551-f0005.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0005.jpg', 'caption': 'PINK1 directly stimulates mitophagy in the absence of mitochondrial damagea, b, Cells were treated with rapalog and analyzed by FACS for lysosomal positive mt-mKeima. Representative data for WT HeLa (a) and pentaKO (b) without or with FLAG/HA-OPTN. c, d, Cell lysates from pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mt-mKeima and (c) WT FLAG/HA-OPTN or mutants, (d) FLAG/HA-p62, WT FLAG/HA-NDP52 or NDP52 mutants as indicated were assessed for receptor expression by immunoblotting. e, f, Cells from c and d were rapalog treated analyzed by FACS for lysosomal positive mt-mKeima. Representative data of two experiments is presented. g, Cell lysates from pentaKOs expressing FRB-Fis1, with or without FLAG/HA-OPTN and WT or kinase-dead (KD) PINK1Δ110-YFP-2xFKBP were assessed for OPTN by immunoblotting. h, FLAG/HA-OPTN pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mt-mKeima transfected and either vector or untagged Parkin were analyzed by FACS. Representative data of two experiments is presented.', 'hash': '33ede88c2479400c5094caf411830a34fec4785ec9bb18bcb540a2ecd713668e'}, {'image_id': 'nihms-707551-f0012', 'image_file_name': 'nihms-707551-f0012.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0012.jpg', 'caption': 'OPTN and NDP52 are redundant in PINK1/Parkin mitophagya, Representative images of WT, pentaKO, OPTN KO, NDP52 KO and N/O (NDP52/OPTN) DKO cells expressing mCherry-Parkin immunostained with anti-DNA and b, quantified for mitophagy. c, Cell lines from a, were analyzed by immunoblotting and d, CoxII levels quantified. e, CoxII levels quantified from pentaKOs expressing mCherry-Parkin (mCh-Parkin) and rescued with WT or mutant GFP-OPTN (See Extended Data Fig. 2g for blots). f, CoxII levels quantified from pentaKOs expressing mCh-Parkin rescued with WT or mutant GFP-NDP52 (See Extended Data Fig. 2k for blots). g, Representative images of WT, TBK1 KO, T/O (TBK1/OPTN) DKO and T/N (TBK1/NDP52) DKO HeLa cells expressing mCherry-Parkin and immunostained with anti-DNA and h, mitophagy was quantified. i, Cells from g were immunoblotted and j, CoxII levels quantified. Quantification in b, d, e, f, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA (***P<0.001, **P<0.005, ns, not significant). >75 cells were measured per confocal sample. OA, Oligomycin and Antimycin A. Scale bars, 10 μm. For untreated and mCherry-Parkin images of a and g, see Extended Data Fig. 3a and f, respectively.', 'hash': 'f05d2cd93487ce4d56439b35f120623f55faa050e70d699d34757768d7acf5fc'}, {'image_id': 'nihms-707551-f0004', 'image_file_name': 'nihms-707551-f0004.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0004.jpg', 'caption': 'Parkin-independent recruitment of receptors to mitochondria through PINK1 activitya, Isolated mitochondria from WT and pentaKOs with or without FRB-Fis1 and with WT or kinase-dead (KD) PINK1Δ110-YFP-2xFKBP were immunoblotted. b–e, Representative images of pentaKOs expressing FRB-Fis1, WT (PINK1-WT) or kinase-dead (PINK1-KD) PINK1Δ110-YFP-2xFKBP and either (b) mCherry-OPTN or mCherry-NDP52, (c) mCherry-p62, (d) mCherry-OPTN-D474N or (e) mCherry-NDP52-ΔZF. Cells were (b) untreated or (c–e) treated with rapalog then immunostained for Tom20. All images are representative of three independent experiments. See Figure 3b for quantification. Scale bars, 10 μm.', 'hash': '8ed53c2a1e980802374bcb09c46c85d7fae2206bab10b589db56479a3b1488d6'}, {'image_id': 'nihms-707551-f0003', 'image_file_name': 'nihms-707551-f0003.jpg', 'image_path': '../data/media_files/PMC5018156/nihms-707551-f0003.jpg', 'caption': 'TBK1 in activates OPTN in PINK1/Parkin mitophagya, Representative images of untreated mCherry-Parkin (mCh-Parkin) cells and merged images of treated cells as indicated immunostained for DNA. See Fig. 2a for anti-DNA/DAPI images of treated samples (n=3). b, Cell lysates from WT, N/O (NDP52/OPTN) DKO, OPTN KO and NDP52 KO cells with or without mCh-Parkin expression were immunoblotted for TBK1 activation. c, Cell lysates from WT and PINK1 KO cells without Parkin expression were immunoblotted for TBK1 activation (S172 phosphorylation). d, Confirmation of T/N (TBK1/NDP52) DKO, T/O (TBK1/OPTN) DKO and TBK1 KO by immunoblotting. e, KO cell lines from d were immunostained for Tom20 (n=3). f, Representative images of untreated mCh-Parkin WT and KO cells, and merged images of treated cells as indicated were immunostained for DNA. See Figure 2g for anti-DNA/DAPI images treated samples (n=3). g, T/N DKO cells rescued with GFP-TBK1 WT or K38M, or GFP-OPTN S177D and were assessed by immunoblotting. h, Cells in g were assessed by immunoblotting. i, Quantification of CoxII levels in h displayed as mean ± s.d. from 3 independent experiments and use one-way ANOVA tests (**P<0.005, ns, not significant). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.', 'hash': '21fb45802ac259775f9d07eed57d0231df8506af94865dca4426140b2fe349d6'}]
|
{'nihms-707551-f0011': ['To clarify autophagy receptor function during mitophagy, genome editing was used to knock out five autophagy receptors in HeLa cells (pentaKO), which do not express endogenous Parkin. DNA sequencing (Supplementary Table 1) and immunoblotting of TAX1BP1, NDP52, NBR1, p62 and OPTN (<xref ref-type="fig" rid="nihms-707551-f0011">Fig. 1a</xref>, lane 6) confirmed their knockout. We analyzed mitophagy in pentaKOs by measuring the degradation of cytochrome C oxidase subunit II (CoxII), a mtDNA encoded inner membrane protein, following mitochondrial damage with oligomycin and antimycin A (OA). After OA treatment, CoxII was degraded in WT cells expressing Parkin, but not in pentaKOs or ATG5 KO HeLa cells, indicating a block in mitophagy (, lane 6) confirmed their knockout. We analyzed mitophagy in pentaKOs by measuring the degradation of cytochrome C oxidase subunit II (CoxII), a mtDNA encoded inner membrane protein, following mitochondrial damage with oligomycin and antimycin A (OA). After OA treatment, CoxII was degraded in WT cells expressing Parkin, but not in pentaKOs or ATG5 KO HeLa cells, indicating a block in mitophagy (<xref ref-type="fig" rid="nihms-707551-f0011">Fig. 1b, c</xref>, , Supplementary Table 1 and Extended Data Fig. 1a). As a second indicator of mitophagy, mitochondrial DNA (mtDNA) nucleoids were quantified by immunofluorescence (<xref ref-type="fig" rid="nihms-707551-f0001">Extended Data Fig. 1b</xref>). After 24 h OA treatment, WT cells were nearly devoid of mtDNA, whereas pentaKOs and ATG5 KOs retained mtDNA (). After 24 h OA treatment, WT cells were nearly devoid of mtDNA, whereas pentaKOs and ATG5 KOs retained mtDNA (<xref ref-type="fig" rid="nihms-707551-f0011">Fig. 1d, e</xref>). Parkin translocated to mitochondria (). Parkin translocated to mitochondria (<xref ref-type="fig" rid="nihms-707551-f0001">Extended Data Fig. 1c</xref>) and Mfn1 and Tom20 were degraded via the proteasome comparably in WT and pentaKOs () and Mfn1 and Tom20 were degraded via the proteasome comparably in WT and pentaKOs (<xref ref-type="fig" rid="nihms-707551-f0011">Fig. 1b</xref>, , <xref ref-type="fig" rid="nihms-707551-f0001">Extended Data Fig. 1d</xref>). mtDNA nucleoids clump following OA treatment in ATG5 KO cells but not in pentaKOs, consistent with a reported role of p62). mtDNA nucleoids clump following OA treatment in ATG5 KO cells but not in pentaKOs, consistent with a reported role of p6210,11.', 'We generated single OPTN, NDP52 KO and NDP52/OPTN double KO (N/O DKO) and NDP52/OPTN/TAX1BP1 triple KO (N/O/Tx TKO) cell lines (Supplementary Table 1, <xref ref-type="fig" rid="nihms-707551-f0011">Fig. 1a</xref>) and found no compensatory change in the expression of the remaining receptors. NDP52 or OPTN KO alone caused no defect in mitophagy, whereas NDP52/OPTN DKO and to a greater extent, NDP52/OPTN/TAX1BP1 TKO inhibited mitophagy () and found no compensatory change in the expression of the remaining receptors. NDP52 or OPTN KO alone caused no defect in mitophagy, whereas NDP52/OPTN DKO and to a greater extent, NDP52/OPTN/TAX1BP1 TKO inhibited mitophagy (<xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2a–d</xref>, , <xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2a, b</xref>). The robust mitophagy observed in OPTN KOs contrasts with a report indicating loss of mitophagy using RNAi-mediated knockdown of OPTN in HeLa cells). The robust mitophagy observed in OPTN KOs contrasts with a report indicating loss of mitophagy using RNAi-mediated knockdown of OPTN in HeLa cells9. Although NDP52 and OPTN redundantly mediate mitophagy, they function non-redundantly in xenophagy13. Their expression levels in human tissues indicate that OPTN or NDP52 may function more prominently in different tissues (<xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2c</xref>).).'], 'nihms-707551-f0001': ['The five endogenous receptors in WT cells (<xref ref-type="fig" rid="nihms-707551-f0001">Extended Data Fig. 1c</xref>) and each receptor re-expressed in pentaKOs () and each receptor re-expressed in pentaKOs (<xref ref-type="fig" rid="nihms-707551-f0001">Extended Data Fig. 1e, f</xref>) translocated to mitochondria after OA treatment. However, in pentaKOs only GFP-NDP52, GFP-OPTN and to a lesser extent, GFP-TAX1BP1, rescued mitophagy () translocated to mitochondria after OA treatment. However, in pentaKOs only GFP-NDP52, GFP-OPTN and to a lesser extent, GFP-TAX1BP1, rescued mitophagy (<xref ref-type="fig" rid="nihms-707551-f0011">Fig. 1f, g</xref>). Another recently reported autophagy receptor, Tollip). Another recently reported autophagy receptor, Tollip12, neither recruited to mitochondria nor rescued mitophagy following OA treatment (<xref ref-type="fig" rid="nihms-707551-f0001">Extended Data Fig. 1g–i</xref>).).'], 'nihms-707551-f0002': ['Mutations in autophagy receptors can lead to diseases such as primary open angle glaucoma (POAG, OPTN; E50K)14, ALS (OPTN; E478G and Q398X)15 and Crohn\'s disease (NDP52; V248A)16. Defects in xenophagy occur when OPTN is mutated to block its phosphorylation by TANK-binding kinase 1 (TBK1; S177A) or ubiquitin binding (D474N)13,17. In pentaKOs, the UBAN-domain disrupting mutants OPTN-Q398X, OPTN-D474N and OPTN-E478G (<xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2d</xref>) failed to translocate to mitochondria () failed to translocate to mitochondria (<xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2e, f</xref>) or rescue mitophagy () or rescue mitophagy (<xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2e</xref>, , <xref ref-type="fig" rid="nihms-707551-f0002">Extended Data 2g</xref>). OPTN-S177A weakly rescued mitophagy and minimally translocated to mitochondria, whereas OPTN-E50K robustly translocated and substantially rescued mitophagy (). OPTN-S177A weakly rescued mitophagy and minimally translocated to mitochondria, whereas OPTN-E50K robustly translocated and substantially rescued mitophagy (<xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2e</xref>, , <xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2e–g</xref>). NDP52-V248A fully recruited to mitochondria and rescued mitophagy, but a mutant lacking the ZF ubiquitin-binding domains (NDP52-ΔZF)). NDP52-V248A fully recruited to mitochondria and rescued mitophagy, but a mutant lacking the ZF ubiquitin-binding domains (NDP52-ΔZF)18 did not (<xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2h–k</xref>, , <xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2f</xref>). Thus, ubiquitin binding by OPTN and NDP52 is necessary for mitophagy and some disease-causing mutations prevent mitophagy.). Thus, ubiquitin binding by OPTN and NDP52 is necessary for mitophagy and some disease-causing mutations prevent mitophagy.', 'a, Representative images of WT, pentaKO, OPTN KO, NDP52 KO and N/O (NDP52/OPTN) DKO cells expressing mCherry-Parkin immunostained with anti-DNA and b, quantified for mitophagy. c, Cell lines from a, were analyzed by immunoblotting and d, CoxII levels quantified. e, CoxII levels quantified from pentaKOs expressing mCherry-Parkin (mCh-Parkin) and rescued with WT or mutant GFP-OPTN (See <xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2g</xref> for blots). for blots). f, CoxII levels quantified from pentaKOs expressing mCh-Parkin rescued with WT or mutant GFP-NDP52 (See <xref ref-type="fig" rid="nihms-707551-f0002">Extended Data Fig. 2k</xref> for blots). for blots). g, Representative images of WT, TBK1 KO, T/O (TBK1/OPTN) DKO and T/N (TBK1/NDP52) DKO HeLa cells expressing mCherry-Parkin and immunostained with anti-DNA and h, mitophagy was quantified. i, Cells from g were immunoblotted and j, CoxII levels quantified. Quantification in b, d, e, f, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA (***P<0.001, **P<0.005, ns, not significant). >75 cells were measured per confocal sample. OA, Oligomycin and Antimycin A. Scale bars, 10 μm. For untreated and mCherry-Parkin images of a and g, see <xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig. 3a and f</xref>, respectively., respectively.'], 'nihms-707551-f0003': ['TBK1 phosphorylation of OPTN at S177 increases its association with LC3 during xenophagy13, and the OPTN E50K mutation increases TBK1/OPTN binding19. TBK1 auto-phosphorylation at Ser172 is indicative of TBK1 activation20 and occurs in a Parkin-dependent manner following 3 h OA treatment, but only in cells expressing OPTN (<xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig. 3b</xref>, lanes 4 and 10). Prolonged OA treatment induces moderate TBK1 phosphorylation in the absence of Parkin but still requires PINK1 (, lanes 4 and 10). Prolonged OA treatment induces moderate TBK1 phosphorylation in the absence of Parkin but still requires PINK1 (<xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig. 3c</xref>). To investigate TBK1 function during mitophagy, we generated TBK1 KO, TBK1/NDP52 (T/N) DKO and TBK1/OPTN (T/O) DKO HeLa cells (). To investigate TBK1 function during mitophagy, we generated TBK1 KO, TBK1/NDP52 (T/N) DKO and TBK1/OPTN (T/O) DKO HeLa cells (<xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig. 3d</xref>, , Supplementary Table 1). Parkin translocated to mitochondria in all lines, however, only TBK1/NDP52 DKOs displayed defective mitophagy (<xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig. 3e</xref>, , <xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2g–j</xref>). Mitophagy in TBK1/NDP52 DKOs was rescued by WT-TBK1 or phospho-mimetic OPTN (OPTN-S177D), but not by kinase-dead TBK1 (TBK1-K38M) (). Mitophagy in TBK1/NDP52 DKOs was rescued by WT-TBK1 or phospho-mimetic OPTN (OPTN-S177D), but not by kinase-dead TBK1 (TBK1-K38M) (<xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig. 3g–i</xref>). Thus, in the absence of NDP52, TBK1 is critical for effective mitophagy via OPTN.). Thus, in the absence of NDP52, TBK1 is critical for effective mitophagy via OPTN.'], 'nihms-707551-f0013': ['Since many autophagy receptors recruit to mitochondria following Parkin activation, why do only some function in mitophagy? Parkin-mediated mitophagy is driven by PINK1\'s phosphorylation of Ser65 of both ubiquitin5–7,21,22 and the UBL domain of Parkin23. Since Ser65 phospho-ubiquitin is structurally unique, it may differentially interact with ubiquitin binding proteins22. To determine whether OPTN is directly recruited to phospho-ubiquitin on mitochondria, we conditionally expressed PINK1 on undamaged mitochondria10 in HeLa cells lacking Parkin (<xref ref-type="fig" rid="nihms-707551-f0013">Fig. 3a</xref>, , <xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4a</xref>). When PINK1Δ110-YFP-2xFKBP is cytosolic, mCherry-OPTN, mCherry-NDP52 and mCherry-p62 are also cytosolic (). When PINK1Δ110-YFP-2xFKBP is cytosolic, mCherry-OPTN, mCherry-NDP52 and mCherry-p62 are also cytosolic (<xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4b, c</xref>). When PINK1Δ110-YFP-2xFKBP is localized to FRB-Fis1 expressing mitochondria with rapalog, where ubiquitin on surface proteins). When PINK1Δ110-YFP-2xFKBP is localized to FRB-Fis1 expressing mitochondria with rapalog, where ubiquitin on surface proteins24 (<xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4a</xref>) can be phosphorylated) can be phosphorylated5,21,25,26, OPTN and NDP52 are recruited (<xref ref-type="fig" rid="nihms-707551-f0013">Fig. 3a, b</xref>), but p62 remains cytosolic (), but p62 remains cytosolic (<xref ref-type="fig" rid="nihms-707551-f0013">Fig. 3b</xref>, , <xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4c</xref>). OPTN/NDP52 recruitment requires PINK1 kinase activity (). OPTN/NDP52 recruitment requires PINK1 kinase activity (<xref ref-type="fig" rid="nihms-707551-f0013">Fig. 3a, b</xref>) and receptor-ubiquitin binding, as OPTN-D474N and NDP52-ΔZF fail to recruit following rapalog treatment () and receptor-ubiquitin binding, as OPTN-D474N and NDP52-ΔZF fail to recruit following rapalog treatment (<xref ref-type="fig" rid="nihms-707551-f0013">Fig. 3b</xref>, , <xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4d, e</xref>). Therefore, PINK1 ubiquitin kinase activity recruits OPTN/NDP52 via ubiquitin binding domains to mitochondria in the absence of Parkin.). Therefore, PINK1 ubiquitin kinase activity recruits OPTN/NDP52 via ubiquitin binding domains to mitochondria in the absence of Parkin.', 'a, Isolated mitochondria from WT and pentaKOs with or without FRB-Fis1 and with WT or kinase-dead (KD) PINK1Δ110-YFP-2xFKBP were immunoblotted. b–e, Representative images of pentaKOs expressing FRB-Fis1, WT (PINK1-WT) or kinase-dead (PINK1-KD) PINK1Δ110-YFP-2xFKBP and either (b) mCherry-OPTN or mCherry-NDP52, (c) mCherry-p62, (d) mCherry-OPTN-D474N or (e) mCherry-NDP52-ΔZF. Cells were (b) untreated or (c–e) treated with rapalog then immunostained for Tom20. All images are representative of three independent experiments. See <xref ref-type="fig" rid="nihms-707551-f0013">Figure 3b</xref> for quantification. Scale bars, 10 μm. for quantification. Scale bars, 10 μm.'], 'nihms-707551-f0005': ['To determine whether the observed autophagy receptor recruitment to mitochondria in the absence of Parkin can induce mitophagy, we developed a sensitive FACS based mitophagy assay. We expressed mitochondrial-targeted mKeima (mt-mKeima, see Online Methods) in WT and pentaKOs also expressing mitochondrial FRB-Fis1 and PINK1Δ110-YFP-2xFKBP. mt-mKeima engulfment into lysosomes results in a spectral shift due to low pH. Only 1% (range 0.89–1.15) of WT or pentaKO cells display mitophagy when PINK1 is cytosolic. However, when PINK1 is recruited to mitochondria with rapalog, mitophagy increases ~7-fold in WT cells and ~8-fold with overexpressed OPTN (Table 1, <xref ref-type="fig" rid="nihms-707551-f0005">Extended Data Fig. 5a</xref>). PentaKOs showed no increase in mitophagy after targeting PINK1 to mitochondria (). PentaKOs showed no increase in mitophagy after targeting PINK1 to mitochondria (Table 1, <xref ref-type="fig" rid="nihms-707551-f0005">Extended Data Fig. 5b</xref>). When rescued with FLAG/HA-OPTN or FLAG/HA-NDP52, pentaKOs displayed an increase in mitophagy of more than 5-fold and 4-fold, respectively (). When rescued with FLAG/HA-OPTN or FLAG/HA-NDP52, pentaKOs displayed an increase in mitophagy of more than 5-fold and 4-fold, respectively (Table 1, <xref ref-type="fig" rid="nihms-707551-f0005">Extended Data Fig. 5b–e</xref>). Rescue with FLAG/HA-p62 or ubiquitin-binding mutants (OPTN-Q398X, OPTN-D474N and NDP52ΔZF) failed to increase mitophagy above baseline, but other mutants (OPTN-E50K, OPTN-S177A and NDP52-V248A) rescued mitophagy (). Rescue with FLAG/HA-p62 or ubiquitin-binding mutants (OPTN-Q398X, OPTN-D474N and NDP52ΔZF) failed to increase mitophagy above baseline, but other mutants (OPTN-E50K, OPTN-S177A and NDP52-V248A) rescued mitophagy (Table 1, <xref ref-type="fig" rid="nihms-707551-f0005">Extended Data Fig. 5c–f</xref>). OPTN-E50K and S177A restored mitophagy as well as or better than WT OPTN (). OPTN-E50K and S177A restored mitophagy as well as or better than WT OPTN (Table 1), differing from their response in the presence of Parkin (<xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2e</xref>) likely due to the lack of robust TBK1 activation in the absence of Parkin () likely due to the lack of robust TBK1 activation in the absence of Parkin (<xref ref-type="fig" rid="nihms-707551-f0003">Extended Data Fig 3b</xref>). Here, enhanced OPTN-E50K binding to TBK1). Here, enhanced OPTN-E50K binding to TBK119 may become advantageous by allowing OPTN phosphorylation by TBK1 in the absence of Parkin thus improving mitophagy. In the absence of TBK1 activation, WT OPTN is likely not phosphorylated at S177 and thus is functionally similar to S177A OPTN. Importantly, ubiquitin kinase activity of PINK1 is required, as kinase-dead (KD) PINK1 did not induce mitophagy (Table 1, <xref ref-type="fig" rid="nihms-707551-f0013">Fig. 3c</xref>, , <xref ref-type="fig" rid="nihms-707551-f0005">Extended Data 5g</xref>). Parkin expression dramatically increased mitophagy in FLAG/HA-OPTN expressing pentaKOs (). Parkin expression dramatically increased mitophagy in FLAG/HA-OPTN expressing pentaKOs (Table 1, <xref ref-type="fig" rid="nihms-707551-f0005">Extended Data Fig. 5h</xref>), supporting the model that PINK1-phosphorylated ubiquitin recruits receptors for mitophagy and Parkin ubiquitination of mitochondrial substrates amplifies this ubiquitin signal.), supporting the model that PINK1-phosphorylated ubiquitin recruits receptors for mitophagy and Parkin ubiquitination of mitochondrial substrates amplifies this ubiquitin signal.'], 'nihms-707551-f0006': ['Comparing mitophagy induced by OA treatment in WT relative to PINK1KO cells confirmed that endogenous PINK1 mediates mitophagy in the absence of Parkin (<xref ref-type="fig" rid="nihms-707551-f0006">Extended Data Fig. 6a, b</xref>). Re-expressing PINK1 in PINK1 KO cells rescued OA-induced mitophagy (). Re-expressing PINK1 in PINK1 KO cells rescued OA-induced mitophagy (<xref ref-type="fig" rid="nihms-707551-f0006">Extended Data Fig. 6c, d</xref>). Furthermore, mCherry-OPTN is recruited to mitochondria in the absence of Parkin in a PINK1-dependent manner following prolonged exposure to OA (). Furthermore, mCherry-OPTN is recruited to mitochondria in the absence of Parkin in a PINK1-dependent manner following prolonged exposure to OA (<xref ref-type="fig" rid="nihms-707551-f0006">Extended Data Fig. 6e, f</xref>).).'], 'nihms-707551-f0007': ['Given that PINK1 ubiquitin kinase activity can recruit OPTN and NDP52, we investigated autophagy receptor binding to phospho-mimetic (S65D) HA-ubiquitin in HeLa cells. Endogenous OPTN and NDP52 preferentially co-immunoprecipitate (co-IP) with HA-ubiquitinS65D (<xref ref-type="fig" rid="nihms-707551-f0007">Extended Data Fig. 7a</xref>). Conversely, p62 was present at equal levels in all co-IPs (). Conversely, p62 was present at equal levels in all co-IPs (<xref ref-type="fig" rid="nihms-707551-f0007">Extended Data Fig. 7a</xref>). Ubiquitin-modified and unmodified forms of OPTN and NDP52 were present in co-IPs, and HA-ubiquitinS65D induced or preserved this modification (). Ubiquitin-modified and unmodified forms of OPTN and NDP52 were present in co-IPs, and HA-ubiquitinS65D induced or preserved this modification (<xref ref-type="fig" rid="nihms-707551-f0007">Extended Data Fig. 7a</xref>). Co-IP samples treated with the deubiquitinase USP2 removed the ubiquitin-modified bands on OPTN and NDP52, yet OPTN and NDP52 retained HA-ubiquitinS65D binding (). Co-IP samples treated with the deubiquitinase USP2 removed the ubiquitin-modified bands on OPTN and NDP52, yet OPTN and NDP52 retained HA-ubiquitinS65D binding (<xref ref-type="fig" rid="nihms-707551-f0007">Extended Data Fig. 7b</xref>). Binding of endogenous receptors in HeLa cell cytosol to ). Binding of endogenous receptors in HeLa cell cytosol to in vitro phosphorylated strep-tagged ubiquitin (<xref ref-type="fig" rid="nihms-707551-f0007">Extended Data Fig. 7c</xref>) showed that OPTN, but not p62, bound better to phospho-ubiquitin () showed that OPTN, but not p62, bound better to phospho-ubiquitin (<xref ref-type="fig" rid="nihms-707551-f0007">Extended Data Fig. 7d, e</xref>). However, recombinant GST-OPTN did not bind better to ). However, recombinant GST-OPTN did not bind better to in vitro phosphorylated K63 linked ubiquitin chains27 indicating that OPTN may need additional factors or modification in vivo to preferentially bind Ser65 phosphorylated ubiquitin.'], 'nihms-707551-f0008': ['Autophagy receptors are thought to primarily function by bridging LC3 and ubiquitinated cargo1,2. In mCherry-Parkin WT cells, GFP-LC3B accumulated in distinct puncta adjacent to mitochondria after OA treatment (<xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Fig. 8a</xref>). Although OA also induced GFP-LC3B puncta in pentaKOs, they were fewer and not near mitochondria (). Although OA also induced GFP-LC3B puncta in pentaKOs, they were fewer and not near mitochondria (<xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Fig. 8a</xref>). Conversely, GFP-LC3B in ATG5 KOs was near mitochondria, but not in puncta (). Conversely, GFP-LC3B in ATG5 KOs was near mitochondria, but not in puncta (<xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Fig. 8a</xref>). LC3B lipidation is retained in pentaKOs, but lost in ATG5 KOs (). LC3B lipidation is retained in pentaKOs, but lost in ATG5 KOs (<xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Fig. 8b</xref>). This indicates that ATG5 is activated downstream of PINK1, but independently of autophagy receptors, and that LC3 lipidation and mitochondrial localization are independent steps of mitophagy.). This indicates that ATG5 is activated downstream of PINK1, but independently of autophagy receptors, and that LC3 lipidation and mitochondrial localization are independent steps of mitophagy.', 'mCherry-Parkin (mCh-Parkin) expressing WT, N/O (NDP52/OPTN) DKO and pentaKOs were quantified for a, GFP-LC3A, LC3B and LC3C translocation to mitochondria, b, GFP-WIPI1 or c, GFP-DFCP1 structures per cell (>100 cells counted for each sample) or d, were immunoblotted using phospho-specific anti-S757 and S317 ULK1 antibodies. e, mCherry-Parkin WT, N/O DKO and pentaKOs stably expressing GFP-ULK1 were quantified for GFP-ULK1 puncta per cell (left graph) and the percentage of those puncta on mitochondria (right graph). f, Representative data of e, cells were immunostained for Tom20 and GFP. g, pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCherry-ULK1 (mCh-ULK1) and myc-tagged receptors, were treated with rapalog then imaged live. h, Quantification of mitochondrial ULK1 puncta in g. i, Quantification of mitochondrial ULK1 puncta in pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCh-ULK1 and myc-OPTN mutants, treated with rapalog then imaged live. Quantification in a, b, c, e, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA. (***P<0.001, **P<0.005, *P<0.05, ns, not significant). For live cell quantification >75 cells counted in a blinded manner. Quantification in h and i were performed after removal of outliers, see Online Methods for details. OA, Oligomycin and Antimycin A. Scale bars, 10 μm. a.u., arbitrary units. See <xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Figs. 8c</xref>, , <xref ref-type="fig" rid="nihms-707551-f0009">9b, c</xref> and and <xref ref-type="fig" rid="nihms-707551-f0010">10d</xref> for representative images of for representative images of a, b, c, and i respectively. See <xref ref-type="fig" rid="nihms-707551-f0009">Extended Data Fig. 9e</xref> untreated samples of untreated samples of f and <xref ref-type="fig" rid="nihms-707551-f0010">Extended Data Fig. 10c</xref> for untreated images of for untreated images of h and i.'], 'nihms-707551-f0014': ['OPTN and NDP52 interact with LC3B and LC3C, respectively, for Salmonella clearance13,28. Beyond that, little is known about the specificity of LC3 family members toward autophagy receptors29 or their involvement in mitophagy. We examined the recruitment of all LC3/GABARAP family members to mitochondria in WT, pentaKO and NDP52/OPTN DKO cells. The OA-induced mitochondrial localization of GFP-LC3s in WT cells was absent in pentaKOs, while only GFP-LC3B recruitment was inhibited in NDP52/OPTN DKOs (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4a</xref>, , <xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Fig. 8c</xref>). GFP-LC3C recruitment was inhibited in NDP52/OPTN/TAX1BP1 TKOs (). GFP-LC3C recruitment was inhibited in NDP52/OPTN/TAX1BP1 TKOs (<xref ref-type="fig" rid="nihms-707551-f0008">Extended Data Fig. 8d, e</xref>), indicating that TAX1BP1 can recruit LC3C during mitophagy. GABARAPs did not recruit to mitochondria, indicating they likely play no substantial role in mitophagy (), indicating that TAX1BP1 can recruit LC3C during mitophagy. GABARAPs did not recruit to mitochondria, indicating they likely play no substantial role in mitophagy (<xref ref-type="fig" rid="nihms-707551-f0009">Extended Data Fig. 9a</xref>).).', 'We also examined the involvement of WIPI1 and DFCP1, two proteins that mediate phagophore biogenesis upstream of LC330, in mitophagy. In WT cells, OA induced foci of both GFP-WIPI1 and GFP-DFCP1, mostly localized on or near mitochondria (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4b, c</xref>, , <xref ref-type="fig" rid="nihms-707551-f0009">Extended Data Fig. 9b, c</xref>). In NDP52/OPTN DKOs, GFP-WIPI1 and GFP-DFCP1 foci were reduced and were almost undetectable in pentaKOs (). In NDP52/OPTN DKOs, GFP-WIPI1 and GFP-DFCP1 foci were reduced and were almost undetectable in pentaKOs (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4b, c</xref>, , <xref ref-type="fig" rid="nihms-707551-f0009">Extended Data Fig. 9b, c</xref>). Despite this, phosphorylation of Beclin1). Despite this, phosphorylation of Beclin131 was normal in both pentaKOs and NDP52/OPTN DKOs (<xref ref-type="fig" rid="nihms-707551-f0009">Extended Data Fig. 9d</xref>), indicating that failure to recruit WIPI1/DFPC1 was not due to defective Vps34 complex. GFP-DFCP1 recruitment in pentaKOs was rescued by expression of FLAG/HA-OPTN or FLAG/HA-NDP52, but not by FLAG/HA-p62 (), indicating that failure to recruit WIPI1/DFPC1 was not due to defective Vps34 complex. GFP-DFCP1 recruitment in pentaKOs was rescued by expression of FLAG/HA-OPTN or FLAG/HA-NDP52, but not by FLAG/HA-p62 (<xref ref-type="fig" rid="nihms-707551-f0010">Extended Data Fig. 10a</xref>).).', 'Though autophagy receptors are thought to function late in autophagy with LC332, the deficit in WIPI1 and DFCP1 recruitment to mitochondria indicates a defect upstream in autophagosome biogenesis. ULK1 phosphorylation by AMPK at S317 and dephosphorylation at S75733, required for activation, occurs comparably in WT, NDP52/OPTN DKO and pentaKO cells (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4d</xref>). Despite this, ULK1 localization to mitochondria). Despite this, ULK1 localization to mitochondria34 following OA is diminished by half in the NDP52/OPTN DKOs and more than 80% in pentaKOs (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4e, f</xref>). FLAG/HA-OPTN or FLAG/HA-NDP52, but not FLAG/HA-p62, rescued GFP-ULK1 localization in pentaKOs (). FLAG/HA-OPTN or FLAG/HA-NDP52, but not FLAG/HA-p62, rescued GFP-ULK1 localization in pentaKOs (<xref ref-type="fig" rid="nihms-707551-f0010">Extended Data Fig. 10b</xref>). Overall, these data indicate that NDP52 and OPTN recruit ULK1 to initiate mitophagy.). Overall, these data indicate that NDP52 and OPTN recruit ULK1 to initiate mitophagy.', 'We next assessed if ubiquitin phosphorylation, independent of Parkin, is also sufficient to recruit ULK1 to mitochondria. Rescue of pentaKOs expressing FRB-Fis1 and PINK1Δ110-YFP-FKBP with myc-OPTN or myc-NDP52 resulted in mitochondrial ULK1 puncta following rapalog treatment (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4g, h</xref>). Myc-OPTN-E50K also rescued ULK1 recruitment to mitochondria, but ALS-associated mutant myc-OPTN-Q398X did not (). Myc-OPTN-E50K also rescued ULK1 recruitment to mitochondria, but ALS-associated mutant myc-OPTN-Q398X did not (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4i</xref>, , <xref ref-type="fig" rid="nihms-707551-f0010">Extended Data Fig. 10d</xref>). ULK1 recruitment was restored by myc-OPTN-F178A (). ULK1 recruitment was restored by myc-OPTN-F178A (<xref ref-type="fig" rid="nihms-707551-f0014">Fig. 4i</xref>, , <xref ref-type="fig" rid="nihms-707551-f0010">Extended Data Fig. 10d</xref>), a mutation that disrupts OPTN association with LC3), a mutation that disrupts OPTN association with LC312, indicating that ULK1 recruitment is not through LC3 interaction and occurs upstream of LC3. Taken together, our data show that PINK1 ubiquitin-kinase activity is sufficient to recruit the autophagy receptors and upstream autophagy machinery to mitochondria to induce mitophagy.', 'All statistical data were calculated and graphed using GraphPad Prism 6. To assess statistical significance, data from three or more independent experiments were analyzed using one-way ANOVA and Tukey\'s post-test with a confidence interval of 95%. All error bars are expressed as mean ± standard deviation (s.d.). In <xref ref-type="fig" rid="nihms-707551-f0014">Fig.4h, i</xref> outliers were removed using ROUT in GraphPad Prism 6 with a Q=1%, 1–2 values from each condition were removed. outliers were removed using ROUT in GraphPad Prism 6 with a Q=1%, 1–2 values from each condition were removed.', 'a, Representative images of WT, pentaKO and ATG5 KO HeLa cells expressing mCherry-Parkin (mCh-Parkin) and GFP-LC3B were immunostained for Tom20 (n=3). b, Cell lysates from mCh-Parkin expressing WT, pentaKO and ATG5 KO cells were immunoblotted. c, Representative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCh-Parkin and either GFP-tagged LC3A, LC3B or LC3C were immunostained for Tom20 (n=3, see <xref ref-type="fig" rid="nihms-707551-f0014">Figure 4a</xref> for quantification). for quantification). d, Representative images of WT and N/O/Tx (NDP52/OPTN/TAX1BP1) TKO cells expressing mCh-Parkin and GFP-LC3C were immunostained for Tom20 (n=3) and e, quantified for GFP-LC3C translocation to mitochondria. Quantification in e is displayed as mean ± s.d. from 3 independent experiments and use one-way ANOVA tests (***P<0.001). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.', 'Representative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCherry-Parkin (mCh-Parkin) and either (a) GFP-tagged GABARAP, GABARAPL1 or GABARAPL2, (b) GFP-WIPI1 or (c) GFP-DFCP1 immunostained for Tom20 (n=3 for each condition, see <xref ref-type="fig" rid="nihms-707551-f0014">Figure 4b, c</xref> for quantification of for quantification of b and c). d, mCh-Parkin cell lines as indicated were subjected to either Phos-Tag SDS-PAGE or standard SDS-PAGE followed by immunoblotting. Arrows indicate the position of phosphorylated Beclin species. e, Representative images of untreated WT, N/O (NDP52/OPTN) DKO and pentaKO cell lines expressing mCh-Parkin and GFP-ULK1 were immunostained for Tom20 and GFP (n=3). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.', 'a, Representative images of pentaKOs expressing mCherry-Parkin (mCh-Parkin), GFP-DFCP1 and the indicated FLAG/HA-tagged autophagy receptors immunostained for HA (n=2). Right-hand panels display co-localization of FLAG/HA-tagged constructs and GFP-DFCP1 by fluorescence intensity line measurement. b, Representative images of pentaKOs expressing mCherry-Parkin and GFP-ULK1 were rescued with FLAG/HA-OPTN, FLAG/HA-NDP52, and FLAG/HA-p62, and immunostained for HA and GFP. Arrows indicate HA-tagged receptor puncta (n=2). Right panels display colocalization of HA and GFP by fluorescence intensity line measurement. c, d, Representative images of pentaKOs stably expressing FRB-Fis1 and transiently expressing PINK1Δ110-YFP-2xFKBP and vector or myc-tagged receptors, were (c) untreated or (d) treated with rapalog and imaged live (n=3, see <xref ref-type="fig" rid="nihms-707551-f0014">Figure 4h, i</xref> for quantification of for quantification of c, d). OA, Oligomycin and Antimycin A. Scale bars, 10 μm. e, Old and new models of PINK1/Parkin mitophagy. The old model is dominated by Parkin ubiquitination of mitochondrial proteins. Here PINK1 plays a small initiator role whose main function is to bring Parkin to the mitochondria. The new model depicts Parkin-dependent and independent pathways leading to robust and low-level mitophagy, respectively. Based on our data, PINK1 is central to mitophagy both before and after Parkin recruitment by phosphorylating UB to recruit both Parkin and autophagy receptors mitochondria, to induce clearance. In the absence of Parkin (right panel), this occurs at a low level due to the relatively low basal UB on mitochondria. When Parkin is present it serves to amplify the PINK1 generated UB-PO4 signal, allowing for robust and rapid mitophagy induction.'], 'nihms-707551-f0010': ['Through genetic knockout of five autophagy receptors we have defined their relative roles in mitophagy and identified their unanticipated upstream involvement in autophagy machinery recruitment. p62 and NBR1 are dispensable for Parkin-mediated mitophagy; OPTN and NDP52 are the primary, yet redundant, receptors. We also uncovered a new and more fundamental role for PINK1 in mitophagy: to directly induce mitophagy through phospho-ubiquitin-mediated recruitment of autophagy receptors. We posit that PINK1 generates the novel and essential signature (phospho-ubiquitin) on mitochondria to induce OPTN and NDP52 recruitment and mitophagy; Parkin acts to increase this signal by generating more ubiquitin chains on mitochondria, which are subsequently phosphorylated by PINK1. Our findings clarify the role of Parkin as an amplifier of the PINK1-generated mitophagy signal, phospho-ubiquitin, which can engage the autophagy receptors to recruit ULK1, DFCP1, WIPI1 and LC3 (see model in <xref ref-type="fig" rid="nihms-707551-f0010">Extended Data Fig. 10e</xref>).).'], 'nihms-707551-f0012': ['a, KO cell lines with or without mCherry-Parkin (mCh-Parkin) expression were immunoblotted and b, CoxII levels were quantified. c, A panel of human tissue lysates was immunoblotted. d, Expression of WT or mutant GFP-OPTN in mCh-Parkin pentaKOs. e, Quantification of cells in f. >100 cells per condition. f, Representative images of mCh-Parkin pentaKOs expressing GFP-OPTN mutants immunostained for Tom20 (n=3). g, pentaKOs expressing mCh-Parkin were rescued with WT or mutant GFP-OPTN, analyzed by immunoblotting. See <xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2e</xref> for quantification of CoxII. for quantification of CoxII. h, Expression of WT or mutant GFP-NDP52 in mCh-Parkin pentaKOs. i, Quantification of cells in j. >100 cells per condition. j, Representative images of mCh-Parkin pentaKOs expressing WT or mutant GFP-NDP52 were immunostained for Tom20 (n=3). k, pentaKOs expressing mCh-Parkin rescued with WT or mutant GFP-NDP52 were analyzed by immunoblotting. See <xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2f</xref> for quantification of CoxII. Quantification in for quantification of CoxII. Quantification in b and i are displayed as mean ± s.d. from 3 independent experiments using one-way ANOVA tests (***P<0.001, ns, not significant) and in e as mean from 2 independent experiments. Scale bars, 10 μm.', 'a, Representative images of untreated mCherry-Parkin (mCh-Parkin) cells and merged images of treated cells as indicated immunostained for DNA. See <xref ref-type="fig" rid="nihms-707551-f0012">Fig. 2a</xref> for anti-DNA/DAPI images of treated samples (n=3). for anti-DNA/DAPI images of treated samples (n=3). b, Cell lysates from WT, N/O (NDP52/OPTN) DKO, OPTN KO and NDP52 KO cells with or without mCh-Parkin expression were immunoblotted for TBK1 activation. c, Cell lysates from WT and PINK1 KO cells without Parkin expression were immunoblotted for TBK1 activation (S172 phosphorylation). d, Confirmation of T/N (TBK1/NDP52) DKO, T/O (TBK1/OPTN) DKO and TBK1 KO by immunoblotting. e, KO cell lines from d were immunostained for Tom20 (n=3). f, Representative images of untreated mCh-Parkin WT and KO cells, and merged images of treated cells as indicated were immunostained for DNA. See <xref ref-type="fig" rid="nihms-707551-f0012">Figure 2g</xref> for anti-DNA/DAPI images treated samples (n=3). for anti-DNA/DAPI images treated samples (n=3). g, T/N DKO cells rescued with GFP-TBK1 WT or K38M, or GFP-OPTN S177D and were assessed by immunoblotting. h, Cells in g were assessed by immunoblotting. i, Quantification of CoxII levels in h displayed as mean ± s.d. from 3 independent experiments and use one-way ANOVA tests (**P<0.005, ns, not significant). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.'], 'nihms-707551-f0004': ['a, Representative images of HeLa cells expressing FRB-Fis1, WT (PINK1-WT) or kinase-dead (PINK1-KD) PINK1Δ110-YFP-2xFKBP and mCherry-OPTN (mCh-OPTN) or mCherry-NDP52 (mCh-NDP52) treated with rapalog. b, Quantification of receptor translocation in cells from a and <xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4c–e</xref>. >100 cells were counted per sample. . >100 cells were counted per sample. c, Cells were treated with rapalog and analyzed by FACS for lysosomal positive mt-mKeima. Representative data for WT or KD PINK1Δ110-YFP-2xFKBP in pentaKOs without or with FLAG/HA-OPTN expression. Quantification in b is displayed as mean ± s.d. from 3 independent experiments and uses one-way ANOVA tests (***P<0.001, *P<0.05, ns, not significant). For images of untreated cells from a, see <xref ref-type="fig" rid="nihms-707551-f0004">Extended Data Fig. 4b</xref>. Scale bars, 10 μm.. Scale bars, 10 μm.']}
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The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy
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Nature
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1440054000
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Poorly water-soluble drugs often suffer from limited or irreproducible clinical response due to their low solubility and dissolution rate. In this study, organic solvent-free solid dispersions (OSF-SDs) containing telmisartan (TEL) were prepared using polyvinylpyrrolidone K30 (PVP K30) and polyethylene glycol 6000 (PEG 6000) as hydrophilic polymers, sodium hydroxide (NaOH) as an alkalizer, and poloxamer 188 as a surfactant by a lyophilization method. In-vitro dissolution rate and physicochemical properties of the OSF-SDs were characterized using the USP I basket method, differential scanning calorimetry (DSC), X-ray diffractometry (XRD) and fourier transform-infrared (FT-IR) spectroscopy. In addition, the oral bioavailability of OSF-SDs in rats was evaluated by using TEL bulk powder as a reference. The dissolution rates of the OSF-SDs were significantly enhanced as compared to TEL bulk powder. The results from DSC, XRD showed that TEL was molecularly dispersed in the OSF-SDs as an amorphous form. The FT-IR results suggested that intermolecular hydrogen bonding had formed between TEL and its carriers. The OSF-SDs exhibited significantly higher AUC0-24 h and Cmax, but similar Tmax as compared to the reference. This study demonstrated that OSF-SDs can be a promising method to enhance the dissolution rate and oral bioavailability of TEL.
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other
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PMC5018156
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30
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[
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] |
Nature. 2015 Aug 20; 524(7565):309-314
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Fluorescence in situ hybridization (FISH)-based karyotype of Theobroma cacao Matina 1-6. A FISH cocktail comprised of two Cent-Tc oligonucleotide probes plus four BAC clones permitted identification of the ten chromosome pairs. (A) Ideogram of the T. cacao Matina 1-6 karyotype. Centromeres are coded in accordance with the color and size of the combined FISH signals for OLI-07 (green pseudo-colored) and OLI-13 (red pseudo-colored). Bacterial artificial chromosome (BAC) probes (red or green) are indicated by paired dots near chromosome termini. The following BACs were used for probes: for Tc03, TcC_Ba057I03 and TcC_Ba027M06; for Tc04, TcC_BB065A03; for Tc06, TcC_Ba018I22. Relative chromosome sizes are not indicated, with the exception of the satellite arm of Tc07, which is shown as a knob. (B) Chromosomes labeled with the FISH cocktail arranged by chromosome number. Chromosomes are discriminated as follows: Tc01 has the second-brightest yellow centromere. Tc02 has the brightest yellow centromere. Tc03, Tc04, Tc06, and Tc07 all have similar centromere labeling (pure green), but are differentiated based on unique BAC probe labeling: Tc03 is labeled at each end by green BAC probes; Tc04 is labeled at one end by a green BAC probe; Tc06 is labeled at one end by a red BAC probe; and Tc09 is not labeled by BAC probes. Tc05 has the second-brightest red centromere; Tc08 has the brightest red centromere with an 'internal' green domain; and Tc09 has the brightest yellow-green centromere and is much longer than Tc10, which has the second-brightest yellow-green centromere. (C) DAPI channel image of chromosomes in (B). The satellite arms of Tc07 are above the centromeres. (D) A FISH image containing a complete chromosome spread. (E) Corresponding DAPI channel image from which chromosomes in (C) were extracted.
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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"fluorescence microscopy",
"bar plot",
"immunoblot"
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[{'image_id': 'gb-2013-14-6-r53-1', 'image_file_name': 'gb-2013-14-6-r53-1.jpg', 'image_path': '../data/media_files/PMC4053823/gb-2013-14-6-r53-1.jpg', 'caption': "Fluorescence in situ hybridization (FISH)-based karyotype of Theobroma cacao Matina 1-6. A FISH cocktail comprised of two Cent-Tc oligonucleotide probes plus four BAC clones permitted identification of the ten chromosome pairs. (A) Ideogram of the T. cacao Matina 1-6 karyotype. Centromeres are coded in accordance with the color and size of the combined FISH signals for OLI-07 (green pseudo-colored) and OLI-13 (red pseudo-colored). Bacterial artificial chromosome (BAC) probes (red or green) are indicated by paired dots near chromosome termini. The following BACs were used for probes: for Tc03, TcC_Ba057I03 and TcC_Ba027M06; for Tc04, TcC_BB065A03; for Tc06, TcC_Ba018I22. Relative chromosome sizes are not indicated, with the exception of the satellite arm of Tc07, which is shown as a knob. (B) Chromosomes labeled with the FISH cocktail arranged by chromosome number. Chromosomes are discriminated as follows: Tc01 has the second-brightest yellow centromere. Tc02 has the brightest yellow centromere. Tc03, Tc04, Tc06, and Tc07 all have similar centromere labeling (pure green), but are differentiated based on unique BAC probe labeling: Tc03 is labeled at each end by green BAC probes; Tc04 is labeled at one end by a green BAC probe; Tc06 is labeled at one end by a red BAC probe; and Tc09 is not labeled by BAC probes. Tc05 has the second-brightest red centromere; Tc08 has the brightest red centromere with an 'internal' green domain; and Tc09 has the brightest yellow-green centromere and is much longer than Tc10, which has the second-brightest yellow-green centromere. (C) DAPI channel image of chromosomes in (B). The satellite arms of Tc07 are above the centromeres. (D) A FISH image containing a complete chromosome spread. (E) Corresponding DAPI channel image from which chromosomes in (C) were extracted.", 'hash': '6125a59e65f94f2af43c21f2c9a059ebe7faaf804f3d4f2ad6255461d13de76a'}, {'image_id': 'gb-2013-14-6-r53-6', 'image_file_name': 'gb-2013-14-6-r53-6.jpg', 'image_path': '../data/media_files/PMC4053823/gb-2013-14-6-r53-6.jpg', 'caption': 'TcMYB113 transcript levels determined by quantitative PCR analysis of RNA from the pericarp of cherelles (young Theobroma cacao fruits) of genotypes with green or red pod color (genotype names are colored accordingly). Bar colors indicate the color of (a,b) the cherelles and (c,d) the alleles analyzed Although standard deviations were calculated, the values obtained were too low to graph at the scale used below. All RNA levels are normalized to control RNA from Pound 7 leaf tissue. (a) TcMYB113 transcript levels in the pericarp of small (10 to 20 mm length) and large (30 to 50 mm length) cherelles of the green-pod genotype Pound 7 and the pericarp of the red-pod genotypes UF 273 Type 1 and Type 2. (b) TcMYB113 transcript levels in the pericarp of green cherelles of the green-pod genotype Gainesville II 316 and the pericarp of green, green plus red. and red cherelles of the red-pod genotype Gainesville II 164. Green cherelles from Gainsville II 164 were obtained from the completely shaded part of the tree sampled. (c) Allele-specific TcMYB113 transcript levels in the pericarp from small (10 to 20 mm length) and large (30 to 50 mm length) cherelles of the green-pod genotype Pound 7 and the pericarp of the red-pod genotypes UF 273 Type 1 and Type 2. (d) TcMYB113 allele-specific transcript levels in the pericarp of green cherelles of the green-pod genotype Gainesville II 316 and inthe pericarp of green (no sun), green plus red (partial sun), and red (full sun) cherelles of the red-pod genotype Gainesville II 164. Green cherelles from Gainsville II 164 were obtained from a completely shaded area of the tree sampled. Red bars in the green-pod Pound 7 is due to background fluorescence.', 'hash': 'c4b3773f96571c816cf3668f934d03638c738b9bc41c5588b05b79c6c306f754'}, {'image_id': 'gb-2013-14-6-r53-3', 'image_file_name': 'gb-2013-14-6-r53-3.jpg', 'image_path': '../data/media_files/PMC4053823/gb-2013-14-6-r53-3.jpg', 'caption': "Statistical significance of association of pod color with markers on chromosome 4 of the parental Theobroma cacao haplotypes. The y-value at each marker is -log10(P-value) with the P-value computed using Fisher's exact test for both haplotypes of each parent, taking as input a 2 × 2 contingency table per marker. The segment between the vertical dashed lines is the genomic region most strongly associated with pod color in all three mapping populations. Thresholds denoted by the dashed red line in each plot were calculated using the Bonferroni correction for multiple comparisons at α = 0.05.", 'hash': 'a8227345c6aa99cd6d98e655ef7565d75f8d2b2a261f3f508bdaa86cbf26cd2b'}, {'image_id': 'gb-2013-14-6-r53-4', 'image_file_name': 'gb-2013-14-6-r53-4.jpg', 'image_path': '../data/media_files/PMC4053823/gb-2013-14-6-r53-4.jpg', 'caption': "Haplotype analysis of trees exhibiting recombination in the chromosome 4 segment associated with pod color. (a) Recombinant trees from population T4 Type 1; (b) recombinant trees from population T4 Type 2; (c) recombinant trees from population MP01. Maternal and paternal haplotypes are shown at the top of each figure. Tree names are colored according to pod-color phenotype. Red represents haplotypes associated with red pod color, and green represents haplotypes associated with green pod color from the two parents, while the yellow marker values represent uncertainty in the haplotype assignment. The black vertical bars surround the most likely region regulating pod-color variation according to the haplotypes of the recombinants (that is, if a recombinant shows only haplotypes for a given marker associated with green pods, but its phenotype is red, this indicates that the marker is not associated with pod color; this is the case for CATIE 1-63 at marker 22,053,861 in (a). The P-values from the Fisher's exact test are shown above each marker for each parent. The P-values are colored by parental phenotype, with the father always being bright red. The location of three candidate genes is indicated by colored dots above the closest markers.", 'hash': '26e4a8ae34d94362b8c441cc3647503b8b2b99e4f724226fc48ab363b3896520'}, {'image_id': 'gb-2013-14-6-r53-5', 'image_file_name': 'gb-2013-14-6-r53-5.jpg', 'image_path': '../data/media_files/PMC4053823/gb-2013-14-6-r53-5.jpg', 'caption': 'mir828 and TAS4-siR81 (-) sequence targets in TcMYB113. The green base pair indicates the single-nucleotide polymorphism (SNP) (20,878,891) that was most significantly associated with pod-color variation (C is associated with green pods and G is associated with red pods).', 'hash': '82b284d37da2f1bccf9e5c19bd0ca51a4bbc32cd10ba594a41b51a167ddd3aea'}, {'image_id': 'gb-2013-14-6-r53-2', 'image_file_name': 'gb-2013-14-6-r53-2.jpg', 'image_path': '../data/media_files/PMC4053823/gb-2013-14-6-r53-2.jpg', 'caption': 'Genomic features of Theobroma cacao Matina 1-6. Shown are overall densities of evidence sets (see Materials and methods) that contributed to T. cacao Matina 1-6 annotation, and the final results as described in the text. Data were plotted for the chromosomes (pseudomolecules) in 50 Kbp sliding windows. Yellow denotes protein homology evidence by alignment to proteins of eight previously annotated plant genomes; blue denotes mapping of transcriptome data from second-generation RNA sequencing; green denotes gene models; red denotes transposons from homology-based and structure-based annotation, as described in the text (see Materials and methods).', 'hash': '157fb55eae12f22029a7656c4df86e3b37df6138762958d3c98049763eaa02ad'}]
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{'gb-2013-14-6-r53-1': ['We performed fluorescence in situ hybridization (FISH)-based karyotyping of mitotic T. cacao chromosomes, using two types of probes: genomic repeats (centromeric repeats) for differential chromosome \'painting\', and bacterial artificial chromosomes (BACs) for chromosome identification. Centromeric repeat-based FISH has been used to identify chromosomes in soybean [16] and maize [17,18]. We identified sequences homologous to a previously identified candidate centromeric repeat [11] by searching unassembled sequence reads acquired during generation of the Matina 1-6 physical map [19]. A ClustalW alignment (not shown) of the 236 sequences with the highest percentage of identity indicated a length for the Matina 1-6 Cent-Tc repeat of 171 bp (see Additional file 2, Figure S1 for consensus), comparable with the centromere monomer length in other plant species [20-22]. We designed specific oligonucleotide probes that target highly conserved or less well conserved centromeric repeat T. cacao (Cent-Tc) regions (see Additional file 2, Figure S1). Probe OLI-07 was present in the majority (74%) of the aligned Cent-Tc repeats, whereas probe OLI-13 was present in 12.5%. When used together in FISH experiments (see Materials and methods section for details) the two probes differentiated the mitotic chromosomes into several distinct color and intensity subgroups (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-1">1</xref>). To identify individual chromosomes, BAC probes (Materials and methods; also see Additional file ). To identify individual chromosomes, BAC probes (Materials and methods; also see Additional file 1, Table S3) derived from low-repeat content regions of each of the 10 pseudomolecules defined by Matina 1-6 physical mapping [19] were individually mapped to Cent-Tc-labeled chromosomes (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-1">1</xref> and data not shown). We then developed a six-component (two Cent-Tc probes plus four BAC probes) FISH \'cocktail\' that simultaneously identified every chromosome in a single mitotic chromosome spread (Figure and data not shown). We then developed a six-component (two Cent-Tc probes plus four BAC probes) FISH \'cocktail\' that simultaneously identified every chromosome in a single mitotic chromosome spread (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-1">1</xref>). Because the Matina 1-6 physical map is anchored by high-density molecular markers that relate the map to the sequenced genome, pseudomolecule-derived BAC probes permit the association of molecular-linkage groups and the pseudomolecules with the chromosomes themselves, thereby enabling chromosome numbering based on pseudomolecule numbering, and generating a single, unified cytogenetic map for Matina 1-6.). Because the Matina 1-6 physical map is anchored by high-density molecular markers that relate the map to the sequenced genome, pseudomolecule-derived BAC probes permit the association of molecular-linkage groups and the pseudomolecules with the chromosomes themselves, thereby enabling chromosome numbering based on pseudomolecule numbering, and generating a single, unified cytogenetic map for Matina 1-6.'], 'gb-2013-14-6-r53-2': ['We prioritized the generation and use of RNA sequencing data from diverse experiments, and from available plant whole-gene sets, for evidence-based annotation. The overall results are shown schematically in Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-2">2</xref>..', 'We used coding and intron spans from protein alignments of eight annotated plant genomes for homology modeling (see Materials and methods; also see Additional file 1, section 3). The contributions of RNA and protein analyses are displayed independently with final models in Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-2">2</xref>..', 'A significant portion of eukaryotic genomes is composed of transposable elements (TEs) [35-37]. Using a structure-based and homology-based strategy, we identified 8,542 intact TEs in the T. cacao Matina 1-6 genome. Those elements, together with numerous truncated elements and other fragments, cover 41.53% of the assembled genome. The overall results are shown schematically in Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-2">2</xref>. In a parallel comparative analysis using the same approach, we identified 5,089 intact TE copies in the . In a parallel comparative analysis using the same approach, we identified 5,089 intact TE copies in the T. cacao Criollo genome [11], and found only 35.40% of the Criollo genome assembly to be comprised of TEs (see Additional file 1, Table S15). Although this estimate represents a nearly 10% increase in TEs over a previous annotation of Criollo (version 1 [11]), the proportion of TEs in the assembled portion of the Criollo genome is notably less than that in the assembled portion of the Matina 1-6 (version 1.1) genome.'], 'gb-2013-14-6-r53-3': ['Using haplotypes can be more powerful than using single-marker methods to detect phenotypic effects of low-frequency variants in genome-wide association studies [41,42]. Haplotype-based methods are also useful for inferring the underlying causal genetic basis for various traits in linkage-mapping populations because, as shown here, it is possible to evaluate the parental inheritance of the associated haplotype more efficiently. We therefore determined parental haplotypes for chromosome 4 in individuals from the three mapping populations studied to help us identify candidate genes that are associated with this trait. We resolved the genotypes of the progeny in each of the mapping populations for chromosome 4 into the two corresponding parental haplotypes, using the output of HAPI-UR [43] (see Materials and methods). To investigate the effect from each parental haplotype separately on pod color, we performed Fisher\'s exact tests separately for each parent, on individuals from each mapping population, using a 2 × 2 contingency table of parental haplotypes for each marker and red and green pod color. This analysis identified a difference between the T4 and MP01 populations; in both T4 populations, haplotypes belonging to one parent, Pound 7, did not affect the color phenotype; whereas in MP01, haplotypes from both parents did affect pod color (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-3">3</xref>). The observation that a single haplotype from each parent associates with red pod color in MP01, but not in T4, is consistent with the mendelian model outlined above, and suggests the existence of a potential common allele in the two T4 mapping populations, even though the parents are different. Our results also suggest that red pod color is dominant over green and that both UF 273 parents are heterozygous for the red allele, whereas the common parent, Pound 7, is homozygous for the recessive green allele.). The observation that a single haplotype from each parent associates with red pod color in MP01, but not in T4, is consistent with the mendelian model outlined above, and suggests the existence of a potential common allele in the two T4 mapping populations, even though the parents are different. Our results also suggest that red pod color is dominant over green and that both UF 273 parents are heterozygous for the red allele, whereas the common parent, Pound 7, is homozygous for the recessive green allele.', 'We then focused on the small region that we had identified on chromosome 4 (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-3">3</xref>) as that most significantly associated with the trait. The locations of the most significant SNP markers vary only slightly between the mapping populations, ranging from 20,987,989 bp to 21,726,996 bp. The haplotype combinations, taking into account SNP 21,126,449 bp, explain the phenotypes in the data with at least 97% accuracy in each population (see Additional file ) as that most significantly associated with the trait. The locations of the most significant SNP markers vary only slightly between the mapping populations, ranging from 20,987,989 bp to 21,726,996 bp. The haplotype combinations, taking into account SNP 21,126,449 bp, explain the phenotypes in the data with at least 97% accuracy in each population (see Additional file 1, Table S21; see Materials and methods for more details on the computation with Fisher\'s exact test).', 'To infer haplotypes from genotype data, we took the consensus phasing results from 101 runs of HAPI-UR (window size of 75 and Ne of 1000) [43]. HAPI-UR does not explicitly relate progeny haplotypes to their respective parental haplotypes. Thus, to assign the haplotype of each progeny to its respective parental haplotype (or haplotypes, if the progeny was recombinant), we first identified which parental haplotype each progeny was most similar to, based on the number of pairwise nucleotide differences (Hamming distance) between them. Each one of the two haplotypes from each progeny was assigned to whichever one of the four parental haplotypes showed the shortest distance of the four pairwise comparisons. There were no instances of the two haplotypes of a progeny being assigned to the same parent. Next, parental assignments for recombinant haplotypes (that is, where the distance to any given parent haplotype was >0) were resolved by identifying which of the two parental haplotypes was represented in the progeny haplotype at each heterozygous marker. Parental assignments at homozygous markers were then inferred by examining the assignments made at the closest heterozygous positions to either side. If the two closest heterozygous assignments agreed, then that assignment was used at the homozygous position; if not, then no assignment was made. By using this method, we implicitly sought to minimize the number of recombination events between the two parental strands as represented in each recombinant progeny haplotype. Parental assignments identified in this way were used in the haplotype-phenotype association test (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-3">3</xref>) and to identify haploblocks from the four parental haplotypes on recombinant progenies (Figure ) and to identify haploblocks from the four parental haplotypes on recombinant progenies (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-4">4</xref>). We also resolved the haplotypes and haploblock (parental) assignments using additional software (iXora [). We also resolved the haplotypes and haploblock (parental) assignments using additional software (iXora [105]) developed by our group and obtained identical results. iXora is a phasing and trait association method that allows precise inference of haplotypes of F1 progeny from mapping and breeding populations derived from non-inbred parents.', 'To test the statistical significance of genotype-phenotype and haplotype-phenotype associations, we applied Fisher\'s exact test independently for each marker using SAS software (SAS Institute, Cary, NC, USA) [71]. The input to the test is a contingency table in which columns denote the phenotypes (red or green pods), and rows denote the genotypes or the haplotypes. We applied the test for the genotypes at each SNP marker from the 6K SNP chip, resulting in a 3 × 2 or 2 × 2 contingency table (see Additional file 2, Figure S7), and also for each parent (using both haplotypes), resulting in a 2 × 2 contingency table per marker for each parent (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-3">3</xref>). The test on the parents separately demonstrates each parental effect on the phenotype.). The test on the parents separately demonstrates each parental effect on the phenotype.'], 'gb-2013-14-6-r53-4': ['To fine-tune the mapping of the region of interest, we examined trees exhibiting a recombination of the haplotypes within the associated region, and added flanking markers to extend the region to 20.35 to 22.05 Mbp. In each T4 population, there were 8 to 15 trees with recombination events in at least one parental haplotype (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-4">4a,b</xref>). The T4 recombinants refined the location of the causal locus to the region between 20,562,635 and 21,726,996 bp on chromosome 4 (Figure ). The T4 recombinants refined the location of the causal locus to the region between 20,562,635 and 21,726,996 bp on chromosome 4 (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-4">4a,b</xref>).).', 'Using MP01 trees that exhibit recombination in this region, the location of the causal locus was further refined (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-4">4c</xref>) to the region between 20,562,635 and 21,126,449 bp on chromosome 4. Indeed, the alleles in ) to the region between 20,562,635 and 21,126,449 bp on chromosome 4. Indeed, the alleles in TcAPO4 and TcMYB6 associated with red pod color in the CCN 51 and TSH 1188 parents were now also associated with green pod color in the recombinants. This result further supports the hypothesis of a single allele underlying pod color (although there is the possibility that different mutations in the same gene could be responsible).', 'The availability of the high-quality Matina 1-6 reference genome permits straightforward identification of candidate genes within the restricted chromosome region identified. Recombination events identified in the T4 Type 1 and Type 2 mapping populations delimit the region regulating pod-color variation to between 20,562,635 and 21,726,996 bp on chromosome 4 (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-4">4a,b</xref>). In this region, three gene models are of particular interest: TCM_019192, homologous to ). In this region, three gene models are of particular interest: TCM_019192, homologous to Arabidopsis MYB113; TCM_019219, homologous to the Vitis vinifera APO4 gene; and TCM_019261, homologous to Arabidopsis MYB6. Both MYB genes encode transcription factors of the R2R3 domain class, which are known to be diverse in plants [44]; whereas APO proteins promote photosystem 1 complex stability and associated chlorophyll accumulation [45].', 'As described above, the MP01 recombinants narrow the region of interest to genes located between 20,562,635 and 21,126,449 bp on chromosome 4 (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-4">4c</xref>). Within this region, only one of the two MYB transcription factors is present: TCM_019192, which is homologous to ). Within this region, only one of the two MYB transcription factors is present: TCM_019192, which is homologous to VvMybA1. We refer to this gene as TcMYB113, and it encodes a protein with 275 amino acids sharing 61% identity with grape VvMYBA1 (see Additional file 2, Figure S8 for protein-sequence comparison). In the Criollo genome [11], TCM_019192 is annotated as two genes: Tc04_t014240 and Tc04_t014250. This difference may be attributed to the strict evidence-based Matina 1-6 version 1.1 annotation procedures that we used (see above).'], 'gb-2013-14-6-r53-5': ['The resequencing data shows specific SNPs in TcMYB113 within coding regions in the alleles associated with red pod color, which are at positions 20,878,747, 20,878,891, 20,878,957, 20,879,122, and 20,879,148. We corroborated this result by Sanger sequencing of the three candidate genes studied and by association mapping (see Additional file 1, Table S23). The association-mapping analysis was performed using 73 SNPs generated via Sanger sequencing (see Materials and methods) and 95 other SNPs from chromosomes 1 to 10 in 54 genotypes with green pods and 17 genotypes with red pods from diverse genetic backgrounds. Without accounting for genetic structure, the most significant P-values from the Fisher test were for the following SNPs (from highest to lowest significance): 20,878,891; 20,875,691, and 20,879,148 (see Additional file 1, Table S23). SNPs at these positions permitted differentiation of alleles that are associated with the green-pod Criollo genotypes from those associated with the red-pod trees of Criollo origin (see Additional file 2, Figure S10). The least significant of these (position 20,879,148; see Additional file 1, Table S23) results in an amino-acid change from serine to asparagine at codon 221 of the TcMYB113 protein in the alleles associated with red pod color. This residue occurs outside of the R2R3 DNA binding domain, but within the C-terminal region that varies substantially between MYB family members, making the structural consequence of this substitution difficult to predict. When genetic structure was taken into account in the association-mapping analysis, the significance of the association between this SNP and pod color was considerably lower (see Additional file 1, Table S23). The second most significant SNP (position 20,875,691) was detected via Sanger sequencing in the TcMYB113 5\' untranslated region (UTR), located 25 bases upstream of the ATG start site (see Additional file 1, Table S23). The function of this SNP is difficult to infer; however, mutations occurring near translation start sites are known to affect protein-translation rates [53]. The SNP that was most significantly associated with pod color (position 20,878,891) is a synonymous mutation found within the coding region of TcMYB113. Intriguingly, this SNP is positioned within a target site for a dicot trans-acting small interfering RNA (tasiRNA) derived from TAS4 (TRANS-ACTING siRNA 4) TAS4-siR81(-) as identified by Luo et al. [54] (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-5">5</xref>; see Additional file ; see Additional file 2, Figure S8). TAS-derived siRNAs post-transcriptionally downregulate protein-coding transcripts in a manner similar to microRNA (miRNA)-directed repression [55,56]. MYB113 regulation in Arabidopsis additionally involves miR828, which acts both to cleave TAS4 to generate the interfering small RNA TAS4-siR81(-) [57] and also, independently, to silence MYB113 [54]. We identified not only the TAS4-siR81 site, but also a conserved miR828 target sequence within TcMYB113 (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-5">5</xref>), suggesting that these regulatory mechanisms are highly conserved in cacao. However, we detected no polymorphism within the miR828 target sequencewhich overlaps with the highly conserved R3 domain (Figure ), suggesting that these regulatory mechanisms are highly conserved in cacao. However, we detected no polymorphism within the miR828 target sequencewhich overlaps with the highly conserved R3 domain (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-5">5</xref>). The activities of both ). The activities of both TAS4 and miR828 ultimately regulate anthocyanin biosynthesis through MYBs in Arabidopsis [54]. Conservation of and natural variation in this regulatory loop is yet to be explored in other plants.'], 'gb-2013-14-6-r53-6': ['Pigmentation intensity has previously been correlated with variation in MYB transcript level [50,58,59]. In parallel with our genetic analyses, we quantified the accumulation of candidate-gene transcripts to assess their involvement with pod color. RNAs from developing young fruits (cherelles) of Pound 7 (a green-pod genotype) and UF 273 Type 1 and Type 2 (red-pod genotypes) were analyzed by quantitative (q)PCR, to compare the relative transcript levels of the T. cacao MYB113, AP04 and MYB6 genes. Relative expression was normalized to leaf cDNA generated from Pound 7. The TcAPO4 and TcMYB6 genes each showed decreased expression in all samples relative to expression in leaf, but there were no differential gene-expression patterns that correlated with pod color. By contrast, TcMYB113 expression in the small cherelle samples for all genotypes showed a modest increase in expression relative to the Pound 7 leaf (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-6">6a</xref>). This suggests that ). This suggests that TcMYB113 gene expression might be tissue-specific, because anthocyanin accumulates in young leaves, including those of Pound 7 [7]. We found TcMYB113 transcript accumulation to be correlated with color intensity in a variety of samples tested. TcMYB113 transcript levels in large UF 273 cherelles (a clone with red pod color ), were 25-fold higher than that in large Pound 7 cherelles (a clone with green pod color), and 250-fold higher than that in Pound 7 leaves (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-6">6a</xref>). Notably higher transcript levels were detected in large cherelles relative to small cherelles (Figure ). Notably higher transcript levels were detected in large cherelles relative to small cherelles (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-6">6a</xref>), and this correlated with the increase in pigmentation intensity during the early stages of fruit development.), and this correlated with the increase in pigmentation intensity during the early stages of fruit development.', 'Because anthocyanin accumulation is influenced by sunlight [58], we also measured transcript levels of TcMYB113 in a genotype (Gainesville II 164) that has red pods at maturity, but exhibits both green and red pigmentation during fruit development according to light exposure. Green cherelles were collected from the shaded branches of a Gainesville II 164 tree, and red cherelles were collected from an area of the same tree that was exposed to sunlight. TcMYB113 transcript levels in Gainesville II 164 were 200-fold and 100-fold higher (relative to the transcript levels in Pound 7 leaf) in the red and the mixed greenred cherelles, respectively, compared with the fully shaded green cherelles (25-fold increase relative to Pound 7 leaf) (Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-6">6b</xref>). Gainesville II 316 (a green-pod genotype) that had been exposed to full sunlight was used for comparison, and its transcript levels (20-fold increase relative to leaf) were comparable with those of the Gainsville II 164 green cherelles. Together, these results further corroborate the involvement of ). Gainesville II 316 (a green-pod genotype) that had been exposed to full sunlight was used for comparison, and its transcript levels (20-fold increase relative to leaf) were comparable with those of the Gainsville II 164 green cherelles. Together, these results further corroborate the involvement of TcMYB113 in determining pod color in cacao, and are a first indication that anthocyanin production in response to light in pods is mediated by TcMYB113 accumulation.', 'To further confirm that TAS4 siRNA plays a role in the downregulation of TcMYB113 transcripts, we repeated the experiments described above, but this time using primers specific to the green and red alleles (Figures <xref ref-type="fig" rid="gb-2013-14-6-r53-6">6c,d</xref>). cDNA from small and large pods of the T4 Type 1 mapping population parents were used as template, as described for the experiments mentioned above. Red-colored bars in the figure represent relative steady-state expression of the red allele, and green bars represent expression of the green allele. These results show a five-fold increase in the expression of the red allele over the green in samples with red pods (UF273 Type 1 and Type 2), whereas in green-colored pods, very little expression of either allele is present. Cumulative expression of both alleles is consistent with the previous expression levels seen for ). cDNA from small and large pods of the T4 Type 1 mapping population parents were used as template, as described for the experiments mentioned above. Red-colored bars in the figure represent relative steady-state expression of the red allele, and green bars represent expression of the green allele. These results show a five-fold increase in the expression of the red allele over the green in samples with red pods (UF273 Type 1 and Type 2), whereas in green-colored pods, very little expression of either allele is present. Cumulative expression of both alleles is consistent with the previous expression levels seen for TcMYB113.', 'Allele-specific transcript levels were also measured in the Gainsville II clone with varying exposure to sunlight. The green-pod Gainsville II 316 showed no steady-state expression of the red allele, and its green-allele expression was similar to that seen in Figure <xref ref-type="fig" rid="gb-2013-14-6-r53-6">6d</xref>. For the red-pod Gainsville II 164, expression of the red allele was consistently higher than that of the green, and increased with increasing exposure to sunlight. Additionally, green-allele accumulation in this clone, although significantly smaller than that of the red allele, still increased with increasing exposure to sunlight. This suggests that . For the red-pod Gainsville II 164, expression of the red allele was consistently higher than that of the green, and increased with increasing exposure to sunlight. Additionally, green-allele accumulation in this clone, although significantly smaller than that of the red allele, still increased with increasing exposure to sunlight. This suggests that TcMYB113 expression is regulated in response to sunlight exposure.']}
|
The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color
|
[
"{'italic': 'Theobroma cacao', '#text': 'L.'}",
"genome",
"Matina 1-6",
"haplotype phasing",
"genetic mapping",
"pod color",
"{'italic': 'MYB113'}"
] |
Genome Biol
|
1370242800
|
[{'@Label': 'BACKGROUND', '@NlmCategory': 'BACKGROUND', '#text': 'Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.'}]
|
[
"Cacao",
"Chromosome Mapping",
"Chromosomes, Plant",
"Color",
"Fruit",
"Gene Expression Regulation, Plant",
"Genes, Plant",
"Genome Size",
"Genome, Plant",
"High-Throughput Nucleotide Sequencing",
"Quantitative Trait Loci",
"Quantitative Trait, Heritable",
"RNA, Small Interfering",
"Transcription Factors",
"Transcription, Genetic"
] |
other
|
PMC4053823
| null |
117
|
[
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Genome Biol. 2013 Jun 3; 14(6):r53
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NO-CC CODE
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Quantification strategy employed to quantify nuclear, perinuclear and cytoplasmic signal using ImarisSchematic representation of the quantification technique used in this manuscript with an example image of a cell expressing the Nup62DG and infected with HIV-1. Cells were stained for the capsid protein p24 and DAPI. a, Nuclear and perinuclear signal was quantified using a DAPI mask generated using the surface function in Imaris that exceeded the boundary of DAPI stain to include the perinuclear signal. As depicted, all signal within this mask was considered nuclear and perinuclear signal. Similarly, all signal outside of this mask was considered cytoplasmic signal. b, To focus on exclusively nuclear signal, an algorithm which reliably identified the nuclear boundary, as indicated by a DAPI stain, was generated in Imaris. Sections close to the upper and lower boundary of the nucleus (in Z) were removed to focus analysis on nuclear events. As in (a) signal within this mask was considered nuclear and all signal outside this mask was considered cytoplasmic and perinuclear.
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nihms-1591050-f0006
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95e538b8f9afd00df1d71735e48b447e159892c97a6e71471edde4ed8e49459e
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
1050,
988
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[{'image_id': 'nihms-1591050-f0012', 'image_file_name': 'nihms-1591050-f0012.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0012.jpg', 'caption': 'Nup358 relocalization induced upon HIV-1 infection absent upon inhibition of HIV-1 reverse transcriptiona, HeLa cells expressing the Nup62DG infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition alone (red) or in the presence of NVP treatment for the first 6 hours (blue) following synchronized infection. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in Figure 2 from three independent experiments. b, HeLa cells and primary human macrophages (MDM) synchronously infected with HIV-1 (WT) and treated with HIV-1 reverse transcriptase inhibitor NVP. Cells fixed 0, 1 and 3 h (shown) post infection and stained for Nup358 (green), HIV-1 capsid protein p24 (red), and DAPI (blue). Middle panel depicts colocalization between Nup358 and p24. Data shown here is representative of three independent experiments. c,d, Percent Nup358 in the cytoplasm and percent p24 colocalizing with Nup358 at the different times post infection in HeLa and MDM. 20 or more cells analyzed in each experiment. Data averaged (mean) from three independent experiment. e, Proximity ligation assay performed in HeLa and MDM after HIV-1 infection and cells fixed 3h post infection. Each red puncta represents a positive PLA signal generated by interaction of Nup358 and p24. f, Average fold increase in PLA signal, relative to uninfected control, from three independent experiments. 20 or more cells analyzed in each experiment. Error bar represents SEM. All statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'hash': 'a7ea2e57455b5a98b05ab9fd1fe06eeb877af6060c93c4152729c2b4fabc72cd'}, {'image_id': 'nihms-1591050-f0004', 'image_file_name': 'nihms-1591050-f0004.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0004.jpg', 'caption': 'Nuclear HIV-1 capsid is required for productive infection.a-e, Cells expressing the Nup62DG construct were infected with VSVg-R7ΔEnvmCherry and NIK were monitored following HD addition (red) as described in Fig 2. To disrupt assembled capsids, cells were incubated with 10 μM PF74 at different time points post synchronized infection as depicted (orange) and was washed off at 24 h. Infectivity was measured as described in Fig 2. Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in Fig 2 from three independent experiments. NT represents no treatment.', 'hash': '80e0c8cb57f605cf6e2442d82a44dc4eca9792885010f2e9df743765ec06b1ec'}, {'image_id': 'nihms-1591050-f0003', 'image_file_name': 'nihms-1591050-f0003.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0003.jpg', 'caption': 'HIV-1 reverse transcription is required for efficient nuclear import and completes in the nucleus of the infected cell.a–c, THP-1 differentiated macrophages (a) primary human macrophages (b) and primary CD4+ T cells (c) expressing the Nup62DG construct were infected with VSVg-R7ΔEnvmCherry and NIK was monitored by HD addition (red) as described in Fig 2. To monitor replication kinetics, the HIV-1 reverse transcriptase inhibitor, Nevirapine (NVP, 5μM), was added at different times post-infection as depicted (green) and was washed off at 24 h. Infectivity was measured as described in Fig 2. Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in Fig 2 from three independent experiments. d, The presence of HIV-1 nucleic acids in the nucleus of THP-1 differentiated macrophages and primary human macrophages was monitored. Cells were infected with VSVg-R7ΔEnvmCherry and were fixed at different time points post synchronized infection. Cells were treated with RNase A and stained for (−) vDNA and (+) vDNA using specific sense and antisense probes. Graph depicts the average number of (−) vDNA positive for (+) vDNA inside the nucleus from three independent experiments. 20 or more cells were analyzed in each experiment. Error bars represent the SEM from three independent experiments. e-f, THP-1 differentiated macrophages (e) and primary human macrophages (f) expressing the Nup62DG construct were infected with VSVg-R7ΔEnvmCherry and NIK was monitored following HD addition alone (red) or in the presence of NVP treatment for the first 6 h (blue) following synchronized infection. Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in Fig 2 from three independent experiments. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'hash': '4e53f1d27165e3dca76c1257944543b4cb4fa887970265f4bd5883fa53dc4522'}, {'image_id': 'nihms-1591050-f0014', 'image_file_name': 'nihms-1591050-f0014.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0014.jpg', 'caption': 'Insensitivity of the capsid mutants N74D and P90A to the Nup62 mediated artificial nuclear pore block suggests heterogeneity in the nuclear pores and usage by WT virusHeLa cells stably expressing the Nup62DG infected with HIV-1 harboring either the WT capsid (blue) or capsid mutants N74D (red) and P90A (orange). NIK measured in these cells by HD addition as described in Fig 2. a, Shown a bar graph from a single experiment. b, Percent inhibition attained upon blocking the nuclear pore for the first 24 h. Data averaged from three independent experiments. Error bar represents SEM.', 'hash': 'dd3e446340d5e9e27378ded4526b250511be2c9ce441075e886bd7b8449f1d05'}, {'image_id': 'nihms-1591050-f0013', 'image_file_name': 'nihms-1591050-f0013.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0013.jpg', 'caption': 'Nuclear p24 signal upon inhibition of reverse transcriptiona, HeLa cells expressing Nup62DG infected with VSVg- R7ΔEnvmCherry and treated with HIV-1 reverse transcriptase inhibitor NVP for 7 hours following synchronized infection. Cells fixed and stained for HIV-1 capsid protein p24 (red), and DAPI (blue). b, Quantification of nuclear HIV-1 p24 signal, performed as described in methods and supplementary Fig 2b. 20 or more cells analyzed in each experiment. Data averaged from three independent experiments. Error bar represents SEM. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'hash': '8ed2cb823cfa8efdc7bd4e19ce2dcfdfba0404d2d618987a2e9f849d05f193cc'}, {'image_id': 'nihms-1591050-f0002', 'image_file_name': 'nihms-1591050-f0002.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0002.jpg', 'caption': 'Monitoring HIV-1 nuclear import kinetics (NIK) reveals faster nuclear import in macrophages and T cells compared to HeLa cells.a, Schematic of how nuclear import kinetics (NIK) were monitored in primary human macrophages, CD4+ T cells and various cell lines expressing the Nup62DG construct. Cells were synchronously infected with VSVg-R7ΔEnvmCherry for 2 h at 13°C before addition of HD drug to block nuclear pore import. HD drug was added at different time points post infection. Cells were incubated with HD until 24 h post-infection and then washed and replaced with normal media. Cells were harvested and infection was monitored by measuring the percentage of mCherry positive cells 48 h post synchronized infection by flow cytometry. For monocyte derived macrophages (MDM), infection was monitored at 96 h. b-d, NIK were monitored in HeLa, THP-1 differentiated macrophages, primary monocyte derived macrophages (MDM),T cell lines, CEM and SupT1, and primary CD4+ T cells. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) from three independent experiments. Data were normalized by setting the baseline (0%) as the maximum inhibition attained by blocking the nuclear pore for 24 h (0h time point) and maximum infection (100%) as the infection attained with no HD treatment (dashed bars). Half time of NIK (t½) was shown in the line graph', 'hash': '35b6d050fc56451fb487eae0e84a160ec5142e568e5e8d7da8b10221dcbe92cc'}, {'image_id': 'nihms-1591050-f0005', 'image_file_name': 'nihms-1591050-f0005.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0005.jpg', 'caption': 'Expression of the Nup62 dimerization construct in cells does not alter normal cell physiologya, HeLa cells were transduced simultaneously with lentiviral vectors driving expression of Nup62DG or mCherry-estrogen receptor alpha (ERα). Imaging fields were selected that contain cells expressing only ERα (marked by asterisk) or both ERα and Nup62DG. Live cell imaging was performed after addition of estradiol (E2) to mediate nuclear translocation of ERα. Images acquired every four minutes for a total of one hour. b, Similar experiment as in (a) except the cells were treated with the homodimerizing drug (HD) to block NPC transport along with E2 treatment. c, Accumulation of mCherry-ERα fluorescence in the nuclear region was monitored in Nup62DG+ and Nup62DG- cells treated with E2 using ImageJ plugin Time Series Analyzer V3. Depicted mean values from three independent experiments (±SEM). d, similar quantification as in (c) in cells treated with E2 and HD. Error bar represents SEM. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'hash': '73d47489c8e442e32cb70936af2d41a31229c0f8b8e0c3037cc0f87e1bd1677f'}, {'image_id': 'nihms-1591050-f0008', 'image_file_name': 'nihms-1591050-f0008.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0008.jpg', 'caption': 'HIV-1 particles retained at the NPC over extended period upon Nup62 dimerization.HeLa cells expressing Nup62DG synchronously infected with Gag-Integrase-Ruby (GIR) labeled HIV-1 particles. 1 hour following synchronized infection, cells were treated with homodimerizing drug (HD) and imaged every 4 minutes for 1 hour. Snapshot of a GIR labeled virus particle (boxed region) at the indicated times during acquisition is shown. Similar pattern observed across 5 independent experiments.', 'hash': '29bd1a82338b541659dd477185de36b800e107b420411d85805064c259046310'}, {'image_id': 'nihms-1591050-f0006', 'image_file_name': 'nihms-1591050-f0006.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0006.jpg', 'caption': 'Quantification strategy employed to quantify nuclear, perinuclear and cytoplasmic signal using ImarisSchematic representation of the quantification technique used in this manuscript with an example image of a cell expressing the Nup62DG and infected with HIV-1. Cells were stained for the capsid protein p24 and DAPI. a, Nuclear and perinuclear signal was quantified using a DAPI mask generated using the surface function in Imaris that exceeded the boundary of DAPI stain to include the perinuclear signal. As depicted, all signal within this mask was considered nuclear and perinuclear signal. Similarly, all signal outside of this mask was considered cytoplasmic signal. b, To focus on exclusively nuclear signal, an algorithm which reliably identified the nuclear boundary, as indicated by a DAPI stain, was generated in Imaris. Sections close to the upper and lower boundary of the nucleus (in Z) were removed to focus analysis on nuclear events. As in (a) signal within this mask was considered nuclear and all signal outside this mask was considered cytoplasmic and perinuclear.', 'hash': '95e538b8f9afd00df1d71735e48b447e159892c97a6e71471edde4ed8e49459e'}, {'image_id': 'nihms-1591050-f0001', 'image_file_name': 'nihms-1591050-f0001.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0001.jpg', 'caption': 'Artificial nuclear pore blockade inhibits HIV-1 infection at the nuclear entry stage.a, Schematics of the Nup62 construct fused to the dimerization domain (DmrB) and 2 copies of eGFP (Nup62DG) used to block active nuclear pore transport after the addition of B/B homodimerizing drug (HD). b, HeLa cells were stably expressing the Nup62DmrBGFP construct and were transfected with estrogen receptor-α (ER-α) fused to mCherry. Twenty-four hours post-transfection, cells were treated with Estradiol (E2) for 30 min in the presence or absence of HD drug. Efficiency of nuclear pore blockade was quantified by counting cells having either nuclear ER-α signal (less efficient) or both nuclear and cytoplasmic ER-α signal (efficient nuclear pore block). Data shown here is representative of three independent experiments. c, Mock or Nup62DG transduced HeLa, THP-1 differentiated macrophages, CEM and SupT1 T cells were synchronously infected with VSVg pseudotyped R7ΔEnvmCherry in the presence or absence of HD drug for the first 24 h of infection. HD drug removed after 24 h, was replaced with normal media and infection was assessed 48 h post synchronized infection by measuring the percent of mCherry positive cells. Shown normalized and averaged data (±SEM) from three independent experiments. d, q-RTPCR quantification of reverse transcription and 2-LTR circles in cells expressing Nup62DG, 24h following HIV-1 infection. Depicted mean of biological triplicates (±SD). Data shown here is representative of three independent experiments. e, HeLa and THP1 differentiated macrophages that stably express the Nup62DG construct were synchronously infected with VSVg-R7ΔEnvmCherry. Cells were treated with HD drug for 4 h, then fixed, and stained for HIV-1 capsid protein, p24, (red) and DAPI (blue) for cell nucleus. Colocalization between Nup62DG with p24 (boxed region) depicted by arrows f,g A quantification process was employed to detect perinuclear and nuclear p24 protein levels as described in the methods and extended data Fig 2. 20 or more cells analyzed in each experiment. Data averaged (±SEM) from three independent experiments. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'hash': 'd260af98ea286c5051269371e8125f56270ccb48ffcb01ab702cbfe739312333'}, {'image_id': 'nihms-1591050-f0010', 'image_file_name': 'nihms-1591050-f0010.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0010.jpg', 'caption': 'HIV-1 reverse transcription completes in the nucleus of infected T cellsa-b, CEM and SupT1 expressing the Nup62DG infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition (red) as described in Fig 2. To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 5μM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in Fig 2. c, Similar experiment as above done on SupT1 cells expressing Nup62DG after infection with R7ΔEnvmCherry pseudotyped with HIV-1 envelope from the HXB2 strain. All bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in Fig 2 from three independent experiments. NT represents no treatment.', 'hash': 'd6b8ae38ea0227eb943e090e715e6e07150d0f7dcbd4d5b5b68b54d0299733ef'}, {'image_id': 'nihms-1591050-f0007', 'image_file_name': 'nihms-1591050-f0007.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0007.jpg', 'caption': 'HIV-1 infection induces Nup62 relocalization and colocalization with HIV-1 cores in the cytoplasma, Nup62 localization in uninfected HeLa cells. b, HeLa cells synchronously infected with VSVg-R7ΔEnvmCherry at 3 h post infection, fixed and stained for Nup62 (green), HIV-1 capsid protein p24 (red), and DAPI (blue) for cell nuclei. Enlarged view of colocalization (boxed region) between Nup62 and HIV-1 capsid protein p24 shown in the bottom panel and indicated by arrows. c, Quantification of cytoplasmic Nup62, as described in methods section and supplementary Fig 2a. 20 or more cells analyzed per sample. Data averaged from three independent experiments. Error bar represents SEM. d, Quantification of the percent p24 colocalizing with Nup62 in HeLa cells. 20 or more cells analyzed per sample. Data averaged from three independent experiments. Error bar represents SEM. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'hash': '5c826aef4094fd1deec7c0229111fc2523680def0a5740d25ee5911b7096b5d9'}, {'image_id': 'nihms-1591050-f0009', 'image_file_name': 'nihms-1591050-f0009.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0009.jpg', 'caption': 'Monitoring reverse transcription with a low dose of RT inhibitor nevirapine to mirror the level of inhibition induced by Nup62DG blockadeTHP1 cells expressing the Nup62DG were differentiated to macrophages and infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition (red) as described in Fig 2. To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 1.6 μM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in Fig 2. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in Fig 2 from three independent experiments. NT represents no treatment.', 'hash': '26a908787a237cca59fce7546c82199fc9cf7457d8c93d7a5947f80d68859795'}, {'image_id': 'nihms-1591050-f0011', 'image_file_name': 'nihms-1591050-f0011.jpg', 'image_path': '../data/media_files/PMC9286700/nihms-1591050-f0011.jpg', 'caption': 'Positive strand HIV1 vDNA colocalize with Negative strand only in the nucleus of infected THP1 differentiated macrophages and primary macrophagesa,b. THP1 differentiated macrophages (a) and monocyte derived macrophages (b) synchronously infected with VSVg- R7ΔEnvmCherry and fixed at different times post synchronized infection. Cells treated with RNase A and stained for (–) VDNA (orange) and (+) vDNA (red) using specific sense and antisense probes. Upon probe staining, these cells were also stained for HIV-1 capsid protein p24 (green) and nuclear Lamin A/C (blue). Depicted a representative image at the indicated time points. Data shown here is representative of three independent experiments. Quantification provided in Fig 3d.', 'hash': '180ffe4f1a9cfed4c9012ee0412d3dde76cfffbacc24f7ed99861bf5e4898bad'}]
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{'nihms-1591050-f0001': ['To overcome these limitations and test this model of infection, we employed an inducible nuclear pore blockade (NPC) that allowed us to determine the spatiotemporal staging of reverse transcription, capsid disassembly and the kinetics of nuclear import of infectious HIV-1 viral particles following a synchronized infection. An inducible NPC blockade was achieved by transducing HIV-1 target cells with a lentiviral vector expressing Nup62 fused to a drug inducible dimerization domain (DmrB) and two copies of GFP (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1a</xref>). Nup62 is a nucleoporin known to localize to the central pore of the NPC, and a previous study has demonstrated that a Nup62-DmrB-GFP (Nup62DG) fusion can allow for inducible ciliary and NPC blockade following addition of a rapamycin analog (HD) that induces homodimerization of the DmrB domain). Nup62 is a nucleoporin known to localize to the central pore of the NPC, and a previous study has demonstrated that a Nup62-DmrB-GFP (Nup62DG) fusion can allow for inducible ciliary and NPC blockade following addition of a rapamycin analog (HD) that induces homodimerization of the DmrB domain12,13. To monitor the efficacy of the Nup62DG construct to block active NPC transport, we monitored the nuclear translocation of estrogen receptor-α (ERα) induced by the addition of estradiol (E2). Consistent with previous observations, expression of this Nup62DG construct effectively prevented nuclear import of estrogen receptor-α (ERα) in the presence of the HD homodimerizing compound and E2 (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1b</xref>, , <xref rid="nihms-1591050-f0005" ref-type="fig">Extended Data Fig 1b</xref>). No difference in the rate of nuclear accumulation of ERα was observed following estradiol (E2) treatment was observed in the absence of HD treatment (). No difference in the rate of nuclear accumulation of ERα was observed following estradiol (E2) treatment was observed in the absence of HD treatment (<xref rid="nihms-1591050-f0005" ref-type="fig">Extended Data Figure 1a</xref> and and <xref rid="nihms-1591050-f0005" ref-type="fig">1c</xref>), demonstrating that Nup62DG expression does not perturb nuclear import when NPC blockade is not induced. Following infection with HIV-1 strain R7ΔEnvmCherry pseudotyped with VSV glycoproteins, cells stably expressing this construct was potently inhibited by addition of HD, while infection was not affected by expression of the construct or addition of HD alone (), demonstrating that Nup62DG expression does not perturb nuclear import when NPC blockade is not induced. Following infection with HIV-1 strain R7ΔEnvmCherry pseudotyped with VSV glycoproteins, cells stably expressing this construct was potently inhibited by addition of HD, while infection was not affected by expression of the construct or addition of HD alone (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1c</xref>, , Supplementary Figure 1). Nuclear pore blockade did not influence reverse transcription but did inhibit the formation of 2-LTR circles, a surrogate marker of nuclear import (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1d</xref>), consistent with a specific block of HIV-1 nuclear import.), consistent with a specific block of HIV-1 nuclear import.', 'Visualization of HIV-1 particles in cells in which nuclear import was blocked revealed accumulation of HIV-1 particles at or near the nuclear membrane, often in complex with the Nup62DG construct (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1e</xref>––<xref rid="nihms-1591050-f0001" ref-type="fig">f</xref>). In the absence of drug treatment, we also observed colocalization of HIV-1 with Nup62DG in the cytoplasm (). In the absence of drug treatment, we also observed colocalization of HIV-1 with Nup62DG in the cytoplasm (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1e</xref>). This was not specific to the Nup62DG construct, as we observed similar relocalization of endogenous Nup62 during infection and colocalization with HIV-1 following infection (). This was not specific to the Nup62DG construct, as we observed similar relocalization of endogenous Nup62 during infection and colocalization with HIV-1 following infection (<xref rid="nihms-1591050-f0007" ref-type="fig">Extended Data Fig 3</xref>), while Nup62 was almost exclusively localized to the NPC in the absence of infection. Live cell imaging revealed that HIV-1 trafficking in complex with Nup62DG away from the nuclear envelope following NPC blockade (), while Nup62 was almost exclusively localized to the NPC in the absence of infection. Live cell imaging revealed that HIV-1 trafficking in complex with Nup62DG away from the nuclear envelope following NPC blockade (<xref rid="nihms-1591050-f0008" ref-type="fig">Extended Data Fig 4</xref>, , Supplementary video 1). These observations are similar to our previous observations of Nup358 relocalization during HIV-1 infection 14 and demonstrates that HIV-1 infection induces dynamic relocalization of numerous NPC components during infection.', 'The observation that functional nuclear import (the nuclear import of infectious HIV-1) is inhibited when reverse transcription is inhibited is consistent with previous studies demonstrating that reverse transcription can induce HIV-1 uncoating in cells 16,17 and in vitro\n28. However, the observation that RT inhibition does not similarly reduce the amount of p24 in the nucleus (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1f</xref>––<xref rid="nihms-1591050-f0001" ref-type="fig">g</xref>) is consistent with other studies suggesting that nuclear import of HIV-1 can occur independently of reverse transcription) is consistent with other studies suggesting that nuclear import of HIV-1 can occur independently of reverse transcription7,26. This observation, taken together with the observation that Nup62 blockade does not completely abolish infection (<xref rid="nihms-1591050-f0001" ref-type="fig">Fig 1c</xref>) and that some CA mutants can enter the nucleus using distinct nuclear import pathways () and that some CA mutants can enter the nucleus using distinct nuclear import pathways (<xref rid="nihms-1591050-f0014" ref-type="fig">Extended Data Fig 10</xref>) suggest that differences in prior studies may be explained by the existence of multiple nuclear import pathways used by HIV-1. Our data suggests that reverse transcription promotes utilization of the primary import pathway utilized during infection, which our data suggests is more sensitive to Nup62 blockade than the alternative import pathways utilized by mutants such as P90A and N64D. The observation that p24 accumulation in the nucleus is not substantially reduced following inhibition of reverse transcription, but does not lead to infection, likely reflects the utilization of other nuclear import pathways not typically utilized during infection, such as those utilized by the P90A and N74D mutants. Consistent with this hypothesis, we have also observed that, similar to the case of RT inhibition () suggest that differences in prior studies may be explained by the existence of multiple nuclear import pathways used by HIV-1. Our data suggests that reverse transcription promotes utilization of the primary import pathway utilized during infection, which our data suggests is more sensitive to Nup62 blockade than the alternative import pathways utilized by mutants such as P90A and N64D. The observation that p24 accumulation in the nucleus is not substantially reduced following inhibition of reverse transcription, but does not lead to infection, likely reflects the utilization of other nuclear import pathways not typically utilized during infection, such as those utilized by the P90A and N74D mutants. Consistent with this hypothesis, we have also observed that, similar to the case of RT inhibition (<xref rid="nihms-1591050-f0012" ref-type="fig">Extended Data Fig 8b</xref>––<xref rid="nihms-1591050-f0012" ref-type="fig">c</xref>), the N74D and P90A mutants do not induce the relocalization of NPC components during infection ), the N74D and P90A mutants do not induce the relocalization of NPC components during infection 14. However, it is unclear why preventing the utilization of the predominant pathway by wt virus would not lead to productive infection in the context. Further characterization of the alternative nuclear import pathway or pathways HIV-1 can utilize during infection, and the development of similar tools to selectively block this pathway, should allow a better understanding of how utilization of distinct nuclear import pathways drives subsequent nuclear steps of infection of HIV-1.'], 'nihms-1591050-f0002': ['We next utilized this selective nuclear pore blockade to determine HIV-1 nuclear import kinetics (NIK) in various target cells. Following a synchronized infection, nuclear pore blockade was induced at various times following infection to determine the rate at which the viral inoculum became insensitive to nuclear pore blockade (<xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2a</xref>). Notably, HIV-1 became resistant to nuclear pore blockade much quicker in monocytic and T cell lines, monocyte derived macrophages (MDMs) and CD4 positive primary T cells than HeLa cells (). Notably, HIV-1 became resistant to nuclear pore blockade much quicker in monocytic and T cell lines, monocyte derived macrophages (MDMs) and CD4 positive primary T cells than HeLa cells (<xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>), with approximately half of the inoculum becoming resistant to nuclear pore blockade ~3.5 hours following a synchronized infection in these cell types (), with approximately half of the inoculum becoming resistant to nuclear pore blockade ~3.5 hours following a synchronized infection in these cell types (<xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2c</xref>––<xref rid="nihms-1591050-f0002" ref-type="fig">d</xref>), compared to ~7 hours in HeLa cells (), compared to ~7 hours in HeLa cells (<xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2b</xref>).).', 'TheNIK assay was performed as outlined in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2a</xref>.Cells stably expressing or transduced with the hNUP62-DmrB-eGFP construct were plated in 48 well plates. Dividing cells were pretreated overnight with 10 μg/ml of Aphidicolin to arrest cell division and ensure viral entry through the nuclear pore complex. Cells were synchronously infected with HIV-1 strain R7ΔEnvmCherry pseudotyped with VSVg or HIV-1 Env glycoproteins by spinoculation at 13°C for 2 h at 1200xg. Following spinoculation, media was replaced with warmed 37°C normal media. Homodimerizing drug (HD) at a final concentration of 1.5 μM was added to block active nuclear pore transport immediately after the synchronized infection (time point 0 h) or added at the indicated time post-infection as depicted in .Cells stably expressing or transduced with the hNUP62-DmrB-eGFP construct were plated in 48 well plates. Dividing cells were pretreated overnight with 10 μg/ml of Aphidicolin to arrest cell division and ensure viral entry through the nuclear pore complex. Cells were synchronously infected with HIV-1 strain R7ΔEnvmCherry pseudotyped with VSVg or HIV-1 Env glycoproteins by spinoculation at 13°C for 2 h at 1200xg. Following spinoculation, media was replaced with warmed 37°C normal media. Homodimerizing drug (HD) at a final concentration of 1.5 μM was added to block active nuclear pore transport immediately after the synchronized infection (time point 0 h) or added at the indicated time post-infection as depicted in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2a</xref>. 24 h post synchronized infection, HD containing media was removed, cells were washed and cultured in normal media. Infectivity was measured 48 h post synchronized infection for all cells expect MDMs, in which case infectivity was measured 96 h post infection. Infectivity determined by measuring the percentage of mCherry positive cells using the BD LSRFortessa flow cytometer (BD Bioscience). The percentage of mCherry positive cells was calculated as the ratio of the double positive population (Nup62eGFP+, R7mCherry+) over the total amount of Nup62GFP+ population (. 24 h post synchronized infection, HD containing media was removed, cells were washed and cultured in normal media. Infectivity was measured 48 h post synchronized infection for all cells expect MDMs, in which case infectivity was measured 96 h post infection. Infectivity determined by measuring the percentage of mCherry positive cells using the BD LSRFortessa flow cytometer (BD Bioscience). The percentage of mCherry positive cells was calculated as the ratio of the double positive population (Nup62eGFP+, R7mCherry+) over the total amount of Nup62GFP+ population (<xref rid="nihms-1591050-f0006" ref-type="fig">Extended Data Fig 2b</xref>).).', 'THP1 cells expressing the Nup62DG were differentiated to macrophages and infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition (red) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 1.6 μM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in . To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 1.6 μM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in . Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref> from three independent experiments. NT represents no treatment. from three independent experiments. NT represents no treatment.', 'a-b, CEM and SupT1 expressing the Nup62DG infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition (red) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 5μM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in . To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 5μM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. . c, Similar experiment as above done on SupT1 cells expressing Nup62DG after infection with R7ΔEnvmCherry pseudotyped with HIV-1 envelope from the HXB2 strain. All bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref> from three independent experiments. NT represents no treatment. from three independent experiments. NT represents no treatment.', 'a, HeLa cells expressing the Nup62DG infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition alone (red) or in the presence of NVP treatment for the first 6 hours (blue) following synchronized infection. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Figure 2</xref> from three independent experiments. from three independent experiments. b, HeLa cells and primary human macrophages (MDM) synchronously infected with HIV-1 (WT) and treated with HIV-1 reverse transcriptase inhibitor NVP. Cells fixed 0, 1 and 3 h (shown) post infection and stained for Nup358 (green), HIV-1 capsid protein p24 (red), and DAPI (blue). Middle panel depicts colocalization between Nup358 and p24. Data shown here is representative of three independent experiments. c,d, Percent Nup358 in the cytoplasm and percent p24 colocalizing with Nup358 at the different times post infection in HeLa and MDM. 20 or more cells analyzed in each experiment. Data averaged (mean) from three independent experiment. e, Proximity ligation assay performed in HeLa and MDM after HIV-1 infection and cells fixed 3h post infection. Each red puncta represents a positive PLA signal generated by interaction of Nup358 and p24. f, Average fold increase in PLA signal, relative to uninfected control, from three independent experiments. 20 or more cells analyzed in each experiment. Error bar represents SEM. All statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'HeLa cells stably expressing the Nup62DG infected with HIV-1 harboring either the WT capsid (blue) or capsid mutants N74D (red) and P90A (orange). NIK measured in these cells by HD addition as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. . a, Shown a bar graph from a single experiment. b, Percent inhibition attained upon blocking the nuclear pore for the first 24 h. Data averaged from three independent experiments. Error bar represents SEM.', 'a–c, THP-1 differentiated macrophages (a) primary human macrophages (b) and primary CD4+ T cells (c) expressing the Nup62DG construct were infected with VSVg-R7ΔEnvmCherry and NIK was monitored by HD addition (red) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. To monitor replication kinetics, the HIV-1 reverse transcriptase inhibitor, Nevirapine (NVP, 5μM), was added at different times post-infection as depicted (green) and was washed off at 24 h. Infectivity was measured as described in . To monitor replication kinetics, the HIV-1 reverse transcriptase inhibitor, Nevirapine (NVP, 5μM), was added at different times post-infection as depicted (green) and was washed off at 24 h. Infectivity was measured as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in . Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref> from three independent experiments. from three independent experiments. d, The presence of HIV-1 nucleic acids in the nucleus of THP-1 differentiated macrophages and primary human macrophages was monitored. Cells were infected with VSVg-R7ΔEnvmCherry and were fixed at different time points post synchronized infection. Cells were treated with RNase A and stained for (−) vDNA and (+) vDNA using specific sense and antisense probes. Graph depicts the average number of (−) vDNA positive for (+) vDNA inside the nucleus from three independent experiments. 20 or more cells were analyzed in each experiment. Error bars represent the SEM from three independent experiments. e-f, THP-1 differentiated macrophages (e) and primary human macrophages (f) expressing the Nup62DG construct were infected with VSVg-R7ΔEnvmCherry and NIK was monitored following HD addition alone (red) or in the presence of NVP treatment for the first 6 h (blue) following synchronized infection. Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref> from three independent experiments. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments. from three independent experiments. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.', 'a-e, Cells expressing the Nup62DG construct were infected with VSVg-R7ΔEnvmCherry and NIK were monitored following HD addition (red) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. To disrupt assembled capsids, cells were incubated with 10 μM PF74 at different time points post synchronized infection as depicted (orange) and was washed off at 24 h. Infectivity was measured as described in . To disrupt assembled capsids, cells were incubated with 10 μM PF74 at different time points post synchronized infection as depicted (orange) and was washed off at 24 h. Infectivity was measured as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref>. Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in . Bar graphs represent data from a single experiment and line graphs represent normalized and average data (±SEM) as described in <xref rid="nihms-1591050-f0002" ref-type="fig">Fig 2</xref> from three independent experiments. NT represents no treatment. from three independent experiments. NT represents no treatment.'], 'nihms-1591050-f0003': ['The rate at which HIV-1 became insensitive to nuclear pore blockade suggested that HIV-1 nuclear import may occur prior to the completion of reverse transcription, which is generally considered to complete prior to nuclear import. Completion of reverse transcription occurs with much slower kinetics than the kinetics of nuclear import observed in cells, particularly in macrophages 15. We therefore examined the rate at which HIV-1 became insensitive to nuclear pore blockade and RT inhibition in MDMs and THP-1 differentiated macrophages to test the hypothesis that nuclear import precedes completion of reverse transcription. These experiments revealed that the virus remains sensitive to RT inhibition for hours after the inoculum had become insensitive to the nuclear pore blockade (<xref rid="nihms-1591050-f0003" ref-type="fig">Fig 3a</xref>--<xref rid="nihms-1591050-f0003" ref-type="fig">b</xref>, , <xref rid="nihms-1591050-f0009" ref-type="fig">Extended Data Fig 5</xref>). CD4+ T cells also remained sensitive to RT inhibition for hours after the viral inoculum became insensitive to NPC blockade (). CD4+ T cells also remained sensitive to RT inhibition for hours after the viral inoculum became insensitive to NPC blockade (<xref rid="nihms-1591050-f0003" ref-type="fig">Fig 3c</xref>). Significant but less substantial differences noted in CEM and SupT1 T cell lines (). Significant but less substantial differences noted in CEM and SupT1 T cell lines (<xref rid="nihms-1591050-f0010" ref-type="fig">Extended Data Fig 6a</xref>––<xref rid="nihms-1591050-f0010" ref-type="fig">b</xref>). Similar results obtained after infection with HIV-1 pseudotyped with CXCR4 tropic (HXB2) HIV-1 glycoproteins (). Similar results obtained after infection with HIV-1 pseudotyped with CXCR4 tropic (HXB2) HIV-1 glycoproteins (<xref rid="nihms-1591050-f0010" ref-type="fig">Extended Data Fig 6c</xref>). Collectively, these data suggest that infectious HIV-1 virions enter the nucleus many hours before reverse transcription is complete. To validate this surprising observation, we ordered hybridization probes specific for each DNA strand formed during reverse transcription (). Collectively, these data suggest that infectious HIV-1 virions enter the nucleus many hours before reverse transcription is complete. To validate this surprising observation, we ordered hybridization probes specific for each DNA strand formed during reverse transcription (Supplementary Figure 2). Consistent with the kinetics of nuclear import and reverse transcription sensitivity observed above, we observe that the puncta positive for both DNA strands did not appear until 9 hours PI and that these puncta were exclusively nuclear in THP-1 differentiated macrophages and MDMs (<xref rid="nihms-1591050-f0003" ref-type="fig">Fig 3d</xref>, , <xref rid="nihms-1591050-f0011" ref-type="fig">Extended Data Fig 7a</xref>--<xref rid="nihms-1591050-f0011" ref-type="fig">b</xref>). These data collectively demonstrate that the viral ribonucleoprotein complex enters the nucleus prior to the completion of reverse transcription.). These data collectively demonstrate that the viral ribonucleoprotein complex enters the nucleus prior to the completion of reverse transcription.', 'Prior studies have exhibited a lack of consensus regarding the role of reverse transcription in HIV-1 nuclear import. While some studies have shown that reverse transcription promotes cytoplasmic uncoating of the viral core prior to nuclear import 16–18, other studies have suggested that HIV-1 nuclear import can occur in the absence of reverse transcription 19. To determine the degree to which cytoplasmic reverse transcription is required for functional nuclear import, we examined the nuclear import kinetics of cells infected in the presence of the RT inhibitor Nevirapine (NVP) for six hours. Although NVP treatment for 6 hours did not significantly impact infection measured at 48 hours in the absence of NPC blockade, we observed that the viral inoculum in the NVP treated infection remained unable to bypass the NPC blockade until NVP was washed out (<xref rid="nihms-1591050-f0003" ref-type="fig">Fig 3e</xref>––<xref rid="nihms-1591050-f0003" ref-type="fig">f</xref>, , <xref rid="nihms-1591050-f0012" ref-type="fig">Extended Data Fig 8a</xref>). Following withdrawal of NVP, nuclear import of the inoculum rapidly recovered and approached the kinetics of nuclear import of untreated infections at later time points. Consistent with these observations, we also observe that NVP treatment prevented the relocalization of NPC components to the cytoplasm (). Following withdrawal of NVP, nuclear import of the inoculum rapidly recovered and approached the kinetics of nuclear import of untreated infections at later time points. Consistent with these observations, we also observe that NVP treatment prevented the relocalization of NPC components to the cytoplasm (<xref rid="nihms-1591050-f0012" ref-type="fig">Extended Data Fig 8b</xref>––<xref rid="nihms-1591050-f0012" ref-type="fig">f</xref>). However, we also observe that RT inhibition reduces but does not eliminate the accumulation of p24 CA in the nucleus (). However, we also observe that RT inhibition reduces but does not eliminate the accumulation of p24 CA in the nucleus (<xref rid="nihms-1591050-f0013" ref-type="fig">Extended Data Fig 9a</xref>––<xref rid="nihms-1591050-f0013" ref-type="fig">b</xref>). These results collectively demonstrate that although reverse transcription can complete in the nucleus, some amount of cytoplasmic reverse transcription is necessary to facilitate productive nuclear import of the virus. The observation that reverse transcription is required to induce the relocalization of NPC components during infection is also consistent with a role for RT in HIV-1 nuclear import.). These results collectively demonstrate that although reverse transcription can complete in the nucleus, some amount of cytoplasmic reverse transcription is necessary to facilitate productive nuclear import of the virus. The observation that reverse transcription is required to induce the relocalization of NPC components during infection is also consistent with a role for RT in HIV-1 nuclear import.', 'a,b. THP1 differentiated macrophages (a) and monocyte derived macrophages (b) synchronously infected with VSVg- R7ΔEnvmCherry and fixed at different times post synchronized infection. Cells treated with RNase A and stained for (–) VDNA (orange) and (+) vDNA (red) using specific sense and antisense probes. Upon probe staining, these cells were also stained for HIV-1 capsid protein p24 (green) and nuclear Lamin A/C (blue). Depicted a representative image at the indicated time points. Data shown here is representative of three independent experiments. Quantification provided in <xref rid="nihms-1591050-f0003" ref-type="fig">Fig 3d</xref>..'], 'nihms-1591050-f0004': ['One reason that reverse transcription has generally been assumed to complete in the cytoplasm is that the strand transfer events which occur during reverse transcription likely require the constrained environment of the capsid core to prevent the premature diffusion of the reverse transcriptase enzyme away from the viral genome. However, most studies of core disassembly or uncoating suggest that this process completes prior to the nuclear import of the viral ribonucleoprotein complex (vRNPC)1,20. Although some CA protein has been shown to remain with the vRNPC in the nucleus5–8, it has previously been difficult to demonstrate that this CA plays a functional role in infection subsequent to driving the final stages of nuclear import. We therefore used PF74, which is known to bind to the interface between CA monomers in assembled CA and inhibit infection21–23. PF74 susceptibility is therefore dependent on assembled CA and continued PF74 susceptibility after infection reveals a continued, functional role for assembled CA at subsequent steps of infection. We therefore used PF74 in the context of NIK assay to determine if assembled CA mediates a functional role in infection following nuclear import. As was observed with RT inhibitors, these experiments revealed that the viral inoculum remains sensitive to PF74 inhibition for hours after the virus becomes insensitive to nuclear pore blockade in all target cells examined (<xref rid="nihms-1591050-f0004" ref-type="fig">Fig 4</xref>). While this effect was more pronounced in MDMs, primary CD4+ T cells and THP-1 cells (). While this effect was more pronounced in MDMs, primary CD4+ T cells and THP-1 cells (<xref rid="nihms-1591050-f0004" ref-type="fig">Fig 4a</xref>––<xref rid="nihms-1591050-f0004" ref-type="fig">b</xref>, , <xref rid="nihms-1591050-f0004" ref-type="fig">Fig 4e</xref>), we observed significant differences in nuclear pore blockade and PF74 sensitivity in SupT1 and CEM cells (), we observed significant differences in nuclear pore blockade and PF74 sensitivity in SupT1 and CEM cells (<xref rid="nihms-1591050-f0004" ref-type="fig">Fig 4c</xref>––<xref rid="nihms-1591050-f0004" ref-type="fig">d</xref>), revealing that assembled CA mediates a functional step in infection subsequent to CA-mediated import of HIV-1.), revealing that assembled CA mediates a functional step in infection subsequent to CA-mediated import of HIV-1.'], 'nihms-1591050-f0014': ['Finally, as CA is known to mediate the nuclear import of HIV-1 and mutations in CA are reported to influence the nuclear import pathway utilized during infection 2,3, we next wanted to determine the degree to which CA mutants influence the nuclear import kinetics of HIV-1. However, infection with HIV-1 containing the N74D capsid, which is defective in binding to CPSF6 and Nup153 3,4,24, and the P90A capsid, which cannot bind Cyclophilin A, exhibited substantially different susceptibility to NPC blockade as compared to WT virus in the same target cells (<xref rid="nihms-1591050-f0014" ref-type="fig">Extended Data Fig 10a</xref>––<xref rid="nihms-1591050-f0014" ref-type="fig">b</xref>). This suggests that these mutants, particularly the P90A mutant, enter the nucleus through distinct NPCs, consistent with a recently proposed model of NPC heterogeneity influencing HIV-1 infection and inhibition by the restriction factor MX2 ). This suggests that these mutants, particularly the P90A mutant, enter the nucleus through distinct NPCs, consistent with a recently proposed model of NPC heterogeneity influencing HIV-1 infection and inhibition by the restriction factor MX2 2.'], 'nihms-1591050-f0007': ['Quantification of p24, Nup358 or Nup62 signal in the cell was performed following the similar quantification method described in our previous study 14.Nuclear and perinuclear signal was determined using surface masks around the nucleus created based on the DAPI channel. To detect perinuclear and nuclear signal, an algorithm was created which reliably overestimates the size of the nucleus, as previously described 14 (<xref rid="nihms-1591050-f0007" ref-type="fig">Extended Data Fig 3a</xref>). The perinuclear algorithm to detect cell nuclei included all z sections acquired. All events within this mask were considered nuclear and perinuclear, while all events outside of this mask were considered cytoplasmic, as determined using the surface function and masking tool in Imaris 8.4.1.). The perinuclear algorithm to detect cell nuclei included all z sections acquired. All events within this mask were considered nuclear and perinuclear, while all events outside of this mask were considered cytoplasmic, as determined using the surface function and masking tool in Imaris 8.4.1.', 'To detect nuclear signal, an algorithm was designed to detect the cell nuclei using the DAPI channel as reference and surface function in Imaris 8.4.1 (<xref rid="nihms-1591050-f0007" ref-type="fig">Extended Data Fig 3b</xref>). z sections close to the top or bottom of the nucleus were excluded to avoid detection of extranuclear signal. Similar to above, all events within the mask were considered nuclear and all events outside the mask were considered perinuclear and cytoplasmic. The amount of perinuclear signal was quantified by subtracting the signal obtained from nuclear and perinuclear signal to signal obtained from nuclear signal.). z sections close to the top or bottom of the nucleus were excluded to avoid detection of extranuclear signal. Similar to above, all events within the mask were considered nuclear and all events outside the mask were considered perinuclear and cytoplasmic. The amount of perinuclear signal was quantified by subtracting the signal obtained from nuclear and perinuclear signal to signal obtained from nuclear signal.'], 'nihms-1591050-f0006': ['a, Schematics of the Nup62 construct fused to the dimerization domain (DmrB) and 2 copies of eGFP (Nup62DG) used to block active nuclear pore transport after the addition of B/B homodimerizing drug (HD). b, HeLa cells were stably expressing the Nup62DmrBGFP construct and were transfected with estrogen receptor-α (ER-α) fused to mCherry. Twenty-four hours post-transfection, cells were treated with Estradiol (E2) for 30 min in the presence or absence of HD drug. Efficiency of nuclear pore blockade was quantified by counting cells having either nuclear ER-α signal (less efficient) or both nuclear and cytoplasmic ER-α signal (efficient nuclear pore block). Data shown here is representative of three independent experiments. c, Mock or Nup62DG transduced HeLa, THP-1 differentiated macrophages, CEM and SupT1 T cells were synchronously infected with VSVg pseudotyped R7ΔEnvmCherry in the presence or absence of HD drug for the first 24 h of infection. HD drug removed after 24 h, was replaced with normal media and infection was assessed 48 h post synchronized infection by measuring the percent of mCherry positive cells. Shown normalized and averaged data (±SEM) from three independent experiments. d, q-RTPCR quantification of reverse transcription and 2-LTR circles in cells expressing Nup62DG, 24h following HIV-1 infection. Depicted mean of biological triplicates (±SD). Data shown here is representative of three independent experiments. e, HeLa and THP1 differentiated macrophages that stably express the Nup62DG construct were synchronously infected with VSVg-R7ΔEnvmCherry. Cells were treated with HD drug for 4 h, then fixed, and stained for HIV-1 capsid protein, p24, (red) and DAPI (blue) for cell nucleus. Colocalization between Nup62DG with p24 (boxed region) depicted by arrows f,g A quantification process was employed to detect perinuclear and nuclear p24 protein levels as described in the methods and <xref rid="nihms-1591050-f0006" ref-type="fig">extended data Fig 2</xref>. 20 or more cells analyzed in each experiment. Data averaged (±SEM) from three independent experiments. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.. 20 or more cells analyzed in each experiment. Data averaged (±SEM) from three independent experiments. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.']}
|
Nuclear pore blockade reveals HIV-1 completes reverse transcription and uncoating in the nucleus
| null |
Nat Microbiol
|
1598943600
|
Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex. Although recent studies have observed the presence of the capsid protein in the nucleus during infection, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.
|
[
"Active Transport, Cell Nucleus",
"CD4-Positive T-Lymphocytes",
"Capsid",
"Capsid Proteins",
"Cell Nucleus",
"Cytoplasm",
"HEK293 Cells",
"HIV Infections",
"HIV-1",
"HeLa Cells",
"Host-Pathogen Interactions",
"Humans",
"Indoles",
"Macrophages",
"Nuclear Pore",
"Phenylalanine",
"Reverse Transcription",
"Virus Replication"
] |
other
|
PMC9286700
| null |
30
|
[
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] |
Nat Microbiol. 2020 Sep 1; 5(9):1088-1095
|
NO-CC CODE
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(a) In vivo NIR fluorescence images of TRAMP-C1 tumor-bearing mice receiving intravenous injection of PBS and different ICG-containing formulations evaluated by IVIS. (b) NIR fluorescence images of the isolated major organs and tumors 48 h post-injection with PBS and different ICG-containing formulations. (c) Average ICG fluorescence intensities of individual organs and tumor from TRAMP-C1 tumor-bearing mice treated with different ICG-containing formulations (n = 5 per group).
|
thnov06p0302g007
|
2
|
538e6fde0079559b106e4485b168a33b922d7d4385ecda756978f68000d991fb
|
thnov06p0302g007.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
734,
491
] |
[{'image_id': 'thnov06p0302g004', 'image_file_name': 'thnov06p0302g004.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g004.jpg', 'caption': '(a) Zeta potential of pristine and cargo-loaded nanoparticles in aqueous solutions. (b) DLS particle size distribution profiles of ICG/DOX-loaded NHTPNs in aqueous solutions. (c) Cumulative ICG release profiles of various cargo-loaded nanoparticles in PBS at 37 oC. (d) Cumulative DOX release profiles of ICG/DOX-loaded NHTPNs and TPNs in aqueous solutions of pH 7.4 and 6.0.', 'hash': '89bc7bc7fd88cf48afe21d0d0c6ae43ea032f4698b6f2921ebd0ac275dfc6679'}, {'image_id': 'thnov06p0302g003', 'image_file_name': 'thnov06p0302g003.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g003.jpg', 'caption': '(a) Normalized maximum absorbance of free ICG and various ICG-containing nanoparticles in PBS at 37 oC. (b) Time-evolved mean hydrodynamic diameters (Dh) of various cargo-loaded nanoparticles in PBS at 37 oC. (c) Temperature profiles and (d) thermal images of free ICG and ICG-containing nanoparticles (ICG concentration = 10 μM) in PBS with 808 nm NIR laser irradiation (1.0 W/cm2).', 'hash': '1912f7a73db444b32b050b050f03a5c61c3678587e043788fe2b4a76e753d17e'}, {'image_id': 'thnov06p0302g002', 'image_file_name': 'thnov06p0302g002.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g002.jpg', 'caption': '(a) DLS particle size distribution profiles of nanoparticles in aqueous solution of pH 7.4. (b) TEM images of (i) pristine NHTPNs, (ii) ICG-loaded NHTPNs, (iii) DOX-loaded NHTPNs, (iv) ICG/DOX-loaded PNs, (v) ICG/DOX-loaded TPNs and (vi) ICG/DOX-loaded NHTPNs. Scale bars are 200 nm. (c) UV/Vis spectra of free ICG, ICG/DOX-loaded NHTPNs, TPNs and PNs in PBS.', 'hash': '1a117edbdb39bce7acd9a1065317665db264ce9ceb1c8737245d47601cc83c4c'}, {'image_id': 'thnov06p0302g005', 'image_file_name': 'thnov06p0302g005.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g005.jpg', 'caption': '(a) Fluorescence images of ICG molecules from TRAMP-C1 cells incubated with free ICG and ICG/DOX-loaded TPNs and NHTPNs, respectively, under different pH conditions attained by IVIS. (b) Intracellular DOX amount of TRAMP-C1 cells incubated with various DOX formulations. (c) Flow cytometric histograms TRAMP-C1 cells treated with ICG/DOX-loaded TPNs and NHTPNs at pH 7.4 and 6.3 for 2 h (DOX concentration = 20 μΜ). (d) Fluorescence images of TRAMP-C1 cells incubated with ICG/DOX-loaded TPN and NHTPNs at pH 7.4 and 6.3 for 1.5 h (DOX concentration = 10 μΜ). Nuclei and F-actin cytoskeleton were stained with Hoechst and F-actin marker, respectively. Scale bar is 50 μm.', 'hash': '51ad394b1fbc5b5217f3b1163dd8dbc3e00f347dba0ff31fc830f1fe39900270'}, {'image_id': 'thnov06p0302i001', 'image_file_name': 'thnov06p0302i001.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302i001.jpg', 'caption': None, 'hash': 'b4a25c80b1a9396d07d3cc08468e352792e5079d3d534f29ff5c8c1ecac2ac3c'}, {'image_id': 'thnov06p0302g010', 'image_file_name': 'thnov06p0302g010.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g010.jpg', 'caption': 'Fluorescence images of tumor sections from the mice bearing TRAMP-C1 tumor 48 h post-injection of various formulations (a) without and (b) with NIR irradiation. Cell nuclei and apoptotic cells were stained with DAPI (blue) and caspase-3 marker (green), respectively. Scale bars are 100 μm.', 'hash': '0541787114ded11acf45e0066b45c493456ba5ea47a32ef7cc79c4f634a7b7c0'}, {'image_id': 'thnov06p0302g008', 'image_file_name': 'thnov06p0302g008.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g008.jpg', 'caption': '(a) Intratumoral distribution of free DOX and ICG/DOX-loaded TPNs and NHTPNs in relation to the loci of angiogenic blood vessels examined by immunofluorescence microscopy. (b) Intratumoral distribution of ICG/DOX-loaded NHTPNs in relation to the loci of tumor hypoxia and TAM by IHC examination of contiguous tumor sections. (c) Intratumoral distribution of free DOX and ICG/DOX-loaded TPNs and NHTPNs in relation to the loci of tumor hypoxia examined by immunofluorescence microscopy. Tumor cell nuclei, angiogenic blood vessels, tumor hypoxia and TAM were stained with DAPI and CD31, HIF 1α and CD11b markers, respectively. Scale bars are 50 μm in (a) and 100 μm in (b and c).', 'hash': '3ece0bb39207db28ec6f15b015aee60b95a79b1a70bb3c4c6e6752b7fd44ffb5'}, {'image_id': 'thnov06p0302g006', 'image_file_name': 'thnov06p0302g006.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g006.jpg', 'caption': '(a) Cell viability of TRAMP-C1 cells incubated with various formulations for 24 h with and without the 5-min NIR laser irradiation, followed by additional 24 h incubation. (b) Cell viability of TRAMP-C1 cells incubated respectively with ICG-loaded NHTPNs and ICG/DOX-loaded NHTPNs (ICG 5.0 μM and DOX 5.5 μM, if applicable) at pH 7.4 and 6.3.', 'hash': 'd1a76c4209d95bec721456e4bc5dc40bd74b5d22a508d5adeb0ab4ef669f8e45'}, {'image_id': 'thnov06p0302g001', 'image_file_name': 'thnov06p0302g001.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g001.jpg', 'caption': 'Illustration of active tumor penetration and uptake of dual drug-loaded nanoparticles with pHe-triggered surface charge transition for the imaging-guided photothermal/chemo combinatorial therapy.', 'hash': 'c9b7878a78368df0170624f8af905077981e8a59346f106da7844decd891b51e'}, {'image_id': 'thnov06p0302g011', 'image_file_name': 'thnov06p0302g011.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g011.jpg', 'caption': '(a) Tumor growth inhibition profiles of the mice bearing TRAMP-C1 tumor injected with various formulations, followed by NIR laser irradiation (5 min, 1.0 W/cm2) 6 h post-injection or without any laser treatment (n = 4 per group). Morphology and size of the tumors of each group isolated from the sacrificed mice at day 15 (the end point) after the treatment. (b) Images of H&E-stained tumor sections harvested from the tumor-bearing mice receiving treatments. Scale bars are 100 μm.', 'hash': 'a455555852bc757dce714ae42fe22d8afd5439b00b9ad3b00bbb9f00df87a7c3'}, {'image_id': 'thnov06p0302g007', 'image_file_name': 'thnov06p0302g007.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g007.jpg', 'caption': '(a) In vivo NIR fluorescence images of TRAMP-C1 tumor-bearing mice receiving intravenous injection of PBS and different ICG-containing formulations evaluated by IVIS. (b) NIR fluorescence images of the isolated major organs and tumors 48 h post-injection with PBS and different ICG-containing formulations. (c) Average ICG fluorescence intensities of individual organs and tumor from TRAMP-C1 tumor-bearing mice treated with different ICG-containing formulations (n = 5 per group).', 'hash': '538e6fde0079559b106e4485b168a33b922d7d4385ecda756978f68000d991fb'}, {'image_id': 'thnov06p0302g009', 'image_file_name': 'thnov06p0302g009.jpg', 'image_path': '../data/media_files/PMC4737719/thnov06p0302g009.jpg', 'caption': '(a) Temperature profiles and (b) infrared thermographic maps at the tumor sites of TRAMP-C1 tumor-bearing mice receiving various formulations and exposed to NIR laser irradiation of 1.0 W/cm2 6 h post-injection.', 'hash': '3c37aaaafc3f9e80a1534f5eec177d73cd52f38d0b43d4020312679e2c11747e'}]
|
{'thnov06p0302g001': ['The preparation of these DDS with pHe-responsive surface charge transition, nevertheless, frequently involves the use of complicate materials, excess organic solvents and multi-step procedures. In addition, most of these nanovehicles transport only a single chemotherapy with limited efficacy 19-21, 23, 25-28. In order to enhance antitumor effect by the imaging-guided photothermal/chemo combined therapy, a practical approach was developed herein to prepare pHe-responsive surface charge-switchable nanoparticles capable of co-delivering a photothermal agent, indocyanine green (ICG), and a chemotherapy drug, DOX (Scheme <xref ref-type="fig" rid="thnov06p0302g001">1</xref>). N-Acetyl histidine (NAcHis) modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (NAcHis-TPGS), prepared by conjugating TPGS with NAcHis, was anchored via the hydrophobic vitamin E moiety at the surfaces of poly(lactic-co-glycolic acid) (PLGA)-based core particles concomitantly loaded with ICG and DOX. Through the small size (ca. 50 nm) in combination with the increase in surface positive charges due to the pH). N-Acetyl histidine (NAcHis) modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (NAcHis-TPGS), prepared by conjugating TPGS with NAcHis, was anchored via the hydrophobic vitamin E moiety at the surfaces of poly(lactic-co-glycolic acid) (PLGA)-based core particles concomitantly loaded with ICG and DOX. Through the small size (ca. 50 nm) in combination with the increase in surface positive charges due to the pHe-triggered protonation of NAcHis residues 29, the payload-carrying nanoparticles rapidly accumulated in acidic tumor tissued in vivo and penetrated into the deep tumor hypoxia regions upon the enhanced uptakes by both TRAMP-C1 cancer cells and tumor-associated macrophages (TAMs). Notably, the imaging-guided near-infrared (NIR)-triggered hyperthermia of the therapeutic nanoparticles accumulated at tumor sites in conjunction with chemotherapy efficiently inhibited tumor growth and recurrence, suggesting that the pHe-sensitive nanovehicles developed in this work are promising in advanced cancer theranostic applications.', 'With the medium pH being adjusted from 7.4 to 5.0, conversions in zeta potentials of pristine and payload-carrying NHTPNs from negative to nearly neutral or slightly positive values were observed (Figure <xref ref-type="fig" rid="thnov06p0302g004">3</xref>a), due to the enhanced protonation of the imidazole groups of the NAcHis-TPGS segments. By contrast, due to the lack of pH-sensitive moiety in TPGS, no significant variation in the zeta potential of cargo-loaded TPNs in response to pH change was observed. While both the pristine and DOX-loaded NHTPNs became positively charged on their surfaces in response to the pH adjustment to 5.0, the same pH stimulation only rendered the surfaces of NHTPNs carrying either ICG alone or both ICG/DOX nearly neutral, yet still mildly negatively charged, due to the sulfonate groups of ICG present mainly on colloidal surfaces. Most of the DOX species were not involved in charge variations as they remained in free base form, rendering themselves hydrophobic and entrapped within the inner core of NHTPNs (Scheme <xref ref-type="fig" rid="thnov06p0302g001">1</xref>). Notably, the mean particle sizes of the cargo-loaded NHTPNs and TPNs remain essentially invariant in the medium pH range 5.0~7.4, as partly illustrated in Figure ). Notably, the mean particle sizes of the cargo-loaded NHTPNs and TPNs remain essentially invariant in the medium pH range 5.0~7.4, as partly illustrated in Figure <xref ref-type="fig" rid="thnov06p0302g004">3</xref>b. This is particularly important that the ICG/DOX-loaded NHTPNs which exhibit neutral surfaces, maintained a particle size of ca 50 nm in Db. This is particularly important that the ICG/DOX-loaded NHTPNs which exhibit neutral surfaces, maintained a particle size of ca 50 nm in Dh when exposed to the weak acidic environment (pH 6.0) close to pHe (Figure <xref ref-type="fig" rid="thnov06p0302g004">3</xref>b), a critical prerequisite for the infiltration into deep tumor tissue in vivo b), a critical prerequisite for the infiltration into deep tumor tissue in vivo 34-36.'], 'thnov06p0302g002': ['The DOX/ICG-loaded nanoparticles coated with either NAcHis-TPGS (NHTPNs) or TPGS (TPNs) exhibited comparable mean hydrodynamic diameters (Dh) of ca 50 nm and mono-modal size distributions (PDI ca 0.11) in aqueous solution (pH 7.4) (Figure <xref ref-type="fig" rid="thnov06p0302g002">1</xref>a and Table 1). The TEM images further revealed that these nanoparticles were well-dispersed and spherical (Figure <xref ref-type="fig" rid="thnov06p0302g002">1</xref>b), indicating that the TPGS adduct retained the amphiphilic property of the parent compound, and thus was able to anchor on the PLGA particles via the hydrophobic vitamin E moieties and to stabilize the nanoparticles in aqueous solution with PEG segments. By contrast, due to the lack of TPGS or its adduct coating on particle surfaces, the resulting ICG/DOX-loaded PNs readily aggregated in aqueous phase as reflected by their enlarged particle size characterized by both DLS (Figure b), indicating that the TPGS adduct retained the amphiphilic property of the parent compound, and thus was able to anchor on the PLGA particles via the hydrophobic vitamin E moieties and to stabilize the nanoparticles in aqueous solution with PEG segments. By contrast, due to the lack of TPGS or its adduct coating on particle surfaces, the resulting ICG/DOX-loaded PNs readily aggregated in aqueous phase as reflected by their enlarged particle size characterized by both DLS (Figure <xref ref-type="fig" rid="thnov06p0302g002">1</xref>a and Table a and Table 1) and TEM (Figure <xref ref-type="fig" rid="thnov06p0302g002">1</xref>b). The coating of either TPGS or its NAcHis conjugate on PLGA particles remarkably enhanced the loading efficiencies of ICG and DOX (Table b). The coating of either TPGS or its NAcHis conjugate on PLGA particles remarkably enhanced the loading efficiencies of ICG and DOX (Table 1), most likely because of the association of ICG and DOX, respectively, with the vitamin E moiety of TPGS through either π-π aromatic and hydrophobic alkyl stacking, although the orientation of these molecules on molecular level is currently not clear. This hypothesis was supported by the observation that, when ICG molecules were incorporated into the nanoparticles coated with TPGS or NAcHis-TPGS, the feature absorption peak of ICG was appreciably shifted from 776 to 794 nm (Figure <xref ref-type="fig" rid="thnov06p0302g002">1</xref>c) by their association with the hydrophobic alkyl chains of TPGS. Similar findings have also been reported elsewhere c) by their association with the hydrophobic alkyl chains of TPGS. Similar findings have also been reported elsewhere 30,31.'], 'thnov06p0302g003': ['To evaluate the aqueous photostability of ICG associated with PLGA-based nanoparticles, the change in absorbance of the ICG-containing nanoparticles in PBS (pH 7.4, I = 0.15 M) at 37 oC was monitored. The maximum absorbance of ICG at each time point relative to that at the beginning (Figure <xref ref-type="fig" rid="thnov06p0302g003">2</xref>a) was obtained from the time-evolved absorption spectra, illustrated in Figure S2. While the normalized absorbance of free ICG decreased considerably over 7 days, only a slight change in absorbance was observed for ICG-containing nanoparticles. The promoted photostability was primarily ascribed to the protective effect of PLGA nanoparticles serving as the ICG dispersion matrices that prevented ICG from rapid self-aggregation and degradation in aqueous phase 30,31. Distinct from a severe aggregation of ICG/DOX-loaded PNs upon 4-day incubation in PBS, the particle sizes of cargo-carrying NHTPNs and TPNs remained essentially unchanged over a time period of 7 days (Figure <xref ref-type="fig" rid="thnov06p0302g003">2</xref>b). The results further demonstrate the effect of the surface elaboration with TPGS or NAcHis-TPGS on the colloidal stability of PLGA nanoparticles in aqueous phase.b). The results further demonstrate the effect of the surface elaboration with TPGS or NAcHis-TPGS on the colloidal stability of PLGA nanoparticles in aqueous phase.', 'The NIR-triggered hyperthermia capability of free ICG and ICG-loaded PLGA nanoparticles was examined by monitoring the temperature of the aqueous solution upon receiving laser irradiation at 808 nm. Upon NIR laser irradiation (power density of 1.0 W/cm2) for 120 s, the temperatures of ICG/DOX-carrying NHTPN and TPN solutions were appreciably raised compared with those of the ICG/DOX-loaded PN and free ICG solutions (Figure <xref ref-type="fig" rid="thnov06p0302g003">2</xref>c and d), due largely to the red-shift in the absorption of ICG embedded within NHTPNs and TPNs that better matched the central wavelength (808 nm) of the diode laser used c and d), due largely to the red-shift in the absorption of ICG embedded within NHTPNs and TPNs that better matched the central wavelength (808 nm) of the diode laser used 32. As expected, the level of temperature increase, a direct measure of NIR-triggered hyperthermia, increased with the concentrations of ICG-loaded NHTPNs and TPNs (Figure S3). The temperatures of all ICG-containing solutions declined gradually 3 min after irradiation due to photo-bleaching and thermal degradation of ICG 32, 33.', 'To study the therapeutic efficacy of ICG-based photo-triggered hyperthermia combined with DOX chemotherapy, the viability of TRAMP-C1 cells treated with NHTPNs carrying single- and dual-modality therapy at pH 7.4 was determined by MTT assay. TRAMP-C1 cells alone exposed to NIR laser irradiation as a control group retained the viability above 95%, being indicative of the harmlessness of NIR laser to cancer cells. While free ICG-treated TRAMP-C1 cells retained a mean viability above 90 %, the viability of TRAMP-C1 cells incubated with ICG-loaded NHTPNs was significantly reduced upon NIR activation (Figure <xref ref-type="fig" rid="thnov06p0302g006">5</xref>aa), indicating that NIR activation of ICG delivered by NHTPNs displayed strong thermal ablation on TRAMP-C1 cells. This is primarily attributed to the superior photo-triggered hyperthermia ability and photostability of the ICG delivered by NHTPNs in comparison with free ICG (Figure <xref ref-type="fig" rid="thnov06p0302g003">2</xref>). Figure ). Figure <xref ref-type="fig" rid="thnov06p0302g006">5</xref>a also shows that, at a DOX concentration of 11 μM without NIR irradiation, the ICG/DOX-loaded NHTPNs exhibited higher cytotoxicity against TRAMP-C1 cells (ca 40 % cell death) than free DOX (15 %), due to the promoted intracellular transport of DOX via endocytic uptake of NHTPNs to cellular nuclei in contrast to the diffusion-based accumulation of free hydrophobic DOX within cell membranes, as shown in Figure a also shows that, at a DOX concentration of 11 μM without NIR irradiation, the ICG/DOX-loaded NHTPNs exhibited higher cytotoxicity against TRAMP-C1 cells (ca 40 % cell death) than free DOX (15 %), due to the promoted intracellular transport of DOX via endocytic uptake of NHTPNs to cellular nuclei in contrast to the diffusion-based accumulation of free hydrophobic DOX within cell membranes, as shown in Figure <xref ref-type="fig" rid="thnov06p0302g006">5</xref>S.S.'], 'thnov06p0302g004': ['Compared to rapid diffusion of free ICG across the dialysis tube in PBS (>80% over 5 h), the ICG liberation from cargo-loaded nanoparticles under the same conditions was considerably retarded, in particular for those nanoparticles elaborated by TPGS or its NAcHis conjugate (Figure <xref ref-type="fig" rid="thnov06p0302g004">3</xref>c), which can be ascribed to the association of ICG with the vitamin E moiety of TPGS or NAcHis-TPGS on particle surfaces as described above. In contrast to the frequently observed strong pH-dependent release of DOX (in salt form) from pH-responsive polymeric delivery carriers, the DOX release profiles of ICG/DOX-loaded NHTPNs and TPNs were slower and independent of medium pH (Figure c), which can be ascribed to the association of ICG with the vitamin E moiety of TPGS or NAcHis-TPGS on particle surfaces as described above. In contrast to the frequently observed strong pH-dependent release of DOX (in salt form) from pH-responsive polymeric delivery carriers, the DOX release profiles of ICG/DOX-loaded NHTPNs and TPNs were slower and independent of medium pH (Figure <xref ref-type="fig" rid="thnov06p0302g004">3</xref>d), as the hydrophobic DOX was localized primarily within the solid PLGA cores of the nanoparticles. It can thus be presumed that the DOX release profile relied in large measure on the degradation rate of PLGA composing the hydrophobic cores of ICG/DOX-loaded nanoparticles.d), as the hydrophobic DOX was localized primarily within the solid PLGA cores of the nanoparticles. It can thus be presumed that the DOX release profile relied in large measure on the degradation rate of PLGA composing the hydrophobic cores of ICG/DOX-loaded nanoparticles.'], 'thnov06p0302g005': ['The effect of pH-responsive surface charge transition of the cargo-carrying NHTPNs on their cellular uptake was studied using TRAMP-C1 cells as a cell model. When the culture pH was adjusted from 7.4 to 6.3, the NIR fluorescence intensity of ICG molecules from TRAMP-C1 cells treated with ICG/DOX-loaded NHTPNs was significantly enhanced compared with the cancer cells incubated with either ICG/DOX-loaded TPNs or free ICG (Figure <xref ref-type="fig" rid="thnov06p0302g005">4</xref>a). The amount of intracellular DOX of TRAMP-C1 cells incubated with ICG/DOX-loaded NHTPNs was ca 1.5-fold enhanced in response to the pH reduction from 7.4 to 6.3 (Figure <xref ref-type="fig" rid="thnov06p0302g005">4</xref>b), while those of cells treated with either free DOX or ICG/DOX-loaded TPNs were virtually pH independent. The pH-dependent increase in cellular uptake of ICG/DOX-loaded NHTPNs was also confirmed by flow cytometric histograms (Figure b), while those of cells treated with either free DOX or ICG/DOX-loaded TPNs were virtually pH independent. The pH-dependent increase in cellular uptake of ICG/DOX-loaded NHTPNs was also confirmed by flow cytometric histograms (Figure <xref ref-type="fig" rid="thnov06p0302g005">4</xref>c). The results strongly suggest that the payload-carrying NHTPNs can effectively promote the intracellular cargo delivery under weak acidic conditions due to the enhanced affinity for cancer cells mediated by the positive charges of NAcHis-TPGS extending into surrounding aqueous phase from the negatively charged surfaces of ICG-embedded PLGA-based nanoparticles. The same findings were attained with another cell model, human breast MCF-7 cancer cells (Figure c). The results strongly suggest that the payload-carrying NHTPNs can effectively promote the intracellular cargo delivery under weak acidic conditions due to the enhanced affinity for cancer cells mediated by the positive charges of NAcHis-TPGS extending into surrounding aqueous phase from the negatively charged surfaces of ICG-embedded PLGA-based nanoparticles. The same findings were attained with another cell model, human breast MCF-7 cancer cells (Figure S4). In agreement with these observations, the fluorescence images show that DOX delivered by NHTPNs at pH 6.3 was found appreciably in the cytoplasm and nuclei of TRAMP-C1 cells, whereas relatively small amounts of DOX transported by either NHTPNs at pH 7.4 or by TPNs at both pH 7.4 and 6.3 were observed intracellularly (Figure <xref ref-type="fig" rid="thnov06p0302g005">4</xref>d). Compared to ICG/DOX-loaded NHTPNs found largely in cytoplasm and cell nuclei, free hydrophobic DOX molecules were mostly localized within cell membrane (Figure d). Compared to ICG/DOX-loaded NHTPNs found largely in cytoplasm and cell nuclei, free hydrophobic DOX molecules were mostly localized within cell membrane (Figure S5), consistent with the observation by Gu\'s group 37. In addition, an appreciably higher cellular uptake of free DOX than that of DOX carried by NHTPNs or TPNs at pH 7.4 was observed (Figure <xref ref-type="fig" rid="thnov06p0302g005">4</xref>b). Such differences are resulting mainly from the distinctive cellular uptake pathways for DOX species transported by NHTPNs (endocytosis) and for free DOX molecules (passive diffusion) b). Such differences are resulting mainly from the distinctive cellular uptake pathways for DOX species transported by NHTPNs (endocytosis) and for free DOX molecules (passive diffusion) 6, 37-40.'], 'thnov06p0302g006': ['Moreover, in the absence of NIR laser irradiation, the comparable cytotoxicity of ICG/DOX-loaded NHTPNs against TRAMP-C1 cells to DOX-loaded NHTPNs indicates that the presence of ICG has little influence on cancer chemotherapy efficacy of DOX released from NHTPNs. With NIR irradiation, the ICG/DOX-loaded NHTPNs showed the highest efficacy in inhibiting the proliferation of TRAMP-C1 cells by combined photothermal/chemo therapy. On the other hand, as revealed in Figure <xref ref-type="fig" rid="thnov06p0302g006">5</xref>b, after NIR laser irradiation, the viability of TRAMP-C1 cells incubated respectively with ICG/DOX-loaded NHTPNs and ICG-loaded NHTPNs at pH 6.3 was appreciably reduced compared to the cells treated at pH 7.4. In the absence of NIR laser irradiation, the enhanced cytotoxicity of ICG/DOX-loaded NHTPNs in response to the change of culture pH from 7.4 to 6.3 was also observed. The results clearly demonstrate the promotion of anticancer effect from payload-containing NHTPNs by virtue of the increased intracellular concentrations of therapeutic agents upon acid-activated cellular uptake.b, after NIR laser irradiation, the viability of TRAMP-C1 cells incubated respectively with ICG/DOX-loaded NHTPNs and ICG-loaded NHTPNs at pH 6.3 was appreciably reduced compared to the cells treated at pH 7.4. In the absence of NIR laser irradiation, the enhanced cytotoxicity of ICG/DOX-loaded NHTPNs in response to the change of culture pH from 7.4 to 6.3 was also observed. The results clearly demonstrate the promotion of anticancer effect from payload-containing NHTPNs by virtue of the increased intracellular concentrations of therapeutic agents upon acid-activated cellular uptake.'], 'thnov06p0302g007': ['The ex vivo NIR fluorescence signal of tumor receiving free ICG was not detected at 48 h post-injection, while the tumors from the NHTPN groups with the loaded cargo of either ICG alone or ICG/DOX exhibited higher fluorescence intensities than that from the group receiving the ICG/DOX-loaded TPNs (Figure <xref ref-type="fig" rid="thnov06p0302g007">6</xref>b and c). Since the particle sizes of the ICG/DOX-loaded NHTPNs and TPNs were essentially identical (ca 50 nm in Db and c). Since the particle sizes of the ICG/DOX-loaded NHTPNs and TPNs were essentially identical (ca 50 nm in Dh as shown in Table 1), the significantly enhanced tumor accumulation of the former is attributed to the increased surface positive charges from protonation of NAcHis (imidazole) residues by the tumor extracellular acidity. This is consistent with the biodistribution profiles of the NHTPNs carrying ICG/DOX (Figure <xref ref-type="fig" rid="thnov06p0302g007">6</xref>b and c), showing that the ex vivo ICG fluorescence intensity was higher in tumor than that in liver. Similar biodistribution of ICG delivered by functionalized nanocarriers has been attained b and c), showing that the ex vivo ICG fluorescence intensity was higher in tumor than that in liver. Similar biodistribution of ICG delivered by functionalized nanocarriers has been attained 30,31. By contrast, owing to the reduced tumor uptake by the lack of NAcHis residues, an appreciably enhanced accumulation of cargo-loaded TPNs in liver rather than in tumor was observed. These results indicate that the NHTPNs can greatly promote the tumor-targeted delivery of ICG and DOX, thus holding promise for improved cancer theranosis. Furthermore, the slightly increased tumor accumulation of ICG/DOX-loaded NHTPNs compared to ICG-loaded NHTPNs was attributed to the small particle size (Figure <xref ref-type="fig" rid="thnov06p0302g002">1</xref>a) and the nearly neutral surfaces of ICG/DOX-loaded NHTPNs (Figure a) and the nearly neutral surfaces of ICG/DOX-loaded NHTPNs (Figure <xref ref-type="fig" rid="thnov06p0302g004">3</xref>a) in tumor weak acidic environment which promote their tumor uptake upon the enhanced penetration ability and affinity with cancer cells.a) in tumor weak acidic environment which promote their tumor uptake upon the enhanced penetration ability and affinity with cancer cells.', 'Due to the enhanced accumulation of ICG-containing NHTPNs and TPNs at TRAMP-C1 tumor sites via the EPR effect (Figure <xref ref-type="fig" rid="thnov06p0302g007">6</xref>), a significant rise in local tumor temperature upon NIR laser irradiation (1.0 W/cm), a significant rise in local tumor temperature upon NIR laser irradiation (1.0 W/cm2) at 6 h post-injection was observed (Figure <xref ref-type="fig" rid="thnov06p0302g009">8</xref>). Because of the low intratumoral ICG concentration, the tumor temperature of free ICG-treated mice under NIR irradiation was only slightly higher than that of the control group injected with either free DOX or PBS. Notably, compared to all other ICG-containing formulations, the extensive accumulation of ICG/DOX-carrying NHTPNs in tumor (Figures <xref ref-type="fig" rid="thnov06p0302g007">6</xref> and and <xref ref-type="fig" rid="thnov06p0302g008">7</xref>) led to the most profound NIR-triggered hyperthermia on tumor within which a temperature as high as 60.2 ) led to the most profound NIR-triggered hyperthermia on tumor within which a temperature as high as 60.2 oC was attained (Figure <xref ref-type="fig" rid="thnov06p0302g009">8</xref>). Hyperthermia above 50 ). Hyperthermia above 50 oC can readily induce irreversible damage to cancer cells, as reflected by the burning scar at the irradiated tumor sites at day 6 post-treatment (Figure S8). The photo-evolved hyperthermia of ICG- and ICG/DOX-loaded NHTPNs within tumor tissues not only led to extensive cell necrosis, but also prominently induced cell apoptosis, as shown by a remarkable increase in the level of caspase-3 protein, a key molecular indicator for cells undergoing the apoptotic pathway (Figure <xref ref-type="fig" rid="thnov06p0302g010">9</xref>). As reported elsewhere 41, 42, 48, hyperthermia can induce sublethal damages and subsequent apoptosis of cancer cells located in the outer peripheral zones of laser-irradiated tumor sites, in addition to the increased blood flow which, in turn, can further facilitate the tumor accumulation of therapeutic nanoparticles.'], 'thnov06p0302g008': ['Although we have confirmed the deep tumor penetration of NHTPNs, whether the payload-carrying NHTPNs can further infiltrate into deep tumor hypoxia regions, with a weak acidic microenvironment of ca pH 6.0 and a distance as far as 150 μm from the neovessels, needs to be verified 22,43. As shown in Figure <xref ref-type="fig" rid="thnov06p0302g008">7</xref>b, the localization of ICG/DOX-loaded NHTPNs within the HIF 1α positive areas implies the presence of NHTPNs in the deep tumor hypoxia. The HIF 1α-identified tumor hypoxia correlated well with the contiguous tumor section stained with a CD11b marker, suggesting that the transport of NHTPNs from vessel periphery into tumor hypoxia may involve cellular cargo pickup via the hypoxia-homing recruitment of TAMs b, the localization of ICG/DOX-loaded NHTPNs within the HIF 1α positive areas implies the presence of NHTPNs in the deep tumor hypoxia. The HIF 1α-identified tumor hypoxia correlated well with the contiguous tumor section stained with a CD11b marker, suggesting that the transport of NHTPNs from vessel periphery into tumor hypoxia may involve cellular cargo pickup via the hypoxia-homing recruitment of TAMs 36, 44-47. By contrast, the access of ICG/DOX-loaded TPNs and free DOX to tumor hypoxia was severely restricted (Figure <xref ref-type="fig" rid="thnov06p0302g008">7</xref>c). It was further postulated that the difference in accessibility of tumor hypoxia to the two nanoparticles as observed in Figure c). It was further postulated that the difference in accessibility of tumor hypoxia to the two nanoparticles as observed in Figure S6 was caused predominantly by the increased surface positive charges of NHTPNs in the tumor acidic microenvironment and thus the cellular midway uptake by TAMs. The results of in vitro cellular uptake study using RAW 264.7 cells (mouse leukemic monocyte macrophage cell line) show a dramatic enhancement in ICG fluorescence intensity for RAW 264.7 cells incubated with cargo-loaded NHTPNs with medium pH adjustment from pH 7.4 to 6.3 (Figure S7), compared with only a limited increase in ICG fluorescence intensity for the cells treated with the TPN counterpart. As postulated, the acidity-triggered surface charge conversion of NHTPNs indeed facilitated their uptake by macrophages. The enhanced internalization of free ICG by RAW 264.7 cells at pH 7.4 (Figure S7) was a result of extensive self-aggregation that facilitated the phagocytic uptake.'], 'thnov06p0302g011': ['The in vivo antitumor efficacies of various formulations in terms of the change of tumor volume of the TRAMP-C1 tumor-bearing mice were evaluated for up to 15 days post intravenous injection. Little change in body weight of the treated mice over time in all groups was observed, revealing that the formulations adopted in this study did not induce severe acute toxicity (Figure S9). The tumor volumes (V) were normalized against their original volumes (Vo) to obtain the relative tumor volumes (V/Vo). As shown in Figure <xref ref-type="fig" rid="thnov06p0302g011">10</xref>a, 15 days after treatment, a 22~26-fold increase in the relative tumor volume of mice treated with either free DOX or ICG with laser irradiation was observed, indicating the failure of tumor growth inhibition due to poor tumor accumulation of ICG and DOX. By contrast, the administration of NHTPNs loaded with either ICG alone or ICG/DOX combined with laser irradiation led to an appreciable suppression on tumor growth up to 7 days post injection, beyond which tumors treated with ICG-loaded NHTPNs gradually enlarged, due to the proliferation of residual cells surviving the single-dose photothermal therapy. For the group treated with ICG/DOX-loaded NHTPNs without irradiation, the single DOX chemotherapy was incapable of inhibiting tumor growth. By combining the NIR-triggered hyperthermia and DOX chemotherapy, nevertheless, the ICG/DOX-loaded NHTPNs significantly retarded tumor growth with only a slight tumor recurrence at the end of treatment. On the other hand, the dual-modality therapy delivered by TPNs failed to effectively inhibit tumor growth due to its poor tumor accumulation and cellular uptake, as demonstrated in Figures a, 15 days after treatment, a 22~26-fold increase in the relative tumor volume of mice treated with either free DOX or ICG with laser irradiation was observed, indicating the failure of tumor growth inhibition due to poor tumor accumulation of ICG and DOX. By contrast, the administration of NHTPNs loaded with either ICG alone or ICG/DOX combined with laser irradiation led to an appreciable suppression on tumor growth up to 7 days post injection, beyond which tumors treated with ICG-loaded NHTPNs gradually enlarged, due to the proliferation of residual cells surviving the single-dose photothermal therapy. For the group treated with ICG/DOX-loaded NHTPNs without irradiation, the single DOX chemotherapy was incapable of inhibiting tumor growth. By combining the NIR-triggered hyperthermia and DOX chemotherapy, nevertheless, the ICG/DOX-loaded NHTPNs significantly retarded tumor growth with only a slight tumor recurrence at the end of treatment. On the other hand, the dual-modality therapy delivered by TPNs failed to effectively inhibit tumor growth due to its poor tumor accumulation and cellular uptake, as demonstrated in Figures <xref ref-type="fig" rid="thnov06p0302g007">6</xref> and and <xref ref-type="fig" rid="thnov06p0302g008">7</xref>. Based on the superior performances of ICG/DOX-loaded NHTPNs in intratumoral distribution and tumor growth inhibition, it is concluded that the photo-evolved hyperthermia not only induces effective thermal ablation of cancer cells but also promotes DOX penetration throughout the tumor, thus substantially inhibiting cancer cell proliferation.. Based on the superior performances of ICG/DOX-loaded NHTPNs in intratumoral distribution and tumor growth inhibition, it is concluded that the photo-evolved hyperthermia not only induces effective thermal ablation of cancer cells but also promotes DOX penetration throughout the tumor, thus substantially inhibiting cancer cell proliferation.', 'In agreement with the results of the in vivo tumor growth inhibition, tumors harvested from the sacrificed mice receiving ICG/DOX-loaded NHTPNs and NIR irradiation were the smallest among the tumors receiving other treatments (Figure <xref ref-type="fig" rid="thnov06p0302g011">10</xref>a). As shown in Table S1, the average TI (ca 73.5 %) of the combinatorial therapy provided by ICG/DOX-loaded NHTPNs with NIR activation was appreciably higher than that without NIR irradiation (26.9 %) and that of ICG/DOX-loaded TPNs with NIR illumination (30.3 %). This further demonstrates that the dual-modality therapy delivered by ICG/DOX-loaded NHTPNs exhibited the highest antitumor efficacy. As illustrated by H&E staining of the tumor sections (Figure <xref ref-type="fig" rid="thnov06p0302g011">10</xref>b), TRAMP-C1 tumor tissue receiving PBS and laser irradiation showed the same histologic features compared to the group without illumination, indicating that the NIR irradiation (1 W/cmb), TRAMP-C1 tumor tissue receiving PBS and laser irradiation showed the same histologic features compared to the group without illumination, indicating that the NIR irradiation (1 W/cm2) by itself was essentially harmless to the tissue. Tumors treated with either free ICG or DOX along with laser irradiation showed no apparent tissue damage primarily because of the lack of tumor targeting and thus the low drug accumulation in tumors. Upon laser irradiation, tumors receiving ICG/DOX-loaded NHTPNs exhibited more severe cell necrosis and extensive hemorrhagic inflammation as compared to those treated with either ICG/DOX-loaded TPNs or ICG-loaded NHTPNs. Compared to the ICG-loaded NHTPN treatment, the damage to tumor tissue receiving ICG/DOX-loaded NHTPNs was significantly enhanced, thereby clearly demonstrating the therapeutic effect of the chemotreatment, most likely with the aid of photo-triggered hyperthermia in enhancing DOX penetration into deep tumor tissue and in accelerating PLGA degradation. Upon the treatment with the photothermal therapy from ICG-loaded NHTPNs, the H&E examination of tumor sections showed only sporadic necrotic regions along with occasional appearances of pyknosis. No apparent abnormality was observed in major organs, particularly in liver of mice treated with all nanoformulations, because the NIR irradiation as a hyperthermia trigger was applied exclusively on tumors only (Figure S10). These results strongly suggest that the imaging-guided photothermal/chemo combinatorial therapy of ICG/DOX-loaded NHTPNs developed herein not only can significantly reduce side effects on normal tissues, but also display a sound anti-tumor efficacy.']}
|
Active Tumor Permeation and Uptake of Surface Charge-Switchable Theranostic Nanoparticles for Imaging-Guided Photothermal/Chemo Combinatorial Therapy
|
[
"surface charge transition",
"deep tumor penetration",
"tumor hypoxia",
"photothermal therapy",
"chemotherapy"
] |
Theranostics
|
1451635200
|
To significantly promote tumor uptake and penetration of therapeutics, a nanovehicle system comprising poly(lactic-co-glycolic acid) (PLGA) as the hydrophobic cores coated with pH-responsive N-acetyl histidine modified D-α-tocopheryl polyethylene glycol succinate (NAcHis-TPGS) is developed in this work. The nanocarriers with switchable surface charges in response to tumor extracellular acidity (pHe) were capable of selectively co-delivering indocyanine green (ICG), a photothermal agent, and doxorubicin (DOX), a chemotherapy drug, to tumor sites. The in vitro cellular uptake of ICG/DOX-loaded nanoparticles by cancer cells and macrophages was significantly promoted in weak acidic environments due to the increased protonation of the NAcHis moieties. The results of in vivo and ex vivo biodistribution studies demonstrated that upon intravenous injection the theranostic nanoparticles were substantially accumulated in TRAMP-C1 solid tumor of tumor-bearing mice. Immunohistochemical examination of tumor sections confirmed the active permeation of the nanoparticles into deep tumor hypoxia due to their small size, pHe-induced near neutral surface, and the additional hitchhiking transport via tumor-associated macrophages. The prominent imaging-guided photothermal therapy of ICG/DOX-loaded nanoparticles after tumor accumulation induced extensive tumor tissue/vessel ablation, which further promoted their extravasation and DOX tumor permeation, thus effectively suppressing tumor growth.
|
[
"Administration, Intravenous",
"Animals",
"Antineoplastic Agents",
"Combined Modality Therapy",
"Disease Models, Animal",
"Doxorubicin",
"Drug Carriers",
"Drug Therapy",
"Hyperthermia, Induced",
"Indocyanine Green",
"Male",
"Mice, Inbred C57BL",
"Nanoparticles",
"Photosensitizing Agents",
"Phototherapy",
"Polyethylene Glycols",
"Skin Neoplasms",
"Succinates",
"Theranostic Nanomedicine",
"Tissue Distribution"
] |
other
|
PMC4737719
| null |
48
|
[
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] |
Theranostics. 2016 Jan 1; 6(3):302-317
|
NO-CC CODE
|
|
Loss of Dnf1 and Dnf2 function in pdr10∆ cells is associated with their increased chitin deposition. a Serial dilutions from overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY427 (dnf1∆ dnf2∆), NRY558 (pdr10∆ dnf1∆ dnf2∆), NRY549 (lem3∆), and NRY551 (pdr10∆ lem3∆) were spotted onto YPD plates at increasing concentrations of Calcofluor White (CW). b Log phase cultures of strains W303-1A, NRY243, NRY427, and NRY558 were stained for chitin and examined by fluorescence microscopy. Bar, 5 μm. c Serial dilutions of strains W303-1A, NRY243, NRY549, and NRY551 were spotted onto YPD plates buffered to pH 3.5 with sodium succinate and containing potassium sorbate. d Serial dilutions of strains W303-1A, NRY243, NRY301 (pdr12∆), and NRY366 (pdr10∆ pdr12∆) were spotted onto YPD plates containing Calcofluor White
|
232_2009_9173_Fig9_HTML
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2
|
390ad71b37e35aa1b61e9d309d5e027deb9bb256af7c39a6b257230bb582d396
|
232_2009_9173_Fig9_HTML.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
484,
597
] |
[{'image_id': '232_2009_9173_Fig10_HTML', 'image_file_name': '232_2009_9173_Fig10_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig10_HTML.jpg', 'caption': 'pdr10∆ cells accumulate excess Pdr12 in the plasma membrane. a Total membranes were prepared from log phase cultures of strains W303-1A (wild type) and NRY243 (pdr10∆). The indicated proteins were detected by immunoblotting, with equal protein loaded for each strain. b Strains NRY915 (wild type) and NRY925 (pdr10∆) were grown to log phase in YPD, and Pdr12-GFP was examined by fluorescence microscopy. A collage of representative images is shown. Pdr12-GFP function was confirmed by sorbate sensitivity. Bar, 6\xa0μm', 'hash': '2a3f53f81624579dbaf24efc65b5cb86cd8b9ade4e2a5e6472d383a7e185b78b'}, {'image_id': '232_2009_9173_Fig8_HTML', 'image_file_name': '232_2009_9173_Fig8_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig8_HTML.jpg', 'caption': 'pdr10∆ cells internalize NBD-PE in a Lem3-dependent manner. Strains W303-1A (wild type), NRY243 (pdr10∆), NRY549 (lem3∆), NRY551 (pdr10∆ lem3∆), NRY427 (dnf1∆ dnf2∆), and NRY558 (pdr10∆ dnf1∆ dnf2∆) were grown to log phase, treated with latrunculin A to block endocytosis, and incubated with the fluorescent reporter lipid NBD-PE or NBD-Cer as described under Materials and Methods. Cells were examined by fluorescence microscopy and differential interference contrast microscopy (DIC). Bar, 6\xa0μm', 'hash': '572af130e5b51bd060d5492f7afe5fd7e25ebf35875517a769b1d282a3205084'}, {'image_id': '232_2009_9173_Fig7_HTML', 'image_file_name': '232_2009_9173_Fig7_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig7_HTML.jpg', 'caption': 'Pdr10 is not required for normal localization of putative PE translocases. Strains NRY914 (wild type, Lem3-GFP), NRY918 (pdr10∆, Lem3-GFP), NRY921 (wild type, Dnf1-GFP), NRY932 (pdr10∆, Dnf1-GFP), NRY923 (wild type, Dnf2-GFP), NRY929 (pdr10∆, Dnf2-GFP), NRY906 (wild type, Pdr5-GFP), and NRY916 (pdr10∆, Pdr5-GFP) were grown to log phase, and the indicated proteins were visualized by fluorescence microscopy. Collages of representative images are shown. Bar, 6\xa0μm', 'hash': 'd223e930ee93ac56474e1732c03c7137c54b33633a09dd03d60a5241ad9eb9fc'}, {'image_id': '232_2009_9173_Fig1_HTML', 'image_file_name': '232_2009_9173_Fig1_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig1_HTML.jpg', 'caption': 'Pdr10 localizes to punctate structures in the plasma membrane and to the detergent-resistant fraction. a Strain NRY912 was grown in YPD for 2\xa0days, and Pdr10-GFP was examined by fluorescence microscopy. Pdr10-GFP function was confirmed by Calcofluor sensitivity. b Strain NRY912 was grown for 2\xa0days as in (a) but was treated with latrunculin A prior to microscopy. Bar for (a) and (b), 6\xa0μm. c Cells were repeatedly exposed to look for movement of Pdr10-GFP puncta (arrowheads). Shorter exposure times and higher gain were used to permit the maximum number of exposures prior to loss of signal due to photobleaching. Bar, 3\xa0μm. d Total membranes were isolated from log phase cells of strain NRY930, extracted with cold Triton X-100, and analyzed by flotation though Optiprep density gradients with subsequent immunoblotting as described under Materials and Methods. Strain NRY930 expresses a PDR10 allele with a C-terminal tandem myc-His6 (mh) tag. The function of the resulting Pdr10-mh protein was confirmed by Calcofluor sensitivity. Hxt1, Gas1, and Pma1 were used as markers for detergent-soluble and detergent-resistant membranes', 'hash': '6115e47009def08782885e99de01597ef32a6952a1293e999a2b5e39943fd702'}, {'image_id': '232_2009_9173_Fig11_HTML', 'image_file_name': '232_2009_9173_Fig11_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig11_HTML.jpg', 'caption': 'pdr10∆ cells accumulate Pdr12 in the detergent-resistant membrane fraction. a Total membranes from log phase cultures of strains W303-1A or NRY904 (wildtype) and NRY243 or NRY908 (pdr10∆) were examined by cold detergent extraction, Optiprep density gradient flotation, and immunoblotting for the indicated proteins. Strains NRY904 and NRY908 contain Lem3 C-terminally tagged with a tandem myc-His6 (mh) tag. In vivo function of the resulting Lem3-mh protein was confirmed by Calcofluor sensitivity. b Immunoblotting data were quantitated, and the percentage of total Pdr12 (left) and Chs3 (right) associated with detergent-resistant and detergent-soluble fractions is presented as the mean value for four independent experiments, with error bars representing the standard deviation. Detergent-resistant and detergent-soluble membranes were defined as fractions 1–3 and 7–9, respectively, in the Optiprep density gradients', 'hash': 'd6f979bc641e80c89ce325e4a5f6e1d4f50b2381800e6daed8ac865c406a917a'}, {'image_id': '232_2009_9173_Fig6_HTML', 'image_file_name': '232_2009_9173_Fig6_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig6_HTML.jpg', 'caption': 'Increased phosphatidylethanolamine is accessible to TNBS labeling in pdr10∆ cells. a Strains W303-1A and NRY243 were grown to log phase, incubated with or without α-factor, and then stained with filipin to visualize sterols by fluorescence microscopy. Bar, 5\xa0μm. b Exposure of endogenous aminophospholipids to TNBS was assayed with cells grown overnight in the presence of 32P-phosphate as described under Materials and Methods. Strains used were W303-1A (wild type), NRY243 (pdr10∆), NRY549 (lem3∆), NRY551 (pdr10∆ lem3∆), NRY427 (dnf1∆ dnf2∆), and NRY558 (pdr10∆ dnf1∆ dnf2∆)', 'hash': '650851236967ee9e0902609503816dea1029e466008d8f976d1d7f1b20fca3b3'}, {'image_id': '232_2009_9173_Fig9_HTML', 'image_file_name': '232_2009_9173_Fig9_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig9_HTML.jpg', 'caption': 'Loss of Dnf1 and Dnf2 function in pdr10∆ cells is associated with their increased chitin deposition. a Serial dilutions from overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY427 (dnf1∆ dnf2∆), NRY558 (pdr10∆ dnf1∆ dnf2∆), NRY549 (lem3∆), and NRY551 (pdr10∆ lem3∆) were spotted onto YPD plates at increasing concentrations of Calcofluor White (CW). b Log phase cultures of strains W303-1A, NRY243, NRY427, and NRY558 were stained for chitin and examined by fluorescence microscopy. Bar, 5\xa0μm. c Serial dilutions of strains W303-1A, NRY243, NRY549, and NRY551 were spotted onto YPD plates buffered to pH 3.5 with sodium succinate and containing potassium sorbate. d Serial dilutions of strains W303-1A, NRY243, NRY301 (pdr12∆), and NRY366 (pdr10∆ pdr12∆) were spotted onto YPD plates containing Calcofluor White', 'hash': '390ad71b37e35aa1b61e9d309d5e027deb9bb256af7c39a6b257230bb582d396'}, {'image_id': '232_2009_9173_Fig5_HTML', 'image_file_name': '232_2009_9173_Fig5_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig5_HTML.jpg', 'caption': 'Pdr10 is not required for bulk endocytosis or actin morphology. a Strains W303-1A (wild type) and NRY243 (pdr10∆) were grown to log phase, incubated with Lucifer Yellow or FM 4-64 as a marker for endocytosis, and examined by fluorescence microscopy. b Strains W303-1A and NRY243 were grown to log phase, permeabilized, and stained with rhodamine-phalloidin to visualize actin by fluorescence microscopy. All bars, 5\xa0μm', 'hash': 'f3971df718856585d70cc5700bccf1af8fba5e03d7e9036eca4b0015a8e84bd4'}, {'image_id': '232_2009_9173_Fig12_HTML', 'image_file_name': '232_2009_9173_Fig12_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig12_HTML.jpg', 'caption': 'Pdr5 and phosphosphingolipids are required for pdr10∆ phenotypes. a Serial dilutions of overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY201 (pdr5∆), NRY230 (pdr5∆ pdr10∆), NRY212 (pdr15∆), NRY236 (pdr10∆ pdr15∆), NRY410 (ipt1∆), NRY534 (pdr10∆ ipt1∆), NRY409 (sur1∆), and NRY526 (pdr10∆ sur1∆) were spotted onto YPD plates containing Calcofluor White (CW; left column) or sorbate buffered with sodium succinate (right column). b Strains W303-1A, NRY243, NRY409, and NRY526 were assayed for PE accessibility to TNBS labeling after overnight labeling with 32P-phosphate as described under Materials and Methods. c Total Pdr12 and Chs3 levels were examined by immunoblotting equal amounts of total membrane proteins isolated from log phase cultures of strains W303-1A, NRY230, and NRY526. d Strains W303-1A, NRY243, NRY409, NRY526, NRY410, and NRY534 were grown to log phase, stained for chitin, and examined by fluorescence microscopy. Bar, 5\xa0μm', 'hash': 'a8edaef9b4301ab2237c3876f3c27361a5c27205d0d1d21f9b943166c4e6a4c6'}, {'image_id': '232_2009_9173_Fig2_HTML', 'image_file_name': '232_2009_9173_Fig2_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig2_HTML.jpg', 'caption': 'Pdr10 is required for normal response to Calcofluor and sorbate. a Spot assays were performed using serial dilutions prepared from overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY201 (pdr5∆), and NRY230 (pdr5∆ pdr10∆). All plates were grown for 2\xa0days at 30°C unless otherwise noted. YPD plates contained either 2\xa0μM ketoconazole (keto), 20\xa0μM rhodamine 6G (Rh. 6G), or 100\xa0ng/ml cycloheximide (CHX). b Halo assays were performed on YPD plates with various detergents. Aliquots from overnight cultures were diluted into YPD top agar as described under Materials and Methods. Top row, strains W303-1A, NRY243, and NRY601 (pdr10∆ chs3∆) were assayed with dodecyl maltoside (DDM). Middle row, the same strains were assayed with dodecyl phosphocholine (DPC). Bottom row, strains YPH499 (wild type) and YHW10\xa0N (pdr10∆) were assayed with DDM. c Spot assays were performed as in A using strains W303-1A, NRY243, and NRY602 (chs3∆), NRY601 and NRY608 (chs6∆), and NRY610 (pdr10∆ chs6∆) on YPD plates (top) or YPD plates containing 6\xa0μg/ml Calcofluor White (CW; middle) or 100\xa0μg/ml Congo Red (CR; bottom). d Similar assays were performed using strains W303-1A (wild type), NRY243 (pdr10∆), NRY301 (pdr12∆), and NRY366 (pdr10∆ pdr12∆) on YPD plates buffered to pH 3.5 with 50\xa0mM sodium succinate with or without 4\xa0mM potassium sorbate', 'hash': 'e67c35ef2b12d96fa7ed7c9f8a5d9adb0bd8aa79e67bcae11b4a91f4e8885abc'}, {'image_id': '232_2009_9173_Fig4_HTML', 'image_file_name': '232_2009_9173_Fig4_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig4_HTML.jpg', 'caption': 'Defective endocytosis of Chs3 causes excess chitin deposition in pdr10∆ cells. a Cells from log phase cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY602 (chs3∆), and NRY601 (pdr10∆ chs3∆) were stained for chitin and examined by fluorescence microscopy. Bar, 4\xa0μm. b Log phase cells from strains W303-1A and NRY251 (pdr10∆) carrying plasmids pRS315-CHS7 and pCRP12 were treated with 3.6\xa0μM α-factor for 35\xa0min in SCD buffered to pH 3.5 with 25\xa0mM sodium succinate and supplemented with adenine. Pheromone was then washed out at time 0, and the localization of Chs3-GFP was monitored by fluorescence microscopy as described under Materials and Methods. Punctate Chs3-GFP staining of the plasma membrane in pdr10∆ cells at late time points is indicated by arrow heads. Bar, 3\xa0μm. c Strains W303-1A, NRY243, and NRY407 (cho1∆) were grown into log phase, stained for chitin, and examined by fluorescence microscopy. Bar, 5\xa0μm. d Serial dilutions of overnight cultures of strains W303-1A, NRY243, and NRY407 were spotted onto YPD plates containing Calcofluor', 'hash': '2c091b62a71bdc1847352c82751bee24bf0162048409522f516c8ad82a626be8'}, {'image_id': '232_2009_9173_Fig3_HTML', 'image_file_name': '232_2009_9173_Fig3_HTML.jpg', 'image_path': '../data/media_files/PMC2687517/232_2009_9173_Fig3_HTML.jpg', 'caption': 'Calcofluor sensitivity and sorbate resistance define a specific pdr10∆ phenotype. Serial dilutions of overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY201 (pdr5∆), NRY212 (pdr15∆), NRY336 (pdr18∆), NRY334 (YOL075c∆), and WKK7 (ste6∆) were spotted onto YPD plates containing 6\xa0μg/ml Calcofluor White (CW; left column) or 4\xa0mM sorbate (right column)', 'hash': '86c80791c70b74d5b1c7fffa86ded1b13b9d7574fe32a13c5cb3bc1ee71cece1'}]
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{'232_2009_9173_Fig6_HTML': ['Cells were grown in 1\xa0ml YPD supplemented with 50–250\xa0μCi 32P-PO43– (10\xa0mCi/ml; Perkin-Elmer) for 15–18\xa0h to label phospholipids to a constant specific activity (Chang et al. 1998). For reaction of intact yeast cells with 2,4,6-trinitrobenzene sulfonate (TNBS), a published procedure (Siegmund et al. 1998) was used with only minor modifications. After radioactive labeling, cells were washed twice (1\xa0ml each) in ice-cold TNBS buffer (120\xa0mM NaHCO3 [pH 8.4], 40\xa0mM NaCl), resuspended in 1\xa0ml of the same buffer, and then aqueous TNBS (31\xa0μl) was added from a concentrated stock (5%; Sigma). After incubation on ice with brief vortex mixing every 20\xa0min over the course of 1\xa0h, the cells were collected by brief centrifugation, washed three times (1\xa0ml each) in ice-cold TNBS buffer, and resuspended in 600\xa0μl CHCl3/CH3OH/0.1\xa0N HCl (1:2:0.8) containing carrier lipid (Chang et al. 1998). To promote lipid extraction after addition of the organic solvent mix, the cells were subjected to vigorous vortexing with glass beads for 3\xa0min, and an additional 200\xa0μl each of CHCl3 and 0.1\xa0N HCl–0.5\xa0M NaCl was added. To achieve good separation of the organic phase from the aqueous phase, the resulting mixture was subjected to centrifugation for 2\xa0min at maximum speed in a microfuge. After withdrawing the organic phase, the lipids present in it were analyzed by two-dimensional thin-layer chromatography (2D-TLC) using CHCl3:MeOH:glacial acetic acid, 65:25:10 (dimension I), and CHCl3:MeOH:88% formic acid, 65:25:10 (dimension II) (Esko and Raetz 1980), with nonradioactive trinitrophenyl-PE (TNP-PE) added as an internal standard. The radioactivity present in each spot on the resulting thin-layer chromatograms was quantitated by using the plates to expose phosphor storage screens, which were then analyzed using PhosphorImager or Typhoon imaging systems and ImageQuant software (Molecular Dynamics). The fraction of PE accessible to TNBS was calculated as TNP-PE/total PE (where total PE\xa0=\xa0TNP-PE\xa0+\xa0unreacted PE). TNP derivatives of phosphatidylserine (PS) were not observed. Comparing across independent experiments, this assay procedure yielded somewhat variable backgrounds, presumably due to slight differences in the fraction of inviable cells in cultures. We have established that there is no statistically significant difference in cell viability in strains cultured together, both by measurement of the ratio of colony-forming units to optical density and by methylene blue staining (Siegmund et al. 1998) of TNBS-labeled, nonradioactive control cultures (>500 cells counted per strain). Therefore, other strains should be measured accurately relative to the background TNBS labeling of wild-type cultures. All such measurements were performed with wild-type controls grown side by side. In every such trial, more PE was accessible in pdr10∆ cells than in the control cultures (n\xa0=\xa07). In reporting the results, we have shown the actual labeled percentages in Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>b for comparison to results of other workers, who have reported such data in this fashion (Pomorski et al. b for comparison to results of other workers, who have reported such data in this fashion (Pomorski et al. 2003), and we have also reported the data as -fold differences in Table\xa02 (which should be more accurate, in light of the variable background).Table\xa02Total phospholipid composition of yeast strainsStraina% Total phospholipid (as incorporated 32P label)TNBS labelingbPSPEPCPISPLWild type7.6\xa0±\xa00.620.2\xa0±\xa01.535.5\xa0±\xa02.124.1\xa0±\xa00.67.3\xa0±\xa00.91.0pdr10∆7.7\xa0±\xa00.520.3\xa0±\xa01.234.9\xa0±\xa01.123.9\xa0±\xa01.17.3\xa0±\xa01.32.5\xa0±\xa00.6dnf1∆ dnf2∆7.5\xa0±\xa00.520.5\xa0±\xa00.536.3\xa0±\xa00.722.9\xa0±\xa00.57.8\xa0±\xa00.62.0\xa0±\xa00.6pdr10∆ dnf1∆ dnf2∆7.7\xa0±\xa00.520.0\xa0±\xa00.535.9\xa0±\xa00.523.8\xa0±\xa00.57.4\xa0±\xa00.52.3\xa0±\xa01.0lem3∆7.9\xa0±\xa00.120.7\xa0±\xa00.735.1\xa0±\xa01.523.6\xa0±\xa01.27.3\xa0±\xa00.42.7\xa0±\xa00.5pdr10∆ lem3∆7.8\xa0±\xa00.220.3\xa0±\xa01.135.0\xa0±\xa00.524.1\xa0±\xa01.97.2\xa0±\xa00.42.7\xa0±\xa01.0pdr5∆9.2\xa0±\xa00.522.7\xa0±\xa00.532.5\xa0±\xa00.519.8\xa0±\xa00.87.4\xa0±\xa00.50.9\xa0±\xa00.2pdr5∆ pdr10∆7.9\xa0±\xa01.020.3\xa0±\xa02.038.7\xa0±\xa01.718.7\xa0±\xa00.76.4\xa0±\xa01.30.3\xa0±\xa00.2pdr12∆7.5\xa0±\xa00.519.7\xa0±\xa00.536.0\xa0±\xa00.524.0\xa0±\xa00.57.7\xa0±\xa00.81.6\xa0±\xa00.6pdr10∆ pdr12∆8.1\xa0±\xa00.519.9\xa0±\xa00.535.0\xa0±\xa01.124.0\xa0±\xa00.57.6\xa0±\xa00.51.5\xa0±\xa00.6sur1∆8.2\xa0±\xa00.519.1\xa0±\xa00.534.2\xa0±\xa00.623.3\xa0±\xa00.510.0\xa0±\xa00.51.1\xa0±\xa00.2pdr10∆ sur1∆8.2\xa0±\xa00.519.7\xa0±\xa00.535.6\xa0±\xa00.722.5\xa0±\xa01.29.2\xa0±\xa00.61.1\xa0±\xa00.2cho1∆<0.111.0\xa0±\xa03.052.7\xa0±\xa06.226.9\xa0±\xa02.85.6\xa0±\xa01.22.7\xa0±\xa00.8Note: PS phosphatidylserine; PE phosphatidylethanolamine; PC phosphatidylcholine; PI phosphatidylinositol; SPL total phosphosphingolipids. Lipids were labeled and analyzed as described under Materials and Methods. Significant changes relative to wild type are in boldfaceaStrains were W303-1A (wild type), NRY243 (pdr10∆), NRY427 (dnf1∆ dnf2∆), NRY558 (pdr10∆ dnf1∆ dnf2∆), NRY549 (lem3∆), NRY551 (pdr10∆ lem3∆), NRY201 (pdr5∆), NRY230 (pdr5∆ pdr10∆), NRY301 (pdr12∆), NRY366 (pdr10∆ pdr12∆), NRY409 (sur1∆), and NRY526 (pdr10∆ sur1∆)bTNBS labeling is reported relative to the wild type', 'It has been proposed that proper maintenance of membrane asymmetry is important for endocytosis at low temperatures and that Pdr5 and another ABC transporter, Yor1, are candidate outward-directed PE translocases (Pomorski et al. 2003). Moreover, pdr5∆ pdr15∆ cells exhibit increased accessibility of PE to the small-molecule reagent TNBS (Schüller et al. 2007), which is able to pass through the cell wall. TNBS labeling has been used to assess exposure of PE and PS on the outer leaflet of the plasma membrane (Gordesky and Marinetti 1973; Pomorski et al. 2003; Siegmund et al. 1998), although subcellular fractionation data raise the possibility that there is considerable leakage of the reagent into the secretory pathway (Pomorski et al. 2003). We analyzed the total phospholipid composition (Table\xa02) and accessibility of aminophospholipids to TNBS (Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>b) in wild-type and b) in wild-type and pdr10∆ cells grown on 32P-phosphate. We found that a pdr10∆ mutation had no discernible effect on overall phospholipid composition (Table\xa02), but caused a significant increase in the fraction of the PE that was accessible to TNBS labeling (Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>b). Thus, b). Thus, pdr10∆ plasma membranes either are significantly more permeable to TNBS or exhibit a defect in maintenance of plasma membrane asymmetry.', 'The ABC transporters with established transport activities that are most closely related to Pdr10, including Pdr5 and Snq2, mediate outward-directed lipid transport and/or export of xenobiotics from the cell (Balzi et al. 1994; Bissinger and Kuchler 1994; Decottignies et al. 1995; Egner et al. 1998; Mahé et al. 1996a; Pomorski et al. 2003; Schüller et al. 2007). If Pdr10 had a similar function, then loss of Pdr10 should reduce, not increase, the fraction of PE that is surface-exposed. In contrast, if the absence of Pdr10 somehow indirectly crippled the function of the inward-directed PE translocases, then the biochemical phenotype observed in pdr10∆ cells would be more readily explained. In support of this, we observed that the level of PE accessible to TNBS labeling in pdr10∆ cells was comparable to that in cells in which the genes for both putative inward-directed plasma membrane PE translocases, DNF1 and DNF2, were deleted (Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>b); similar results were also obtained for cells lacking the b); similar results were also obtained for cells lacking the LEM3 gene, which encodes an integral membrane protein that is required for exit of Dnf1 and Dnf2 from the endoplasmic reticulum (Saito et al. 2004). Furthermore, no additive increase in exposure of PE was observed in pdr10∆ lem3∆ or pdr10∆ dnf1∆ dnf2∆ strains (Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>b). The overall phospholipid compositions of all of these strains were quite comparable (Table\xa0b). The overall phospholipid compositions of all of these strains were quite comparable (Table\xa02). The absence of any additive or synergistic effect of the dnf1∆dnf2∆ (or lem3∆) mutations in a pdr10∆ mutant demonstrated that pdr10∆ phenocopied dnf1∆ dnf2∆. That is, regardless of whether TNBS labeling is measuring PE exposure on the outer leaflet or permeability, the loss of Pdr10 function mimicked the loss of Dnf1 and Dnf2 function in these cells.'], '232_2009_9173_Fig1_HTML': ['To confirm that the product of the PDR10 gene is a protein that is expressed at a detectable level and, if so, to establish its subcellular localization, a strain was constructed in which the green fluorescent protein (GFP) was fused in-frame to the C-terminal end of the open reading frame encoded by the chromosomal PDR10 locus. We found that Pdr10-GFP localized to discrete, punctate structures in the plasma membrane (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>a). This chimera was most easily visualized in cells that had entered stationary phase (after 2\xa0days of growth); however, the same pattern was observed in exponentially growing cells, only with a much weaker signal (data not shown). This finding is in agreement with global microarray analysis showing that transcription of a). This chimera was most easily visualized in cells that had entered stationary phase (after 2\xa0days of growth); however, the same pattern was observed in exponentially growing cells, only with a much weaker signal (data not shown). This finding is in agreement with global microarray analysis showing that transcription of PDR10 is induced when cells undergo the diauxic shift from fermentative to nonfermentative growth as glucose becomes limiting (Roberts and Hudson 2006) or when cells are treated with rapamycin, which mimics conditions of nutrient limitation (Huang et al. 2004). Collectively, these findings suggest that the demand for this ABC transporter is greatest when cells are undergoing the stress of maintaining viability as growth rate slows down. Moreover, because our Pdr10-GFP construct was expressed from the endogenous PDR10 promoter, transcription factors other than (or in addition to) Pdr1 and Pdr3 likely mediate PDR10 expression under the stress of nutrient depletion.Fig.\xa01\u2003Pdr10 localizes to punctate structures in the plasma membrane and to the detergent-resistant fraction. a Strain NRY912 was grown in YPD for 2\xa0days, and Pdr10-GFP was examined by fluorescence microscopy. Pdr10-GFP function was confirmed by Calcofluor sensitivity. b Strain NRY912 was grown for 2\xa0days as in (a) but was treated with latrunculin A prior to microscopy. Bar for (a) and (b), 6\xa0μm. c Cells were repeatedly exposed to look for movement of Pdr10-GFP puncta (arrowheads). Shorter exposure times and higher gain were used to permit the maximum number of exposures prior to loss of signal due to photobleaching. Bar, 3\xa0μm. d Total membranes were isolated from log phase cells of strain NRY930, extracted with cold Triton X-100, and analyzed by flotation though Optiprep density gradients with subsequent immunoblotting as described under Materials and Methods. Strain NRY930 expresses a PDR10 allele with a C-terminal tandem myc-His6 (mh) tag. The function of the resulting Pdr10-mh protein was confirmed by Calcofluor sensitivity. Hxt1, Gas1, and Pma1 were used as markers for detergent-soluble and detergent-resistant membranes', 'The markedly punctate distribution in the plasma membrane displayed by Pdr10-GFP has been observed for several other integral plasma membrane proteins. In some cases, these puncta are mobile because they represent proteins (for example, the glucan synthase Fks1) associated with sites where actin-mediated endocytosis occurs (“actin patches”) (Utsugi et al. 2002). In other cases, the puncta are immobile because they represent proteins (for example, the arginine permease Can1 and the tetraspanin Sur7) associated with stable, static plasma membrane subdomains such as the MCC compartment (Grossmann et al. 2007; Malinska et al. 2003; Young et al. 2002). Therefore, we examined whether the Pdr10-GFP puncta were stable over time and whether they were affected by inhibition of actin function. We found that treatment of cells with latrunculin A did not disrupt the punctate localization displayed by Pdr10-GFP (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>b). Some of the Pdr10-GFP puncta in a typical cell were immobile for at least 20\xa0min (Fig.\xa0b). Some of the Pdr10-GFP puncta in a typical cell were immobile for at least 20\xa0min (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>c). However, other Pdr10-GFP puncta seemed more mobile, particularly in the bud. There is evidence that the mother and bud compartments have distinct properties. For example, PE exposure is reportedly greater at the bud tip than in the mother cell (Iwamoto et al. c). However, other Pdr10-GFP puncta seemed more mobile, particularly in the bud. There is evidence that the mother and bud compartments have distinct properties. For example, PE exposure is reportedly greater at the bud tip than in the mother cell (Iwamoto et al. 2004), and the collar of septin filaments at the bud neck acts as a diffusion barrier to confine certain plasma membrane proteins uniquely in the bud (Takizawa et al. 2000). However, the presence of mobile patches in the mother cell as well indicates that there is a mobile population of Pdr10 throughout the cell. Pdr10-GFP did not behave like an actin patch-associated protein, but the presence of both mobile and immobile populations argues against its exclusive localization to previously defined subdomains of the yeast plasma membrane.', 'Can1 and other structurally related permeases not only display a punctate distribution in the plasma membrane, but also are found in the detergent-resistant membrane (DRM) fraction (sometimes equated with lipid rafts) when cells are subjected to biochemical fractionation (Malinska et al. 2003, 2004). To permit facile examination of the behavior of Pdr10 during biochemical fractionation, we tagged the chromosomal allele of PDR10 at its C-terminus with c-Myc and His6 epitopes in tandem (the mh tag) under control of the native PDR10 promoter. We then prepared total membranes from such cells, extracted them with cold detergent (Triton X-100), and fractionated them by density gradient flotation. When subjected to this procedure, the residual lipid associated with the DRM fraction causes it to appear at the top of the gradient, while detergent-soluble or aggregated proteins exhibit higher apparent density and are found at the bottom of the gradient (Bagnat et al. 2000; Malinska et al. 2003, 2004). Previous studies using this assay have identified the plasma membrane H+-ATPase Pma1 and the glycosylphosphatidylinositol (GPI)-anchored protein Gas1 as markers for the DRM fraction (Bagnat et al. 2000; Malinska et al. 2003). Markers for the detergent-soluble fraction of the plasma membrane have proven more controversial: the hexose transporter Hxt1 has been reported to primarily localize to the detergent-soluble fraction by Tanner and colleagues (Malinska et al. 2003, 2004), using anti-Hxt1 antisera for detection, but Hxt1-GFP has instead been found in the detergent-resistant fraction by Lauwers and André (2006) using anti-GFP antisera for detection. These studies also use different strain backgrounds, which may explain the discrepancy. We find that Hxt1 is primarily (~90%) in the detergent-soluble fraction (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>d), in good agreement with the results reported by Tanner and colleagues (Malinska et al. d), in good agreement with the results reported by Tanner and colleagues (Malinska et al. 2003, 2004).', 'To detect the inabundant Pdr10 in flotation gradients, we found that it was necessary to load substantially more material than has typically been reported. Unfortunately, the greater amounts loaded resulted in loss of resolution in the small Optiprep (iodixanol) density gradients pioneered by Simons and colleagues (Bagnat et al. 2000). We therefore developed a modified procedure using a larger gradient with a consequently longer centrifugation (described in detail under Materials and Methods). The DRM marker Gas1 exhibits essentially identical behavior in both gradients, as does the chitin synthase Chs3 (data not shown). However, while more than 90% of Pma1 was found in the DRM fraction in the smaller gradients, a substantial amount of Pma1 was instead associated with the soluble fraction in these expanded gradients, with only 25%–50% of Pma1 remaining in the DRM fraction (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>d). We have not attempted to ascertain whether this reflects slow solubilization or aggregation of Pma1 in the longer ultracentrifugation procedure we employ, because Gas1 and Hxt1 serve as robust markers for the detergent-resistant and detergent-soluble fractions in this gradient.d). We have not attempted to ascertain whether this reflects slow solubilization or aggregation of Pma1 in the longer ultracentrifugation procedure we employ, because Gas1 and Hxt1 serve as robust markers for the detergent-resistant and detergent-soluble fractions in this gradient.', 'Using this procedure, we were able to detect Pdr10-mh with antisera directed against the c-myc epitope (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>d). Tellingly, Pdr10-mh displayed a distribution quite similar to that of Pma1, with substantial Pdr10-mh residing in the DRM fraction. The presence of both detergent-soluble and detergent-resistant Pdr10 is reminiscent of the presence of immobile and mobile populations of Pdr10-GFP as seen by fluorescence microscopy, but the nature of the DRM experiment precludes assignment of the in vitro populations to in vivo populations (Lichtenberg et al. d). Tellingly, Pdr10-mh displayed a distribution quite similar to that of Pma1, with substantial Pdr10-mh residing in the DRM fraction. The presence of both detergent-soluble and detergent-resistant Pdr10 is reminiscent of the presence of immobile and mobile populations of Pdr10-GFP as seen by fluorescence microscopy, but the nature of the DRM experiment precludes assignment of the in vitro populations to in vivo populations (Lichtenberg et al. 2005).', 'When membranes are subjected to biochemical fractionation, a readily detectable amount of Pdr10 is associated with the DRM fraction (Fig.\xa0<xref rid="232_2009_9173_Fig1_HTML" ref-type="fig">1</xref>d). Hence, we also examined the distribution of Pdr12 in this assay. Strikingly, we found that approximately 60% of the total pool of Pdr12 was associated with the DRM fraction in membranes from d). Hence, we also examined the distribution of Pdr12 in this assay. Strikingly, we found that approximately 60% of the total pool of Pdr12 was associated with the DRM fraction in membranes from pdr10∆ cells, whereas in the membranes from wild-type cells approximately one-third of the total Pdr12 was present in the DRM fraction and more than 50% was present in the detergent-soluble fraction (Fig.\xa0<xref rid="232_2009_9173_Fig11_HTML" ref-type="fig">11</xref> and Table\xa0 and Table\xa03). The Pdr10-dependent shift observed for Pdr12 was in contrast to Chs3 and Gas1 (Bagnat et al. 2000; Nuoffer et al. 1991), whose distributions were unaltered by loss of Pdr10 (Fig.\xa0<xref rid="232_2009_9173_Fig11_HTML" ref-type="fig">11</xref> and Table\xa0 and Table\xa03). Association of Lem3 and Pdr5 with the DRM fraction was slightly higher in pdr10∆ cells than in the wild type (Table\xa03), but this effect was too small to be statistically significant. Thus, our data indicate a novel role for Pdr10 as a negative regulator of the partitioning of Pdr12 into the DRM fraction; that is, Pdr10 must regulate the local environment of Pdr12 in some manner that alters its solubilization properties in the presence of detergent.Fig.\xa011pdr10∆ cells accumulate Pdr12 in the detergent-resistant membrane fraction. a Total membranes from log phase cultures of strains W303-1A or NRY904 (wildtype) and NRY243 or NRY908 (pdr10∆) were examined by cold detergent extraction, Optiprep density gradient flotation, and immunoblotting for the indicated proteins. Strains NRY904 and NRY908 contain Lem3 C-terminally tagged with a tandem myc-His6 (mh) tag. In vivo function of the resulting Lem3-mh protein was confirmed by Calcofluor sensitivity. b Immunoblotting data were quantitated, and the percentage of total Pdr12 (left) and Chs3 (right) associated with detergent-resistant and detergent-soluble fractions is presented as the mean value for four independent experiments, with error bars representing the standard deviation. Detergent-resistant and detergent-soluble membranes were defined as fractions 1–3 and 7–9, respectively, in the Optiprep density gradientsTable\xa03Association of membrane proteins with the detergent-resistant membrane fractionaProteinWild typepdr10∆% DRM% Soluble% DRM% SolublePdr1233\xa0±\xa01154\xa0±\xa01061\xa0±\xa0628\xa0±\xa011Chs317\xa0±\xa0962\xa0±\xa0619\xa0±\xa0956\xa0±\xa010Lem3-mh19\xa0±\xa0575\xa0±\xa0724\xa0±\xa0472\xa0±\xa01Gas147\xa0±\xa0537\xa0±\xa0145\xa0±\xa0236\xa0±\xa02Pdr557\xa0±\xa01123\xa0±\xa01069\xa0±\xa0914\xa0±\xa08Note: Membranes were prepared and analyzed by cold Triton X-100 extraction, Optiprep density gradient flotation, and quantitative immunoblotting as described under Materials and Methods. For Pdr12 and Chs3, results are based on four independent experiments. All other proteins were examined at least twice. Strains were W303-1A or NRY904 (wild type with or without chromosomally mh-tagged Lem3) and NRY243 or NRY908 (pdr10∆ with or without chromosomally mh-tagged Lem3)'], '232_2009_9173_Fig2_HTML': ['Among the ABC transporters encoded in the S. cerevisiae genome, Pdr10 exhibits the closest homology to Pdr5 (67% identity), Pdr15 (68% identity), and Snq2 (40% identity). All three of the latter are capable of mediating the export of various drugs, antimetabolites, membrane perturbing agents, and other noxious compounds (Balzi et al. 1994; Bissinger and Kuchler 1994; Decottignies et al. 1995; Schüller et al. 2007; Servos et al. 1993; Wolfger et al. 2004). Therefore, we examined whether deletion of the PDR10 gene would confer enhanced sensitivity to a spectrum of cytotoxic compounds. We also examined whether loss of Pdr10 would further enhance (or otherwise alter) the drug sensitivity of a pdr5∆ mutant. We found that, in contrast to pdr5∆ cells, pdr10∆ cells were not abnormally sensitive to ketoconazole, rhodamine 6G, or cycloheximide (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>a), nor did a a), nor did a pdr5∆ pdr10∆ double mutant differ in its sensitivity from a pdr5∆ single mutant (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>a). The absence of Pdr10 similarly had no effect on the toxicity of itraconazole, oligomycin, amphotericin B, or Crystal Violet, with or without Pdr5 (data not shown). Hence, Pdr10 does not appear to contribute significantly to the capacity of the cell to export such growth-inhibitory compounds.a). The absence of Pdr10 similarly had no effect on the toxicity of itraconazole, oligomycin, amphotericin B, or Crystal Violet, with or without Pdr5 (data not shown). Hence, Pdr10 does not appear to contribute significantly to the capacity of the cell to export such growth-inhibitory compounds.Fig.\xa02Pdr10 is required for normal response to Calcofluor and sorbate. a Spot assays were performed using serial dilutions prepared from overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY201 (pdr5∆), and NRY230 (pdr5∆ pdr10∆). All plates were grown for 2\xa0days at 30°C unless otherwise noted. YPD plates contained either 2\xa0μM ketoconazole (keto), 20\xa0μM rhodamine 6G (Rh. 6G), or 100\xa0ng/ml cycloheximide (CHX). b Halo assays were performed on YPD plates with various detergents. Aliquots from overnight cultures were diluted into YPD top agar as described under Materials and Methods. Top row, strains W303-1A, NRY243, and NRY601 (pdr10∆ chs3∆) were assayed with dodecyl maltoside (DDM). Middle row, the same strains were assayed with dodecyl phosphocholine (DPC). Bottom row, strains YPH499 (wild type) and YHW10\xa0N (pdr10∆) were assayed with DDM. c Spot assays were performed as in A using strains W303-1A, NRY243, and NRY602 (chs3∆), NRY601 and NRY608 (chs6∆), and NRY610 (pdr10∆ chs6∆) on YPD plates (top) or YPD plates containing 6\xa0μg/ml Calcofluor White (CW; middle) or 100\xa0μg/ml Congo Red (CR; bottom). d Similar assays were performed using strains W303-1A (wild type), NRY243 (pdr10∆), NRY301 (pdr12∆), and NRY366 (pdr10∆ pdr12∆) on YPD plates buffered to pH 3.5 with 50\xa0mM sodium succinate with or without 4\xa0mM potassium sorbate', 'We also challenged pdr10∆ cells with a number of detergents. As judged by a halo assay, a pdr10∆ mutant strain in the W303 genetic background reproducibly exhibited modestly elevated resistance to detergents containing C12 alkyl chains, including dodecyl maltoside and dodecyl phosphocholine (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>b, upper two rows). However, a b, upper two rows). However, a pdr10∆ mutant in the YPH499 genetic background did not exhibit this effect (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>b, lowest row). We therefore used b, lowest row). We therefore used pdr10∆ cells derived from the W303 lineage for further study.', 'In addition to changes in membrane composition, changes in cell wall structure can affect the detergent sensitivity displayed by S. cerevisiae cells; indeed, cell wall alterations can affect the sensitivity of yeast cells even to a strong anionic detergent like SDS (Bickle et al. 1998; Machi et al. 2004; Santos and Snyder 2000). The yeast cell wall is composed of three types of polymers: mannoproteins, glucan, and chitin (Klis et al. 2006; Lesage and Bussey 2006). We found (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>c) that c) that pdr10∆ mutants were abnormally sensitive to two agents, Calcofluor White and Congo Red, that inhibit the growth of cells that have abnormally high amounts of chitin (Roncero et al. 1988). In contrast, otherwise isogenic wild-type cells (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>c), or cells expressing either Pdr10-GFP or Pdr10-mh from the chromosomal c), or cells expressing either Pdr10-GFP or Pdr10-mh from the chromosomal PDR10 locus (data not shown), did not display such hypersensitivity (and, thus, both Pdr10-GFP and Pdr10-mh are functional). If the sensitivity of the pdr10∆ cells is due to abnormal chitin, then we expected that sensitivity to Calcofluor White might be ameliorated by compromising chitin production. The chitin synthase responsible for producing the majority of the chitin generated in a vegetatively growing cell is Chs3, but cells can survive in its absence due to the function of the homologous chitin synthase Chs2 (Cabib 2004). Chs3 is normally delivered to the plasma membrane via the action of dedicated escort factors, including Chs6 (Valdivia et al. 2002; Valdivia and Schekman 2003). Consistent with the conclusion that the Calcofluor White sensitivity of pdr10∆ cells arises from elevated cell wall chitin, we found that pdr10∆ chs3∆ and pdr10∆ chs6∆ double mutants were not hypersensitive to Calcofluor White (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>c). Likewise, in the W303 strain background, a c). Likewise, in the W303 strain background, a pdr10∆chs3∆ double mutant was not detergent resistant (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>b, upper rows). These findings indicate that both the Calcofluor White sensitivity and the detergent resistance displayed by b, upper rows). These findings indicate that both the Calcofluor White sensitivity and the detergent resistance displayed by pdr10∆ cells are largely due to enhanced chitin production by the integral membrane protein Chs3 in the absence of this ABC transporter.', 'Although Pdr10 shares its highest degree of homology with Pdr5 and Pdr15, it is also closely related to other ABC transporters in the S. cerevisiae genome, including Pdr12 (36% identity to Pdr10). Pdr12 is located in the plasma membrane and is responsible for the export of weak organic acids (Piper et al. 1998). Consequently, pdr12∆ mutants are hypersensitive to the growth inhibitory action of weak organic acids used as food preservatives (e.g., sorbate or benzoate) (Piper et al. 1998). We found that pdr10∆ cells exhibited weak resistance to sorbate (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>d); however, this behavior required the function of Pdr12, because the modest sorbate resistance observed was eliminated in a d); however, this behavior required the function of Pdr12, because the modest sorbate resistance observed was eliminated in a pdr10∆ pdr12∆ double mutant (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>d). Thus, here, too, the absence of Pdr10 somehow promoted the function of a sister integral membrane protein, Pdr12.d). Thus, here, too, the absence of Pdr10 somehow promoted the function of a sister integral membrane protein, Pdr12.', 'Cells lacking Pdr10 also exhibit a modest increase in their resistance to sorbate that requires the sorbate transporter, Pdr12 (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>d). We were interested in whether this was also a phenotype of d). We were interested in whether this was also a phenotype of dnf1∆ dnf2∆ cells or lem3∆ cells. Interestingly, we found not only that lem3∆ cells were not sorbate resistant, but that a lem3∆ mutation eliminated the modest sorbate resistance conferred by the pdr10∆ mutation; no such epistasis was found with the dnf1∆ dnf2∆ double mutant (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>c and data not shown). Thus, the apparent increase in Pdr12 action found in cells lacking Pdr10 does not mimic a defect found in c and data not shown). Thus, the apparent increase in Pdr12 action found in cells lacking Pdr10 does not mimic a defect found in dnf1∆ dnf2∆ or lem3∆ cells but, rather, requires the presence of functional Lem3.', 'Cells lacking Pdr10 exhibit increased sensitivity to the chitin-directed agent, Calcofluor White (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>c), and that is not further enhanced by the absence of Dnf1 and Dnf2 (Fig.\xa0c), and that is not further enhanced by the absence of Dnf1 and Dnf2 (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>a). Strikingly, as observed for the sorbate resistance of a). Strikingly, as observed for the sorbate resistance of pdr10∆ cells, we found that loss of Pdr12 eliminated the Calcofluor White sensitivity of pdr10∆ (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>d). We also found that the introduction of a d). We also found that the introduction of a pdr12∆ mutation into pdr10∆ cells substantially reduced the accessibility of PE to TNBS labeling (Table\xa02). Collectively, these findings indicate complex interactions among Pdr10, Pdr12, and Lem3, which produce secondary effects on Chs3 endocytosis.'], '232_2009_9173_Fig3_HTML': ['Taken together, these results suggested that the function of Pdr10 contributes to the normal expression, and/or sorting and trafficking, and/or catalytic competence of Chs3 and Pdr12. To determine whether this phenotype was unique to the loss of Pdr10, we examined the Calcofluor White and sorbate sensitivity of yeast strains lacking other members of the S. cerevisiae ABC transporter family known to reside in the plasma membrane. Although comparable sorbate resistance was observed in pdr15∆ cells and comparable Calcofluor White sensitivity was observed in cells lacking the putative, but uncharacterized, ABC transporter encoding by the YOL075c locus, the combination of Calcofluor White sensitivity and sorbate resistance was only observed in pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig3_HTML" ref-type="fig">3</xref>). Thus, Pdr10 uniquely has a role in maintaining the proper level, maturation, localization, and/or function of both Chs3 and Pdr12.). Thus, Pdr10 uniquely has a role in maintaining the proper level, maturation, localization, and/or function of both Chs3 and Pdr12.Fig.\xa03Calcofluor sensitivity and sorbate resistance define a specific pdr10∆ phenotype. Serial dilutions of overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY201 (pdr5∆), NRY212 (pdr15∆), NRY336 (pdr18∆), NRY334 (YOL075c∆), and WKK7 (ste6∆) were spotted onto YPD plates containing 6\xa0μg/ml Calcofluor White (CW; left column) or 4\xa0mM sorbate (right column)'], '232_2009_9173_Fig4_HTML': ['To distinguish among these possibilities, we first examined the effect of a pdr10∆ mutation on the function and localization of Chs3. To confirm that pdr10∆ cells do produce abnormally high levels of chitin, we examined cells by fluorescence microscopy after staining with Calcofluor White. Indeed, we found that, compared to otherwise isogenic PDR10+ control cells, pdr10∆ cells exhibited brighter fluorescence and therefore excess chitin, while cell wall chitin was almost entirely eliminated when Chs3 was absent (Fig.\xa0<xref rid="232_2009_9173_Fig4_HTML" ref-type="fig">4</xref>a). Elevated chitin production could arise from increased Chs3, more active Chs3, or aberrant localization of Chs3. As mentioned already, normal anterograde trafficking of Chs3 to the plasma membrane requires additional protein factors, including Chs6 (Valdivia and Schekman a). Elevated chitin production could arise from increased Chs3, more active Chs3, or aberrant localization of Chs3. As mentioned already, normal anterograde trafficking of Chs3 to the plasma membrane requires additional protein factors, including Chs6 (Valdivia and Schekman 2003), and we found that, like a chs3∆ mutation, a chs6∆ mutation also eliminated the Calcofluor White sensitivity of pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig2_HTML" ref-type="fig">2</xref>c). Therefore, the elevated chitin production of c). Therefore, the elevated chitin production of pdr10∆ cells does not occur because the normal pathway for transport of Chs3 to the plasma membrane is bypassed when Pdr10 is absent.Fig.\xa04Defective endocytosis of Chs3 causes excess chitin deposition in pdr10∆ cells. a Cells from log phase cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY602 (chs3∆), and NRY601 (pdr10∆ chs3∆) were stained for chitin and examined by fluorescence microscopy. Bar, 4\xa0μm. b Log phase cells from strains W303-1A and NRY251 (pdr10∆) carrying plasmids pRS315-CHS7 and pCRP12 were treated with 3.6\xa0μM α-factor for 35\xa0min in SCD buffered to pH 3.5 with 25\xa0mM sodium succinate and supplemented with adenine. Pheromone was then washed out at time 0, and the localization of Chs3-GFP was monitored by fluorescence microscopy as described under Materials and Methods. Punctate Chs3-GFP staining of the plasma membrane in pdr10∆ cells at late time points is indicated by arrow heads. Bar, 3\xa0μm. c Strains W303-1A, NRY243, and NRY407 (cho1∆) were grown into log phase, stained for chitin, and examined by fluorescence microscopy. Bar, 5\xa0μm. d Serial dilutions of overnight cultures of strains W303-1A, NRY243, and NRY407 were spotted onto YPD plates containing Calcofluor', 'In vegetatively growing cells, Chs3 cycles between the trans-Golgi network and endosomal compartments during most of the cell cycle (Valdivia et al. 2002) but displays significant accumulation at the plasma membrane primarily at the small-bud stage (Chuang and Schekman 1996). The intracellular pool of Chs3 can also be redistributed to the plasma membrane after treatment of cells with mating pheromone, an effect we exploited to trap Chs3 at the plasma membrane in wild-type and pdr10∆ cells. Upon washout of pheromone, cells re-entered the cell cycle and Chs3 moved to the bud neck, as expected (Fig.\xa0<xref rid="232_2009_9173_Fig4_HTML" ref-type="fig">4</xref>b). At later time points, Chs3 was efficiently internalized in wild-type cells, whereas in b). At later time points, Chs3 was efficiently internalized in wild-type cells, whereas in pdr10∆ cells a small amount of Chs3-GFP persisted in discrete patches at the plasma membrane, even though the majority was intracellular. Thus, in pdr10∆ cells, endocytosis of Chs3 was somewhat less efficient than in wild-type cells (although this block was by no means complete). The prolonged residence time of Chs3 at the plasma membrane in pdr10∆ cells could well explain the increased amount of chitin produced in these cells.', 'If the partial defect in endocytosis of Chs3 in a pdr10∆ mutant is responsible for its elevated chitin production and thus its increased sensitivity to Calcofluor White, then other mutations or conditions that influence the efficiency of Chs3 endocytosis should elicit the same phenotype. Interestingly, we found that a cho1∆ strain, which lacks phosphatidylserine synthase (Atkinson et al. 1980a, b; Kiyono et al. 1987; Kohlwein et al. 1988; Letts et al. 1983), displayed even greater chitin deposition (Fig.\xa0<xref rid="232_2009_9173_Fig4_HTML" ref-type="fig">4</xref>c) and an even higher sensitivity to Calcofluor White than the c) and an even higher sensitivity to Calcofluor White than the pdr10∆ strain (Fig.\xa0<xref rid="232_2009_9173_Fig4_HTML" ref-type="fig">4</xref>d). Quantitation of chitin deposition indicated that d). Quantitation of chitin deposition indicated that pdr10∆ cells had approximately 1.9-fold more chitin in their cell walls than wild-type cells, while cho1∆ cells had approximately 3.8-fold more chitin. We have not yet examined directly the localization of Chs3-GFP in cho1∆ cells; however, these data suggested that membrane composition might be important in Chs3 endocytosis and thus that the absence of Pdr10 might influence plasma membrane lipid distribution. We therefore examined sterol localization in wildtype and pdr10∆ cells by staining with filipin (Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>a), finding little difference between those two strains. Simons and colleagues have reported that treatment with mating pheromone polarizes sterols to the site of polarized growth (the shmoo tip) (Bagnat and Simons a), finding little difference between those two strains. Simons and colleagues have reported that treatment with mating pheromone polarizes sterols to the site of polarized growth (the shmoo tip) (Bagnat and Simons 2002), and we find that this process proceeds normally in pdr10∆ cells as well (Fig.\xa0<xref rid="232_2009_9173_Fig6_HTML" ref-type="fig">6</xref>a).a).Fig.\xa06Increased phosphatidylethanolamine is accessible to TNBS labeling in pdr10∆ cells. a Strains W303-1A and NRY243 were grown to log phase, incubated with or without α-factor, and then stained with filipin to visualize sterols by fluorescence microscopy. Bar, 5\xa0μm. b Exposure of endogenous aminophospholipids to TNBS was assayed with cells grown overnight in the presence of 32P-phosphate as described under Materials and Methods. Strains used were W303-1A (wild type), NRY243 (pdr10∆), NRY549 (lem3∆), NRY551 (pdr10∆ lem3∆), NRY427 (dnf1∆ dnf2∆), and NRY558 (pdr10∆ dnf1∆ dnf2∆)'], '232_2009_9173_Fig5_HTML': ['We were interested in determining whether this modest endocytic defect of pdr10∆ cells was specific for Chs3 or was more general. We therefore examined endocytic uptake of two fluorescent dyes, Lucifer Yellow and FM 4-64 (Fig.\xa0<xref rid="232_2009_9173_Fig5_HTML" ref-type="fig">5</xref>a). These dyes serve as markers for fluid phase endocytosis and bulk membrane internalization, respectively, with both being internalized to the vacuole. Uptake of both of these markers was normal in a). These dyes serve as markers for fluid phase endocytosis and bulk membrane internalization, respectively, with both being internalized to the vacuole. Uptake of both of these markers was normal in pdr10∆ cells, demonstrating that the partial block in Chs3 endocytosis is not a general effect on all endocytic processes. We also examined actin morphology in these cells (Fig.\xa0<xref rid="232_2009_9173_Fig5_HTML" ref-type="fig">5</xref>b), because an intact actin cytoskeleton is a prerequisite for endocytosis in yeast (Ayscough et al. b), because an intact actin cytoskeleton is a prerequisite for endocytosis in yeast (Ayscough et al. 1997; Kubler and Riezman 1993). Deletion of PDR10 had no apparent effect on actin localization or on its polarization in small-budded cells (Fig.\xa0<xref rid="232_2009_9173_Fig5_HTML" ref-type="fig">5</xref>b).b).Fig.\xa05Pdr10 is not required for bulk endocytosis or actin morphology. a Strains W303-1A (wild type) and NRY243 (pdr10∆) were grown to log phase, incubated with Lucifer Yellow or FM 4-64 as a marker for endocytosis, and examined by fluorescence microscopy. b Strains W303-1A and NRY243 were grown to log phase, permeabilized, and stained with rhodamine-phalloidin to visualize actin by fluorescence microscopy. All bars, 5\xa0μm'], '232_2009_9173_Fig7_HTML': ['We were interested in whether this might reflect a direct role for Pdr10 in the synthesis, sorting, or activity of the inward-directed translocases. Therefore, we examined the level of expression and the localization of Dnf1, Dnf2, and Lem3 in pdr10∆ cells by constructing functional chromosomal GFP fusions (tests for functionality described under Materials and Methods). Dnf1-GFP, Dnf2-GFP, and Lem3-GFP were all readily detectable by fluorescence microscopy, present at equivalent levels, and exhibited the expected punctate pattern in the plasma membrane in both wild-type and pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig7_HTML" ref-type="fig">7</xref>). There were no discernible differences in the localization of any of these three proteins between wild-type and ). There were no discernible differences in the localization of any of these three proteins between wild-type and pdr10∆ cells. Likewise, a functional Pdr5-GFP chimera expressed from the chromosomal PDR5 locus was localized to the plasma membrane indistinguishably in wild-type and pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig7_HTML" ref-type="fig">7</xref>). Putative inward- and outward-directed translocases were thus present at normal levels and were properly localized to the plasma membrane in ). Putative inward- and outward-directed translocases were thus present at normal levels and were properly localized to the plasma membrane in pdr10∆ cells.Fig.\xa07Pdr10 is not required for normal localization of putative PE translocases. Strains NRY914 (wild type, Lem3-GFP), NRY918 (pdr10∆, Lem3-GFP), NRY921 (wild type, Dnf1-GFP), NRY932 (pdr10∆, Dnf1-GFP), NRY923 (wild type, Dnf2-GFP), NRY929 (pdr10∆, Dnf2-GFP), NRY906 (wild type, Pdr5-GFP), and NRY916 (pdr10∆, Pdr5-GFP) were grown to log phase, and the indicated proteins were visualized by fluorescence microscopy. Collages of representative images are shown. Bar, 6\xa0μm'], '232_2009_9173_Fig8_HTML': ['To assess whether inward-directed translocase activity was still present in pdr10∆ cells, we examined the uptake of an exogenously supplied fluorescent PE surrogate, NBD-PE (Kean et al. 1993), in wild-type and pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig8_HTML" ref-type="fig">8</xref>). It has been reported previously that it is possible to observe specific, translocase-dependent internalization of NBD-labeled lipids at low temperature, which prevented background internalization due to endocytosis (Grant et al. ). It has been reported previously that it is possible to observe specific, translocase-dependent internalization of NBD-labeled lipids at low temperature, which prevented background internalization due to endocytosis (Grant et al. 2001). However, in our hands, both wild-type and pdr10∆ cells were still able to internalize modest amounts of NBD-PE to discrete intracellular puncta at low temperature, indicating either that endocytosis was not completely blocked in our hands or that a low level of membrane leakage was occurring (data not shown). We therefore examined NBD-PE uptake in the presence of latrunculin A, which disrupts the actin cytoskeleton and hence blocks endocytosis (Ayscough et al. 1997; Pomorski et al. 2003). We found that pdr10∆ cells exhibited no defect compared to wild-type cells. dnf1∆ dnf2∆ cells also exhibited no defect in NBD-PE internalization under these conditions (Fig.\xa0<xref rid="232_2009_9173_Fig8_HTML" ref-type="fig">8</xref>), perhaps because the homologous P-type ATPase Drs2 can relocalize to the plasma membrane upon latrunculin treatment (Liu et al. ), perhaps because the homologous P-type ATPase Drs2 can relocalize to the plasma membrane upon latrunculin treatment (Liu et al. 2007). Strikingly, however, lem3∆ cells were unable to internalize NBD-PE under these conditions, with or without the presence of Pdr10. These data demonstrate that Lem3 must have functions other than its role in the biogenesis of Dnf1 and Dnf2, perhaps in forward transport of Drs2 to the plasma membrane. Neither pdr10∆ nor lem3∆ had any effect on uptake of a fluorescent ceramide surrogate, NBD-Cer (Fig.\xa0<xref rid="232_2009_9173_Fig8_HTML" ref-type="fig">8</xref>). Taken together, these results demonstrate the presence of normal amounts of Dnf1, Dnf2, and Lem3 in ). Taken together, these results demonstrate the presence of normal amounts of Dnf1, Dnf2, and Lem3 in pdr10∆ plasma membranes and demonstrate that pdr10∆ cells are still capable of PE internalization. It is known that mutations in a number of genes implicated in vesicular trafficking produce altered plasma membrane asymmetry as a secondary effect (Chen et al. 2006; Parsons et al. 2006), so it seems likely that increased accessibility of PE to TNBS labeling in pdr10∆ cells is such an indirect consequence.Fig.\xa08pdr10∆ cells internalize NBD-PE in a Lem3-dependent manner. Strains W303-1A (wild type), NRY243 (pdr10∆), NRY549 (lem3∆), NRY551 (pdr10∆ lem3∆), NRY427 (dnf1∆ dnf2∆), and NRY558 (pdr10∆ dnf1∆ dnf2∆) were grown to log phase, treated with latrunculin A to block endocytosis, and incubated with the fluorescent reporter lipid NBD-PE or NBD-Cer as described under Materials and Methods. Cells were examined by fluorescence microscopy and differential interference contrast microscopy (DIC). Bar, 6\xa0μm'], '232_2009_9173_Fig9_HTML': ['Dnf1 and Dnf2 have been implicated in endocytosis at low temperatures (Furuta et al. 2007; Pomorski et al. 2003), in keeping with a broader role for members of this transporter family in vesicular trafficking (Chen et al. 2006). Given that the PE exposure caused by a pdr10∆ mutation was quite comparable to that caused by dnf1∆ dnf2∆ double deletion, the mild defect in endocytic trafficking we observed for Chs3 in pdr10∆ cells might also be associated with dnf1∆ dnf2∆ or lem3∆ cells. Indeed, we found that cells lacking both Dnf1 and Dnf2 were sensitive to Calcofluor White (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>a), exhibited increased chitin in their cell wall (Fig.\xa0a), exhibited increased chitin in their cell wall (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>b), and were equivalent to b), and were equivalent to pdr10∆ cells in both respects. Moreover, a pdr10∆ dnf1∆ dnf2∆ triple mutant strain did not display any increase in Calcofluor sensitivity or chitin deposition compared to the otherwise isogenic pdr10∆ single mutant or the dnf1∆ dnf2∆ double mutant (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>a, b), with all mutant strains exhibiting a 1.8- to 1.9-fold increase in chitin deposition. This lack of synergy was not due to any limitation in the capacity of these assays to detect such potentiation, because a, b), with all mutant strains exhibiting a 1.8- to 1.9-fold increase in chitin deposition. This lack of synergy was not due to any limitation in the capacity of these assays to detect such potentiation, because cho1∆ cells exhibited much higher Calcofluor White sensitivity and chitin deposition than pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig4_HTML" ref-type="fig">4</xref>). Moreover, ). Moreover, lem3∆ cells were also more sensitive to Calcofluor White than pdr10∆ or dnf1∆ dnf2∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig9_HTML" ref-type="fig">9</xref>a), which is reminiscent of the more profound effect of this mutation on NBD-PE uptake (Fig.\xa0a), which is reminiscent of the more profound effect of this mutation on NBD-PE uptake (Fig.\xa0<xref rid="232_2009_9173_Fig8_HTML" ref-type="fig">8</xref>). Taken together, these results show that ). Taken together, these results show that pdr10∆ cells exhibit a modest defect in endocytosis of Chs3, that this mimics a phenotype of dnf1∆ dnf2∆ cells, and that Pdr10 is not required for expression or localization of Dnf1, Dnf2, and Lem3. We have not been able to elucidate the molecular basis for this defect in Chs3 trafficking.Fig.\xa09Loss of Dnf1 and Dnf2 function in pdr10∆ cells is associated with their increased chitin deposition. a Serial dilutions from overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY427 (dnf1∆ dnf2∆), NRY558 (pdr10∆ dnf1∆ dnf2∆), NRY549 (lem3∆), and NRY551 (pdr10∆ lem3∆) were spotted onto YPD plates at increasing concentrations of Calcofluor White (CW). b Log phase cultures of strains W303-1A, NRY243, NRY427, and NRY558 were stained for chitin and examined by fluorescence microscopy. Bar, 5\xa0μm. c Serial dilutions of strains W303-1A, NRY243, NRY549, and NRY551 were spotted onto YPD plates buffered to pH 3.5 with sodium succinate and containing potassium sorbate. d Serial dilutions of strains W303-1A, NRY243, NRY301 (pdr12∆), and NRY366 (pdr10∆ pdr12∆) were spotted onto YPD plates containing Calcofluor White'], '232_2009_9173_Fig10_HTML': ['To see whether a clearer molecular function could be assigned to Pdr10, we examined whether loss of Pdr10 affects the level and/or localization of Pdr12. When equivalent amounts of plasma membrane protein were resolved by SDS-PAGE and analyzed by immunoblotting, we found that, compared to otherwise isogenic control cells, pdr10∆ mutant cells exhibited a reproducible, approximately twofold increase in the amount of Pdr12 relative to wild-type cells, whereas there was no significant difference between wild-type and pdr10∆ cells in the content of numerous other membrane proteins in the same samples (Fig.\xa0<xref rid="232_2009_9173_Fig10_HTML" ref-type="fig">10</xref>a). Visualization of functional Pdr12-GFP expressed from the a). Visualization of functional Pdr12-GFP expressed from the PDR12 locus in wild-type and pdr10∆ cells confirmed that Pdr12-GFP expression was elevated in the pdr10∆ mutant cells (Fig.\xa0<xref rid="232_2009_9173_Fig10_HTML" ref-type="fig">10</xref>b), but we could distinguish no obvious difference in localization. In both wild-type and b), but we could distinguish no obvious difference in localization. In both wild-type and pdr10∆ cells, the Pdr12-GFP was observed throughout the plasma membrane in a readily observable punctate pattern. Approximately 30% of cells in both strains also exhibited some intracellular punctate staining (data not shown), with most such cells also exhibiting plasma membrane Pdr12-GFP. The level of PDR12 mRNA in pdr10∆ cells was not different from that in PDR10+ control cells, as judged by global transcriptome analysis using hybridization to DNA microarrays (S. Shtivelman, N. C. Rockwell, and J. Thorner, unpublished data). Thus, the increase in Pdr12 found in pdr10∆ cells could not be attributed to any change in transcription, and therefore the elevation of plasma membrane Pdr12 in pdr10∆ cells most likely results from a reduced rate of turnover rather than a faster rate of synthesis. The latter might occur if compartmentation of Pdr12 was altered in pdr10∆ cells in a manner that was not revealed at the level of fluorescence microscopy.Fig.\xa010pdr10∆ cells accumulate excess Pdr12 in the plasma membrane. a Total membranes were prepared from log phase cultures of strains W303-1A (wild type) and NRY243 (pdr10∆). The indicated proteins were detected by immunoblotting, with equal protein loaded for each strain. b Strains NRY915 (wild type) and NRY925 (pdr10∆) were grown to log phase in YPD, and Pdr12-GFP was examined by fluorescence microscopy. A collage of representative images is shown. Pdr12-GFP function was confirmed by sorbate sensitivity. Bar, 6\xa0μm', 'One could also envision the redistribution of Pdr12 into the DRM fraction as arising from an endocytic defect, such that an intracellular pool of Pdr12 would effectively relocalize to the plasma membrane in the absence of Pdr10. The proposed link between endocytosis and exit from the DRM fraction (Lauwers and Andre 2006; Lauwers et al. 2007) would imply that such an intracellular pool would become available for partitioning into the DRM fraction upon such relocalization. In this model, the plasma membrane Pdr12 would be in equilibrium between a microenvironment that gives rise to a DRM fraction and a microenvironment that does not, while intracellular Pdr12 would instead be entirely associated with the detergent-soluble fraction. The absence of Pdr12 endocytosis in pdr10∆ cells would then result in the total Pdr12 population equilibrating between these two microenvironments, rather than just a plasma membrane population doing so. Were this to be the case, the association of approximately 60% of total Pdr12 with the DRM fraction in pdr10∆ cells would reflect the intrinsic affinity of Pdr12 for this fraction, while no intracellular Pdr12 would be found in the DRM fraction. The amount of Pdr12 in the DRM fraction would thus vary between 0% and 60%. The association of approximately 30% of total Pdr12 with the DRM fraction in wild-type cells would thus imply the presence of approximately 50% of total Pdr12 in an intracellular pool. However, we demonstrated that a functional Pdr12-GFP chimera is predominantly localized to the plasma membrane (Fig.\xa0<xref rid="232_2009_9173_Fig10_HTML" ref-type="fig">10</xref>b). Only\xa0≤\xa030% of cells exhibited any detectable intracellular Pdr12-GFP, and the vast majority of such cells also displayed plasma membrane Pdr12-GFP. Moreover, this localization pattern was equivalent in both wild-type and b). Only\xa0≤\xa030% of cells exhibited any detectable intracellular Pdr12-GFP, and the vast majority of such cells also displayed plasma membrane Pdr12-GFP. Moreover, this localization pattern was equivalent in both wild-type and pdr10∆ cells. Therefore, the intracellular population of Pdr12 is likely to be much less than 50% of the total, and there is no evidence that pdr10∆ cells lack this population. These observations are inconsistent with the idea that Pdr12 accumulation in the DRM fraction of pdr10∆ membranes arises from relocalization of Pdr12 to the plasma membrane. We favor, therefore, the idea that the composition of plasma membrane in cells lacking Pdr10 is itself different from that of the wild-type plasma membrane in a way that results in overincorporation of Pdr12 into the DRM fraction.'], '232_2009_9173_Fig12_HTML': ['We were also interested in whether Pdr10 function was redundant with that of its two closest homologues, Pdr5 and Pdr15. As one means to examine possible redundancy of this sort among Pdr10, Pdr5, and Pdr15, we examined their genetic interactions using Calcofluor White sensitivity and sorbate resistance. A pdr5∆ mutation eliminated both the Calcofluor White sensitivity and the mild sorbate resistance of pdr10∆ cells (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>a). a). pdr5∆ pdr10∆ cells also exhibited significantly lower TNBS labeling of PE (Table\xa02) and normal levels of Pdr12 (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>c). In contrast, a c). In contrast, a pdr15∆ mutation had no discernible effect on pdr10∆ phenotypes (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>a).a).Fig.\xa012Pdr5 and phosphosphingolipids are required for pdr10∆ phenotypes. a Serial dilutions of overnight cultures of strains W303-1A (wild type), NRY243 (pdr10∆), NRY201 (pdr5∆), NRY230 (pdr5∆ pdr10∆), NRY212 (pdr15∆), NRY236 (pdr10∆ pdr15∆), NRY410 (ipt1∆), NRY534 (pdr10∆ ipt1∆), NRY409 (sur1∆), and NRY526 (pdr10∆ sur1∆) were spotted onto YPD plates containing Calcofluor White (CW; left column) or sorbate buffered with sodium succinate (right column). b Strains W303-1A, NRY243, NRY409, and NRY526 were assayed for PE accessibility to TNBS labeling after overnight labeling with 32P-phosphate as described under Materials and Methods. c Total Pdr12 and Chs3 levels were examined by immunoblotting equal amounts of total membrane proteins isolated from log phase cultures of strains W303-1A, NRY230, and NRY526. d Strains W303-1A, NRY243, NRY409, NRY526, NRY410, and NRY534 were grown to log phase, stained for chitin, and examined by fluorescence microscopy. Bar, 5\xa0μm', 'Both sur1∆ and ipt1∆ mutations were epistatic to pdr10∆ for both Calcofluor White sensitivity and sorbate resistant (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>a). Thus, deletion of genes required for synthesis of mature sphingolipid headgroups ablated the phenotypes of a). Thus, deletion of genes required for synthesis of mature sphingolipid headgroups ablated the phenotypes of pdr10∆ cells, even though pdr10∆ cells did not exhibit significant changes either in the level of total phosphosphingolipid (Table\xa02) or in the composition of phosphosphingolipids (Table\xa04). However, sur1∆ and ipt1∆ cells exhibited significant changes in sphingolipid composition whether Pdr10 was present or absent, as expected (Table\xa04). pdr10∆ sur1∆ cells did not display abnormally high TNBS labeling of PE (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>b and Table\xa0b and Table\xa02) and contained normal levels of Pdr12 (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>c). Furthermore, both c). Furthermore, both sur1∆ and ipt1∆ eliminated the elevated chitin deposition caused by a pdr10∆ mutation (Fig.\xa0<xref rid="232_2009_9173_Fig12_HTML" ref-type="fig">12</xref>d). Taken together, these results demonstrate that the aberrant accumulation of Pdr12 and defective endocytosis of Chs3 that occur in d). Taken together, these results demonstrate that the aberrant accumulation of Pdr12 and defective endocytosis of Chs3 that occur in pdr10∆ cells require the presence of mannosylphosphosphingolipids.Table\xa04Composition of mature sphingolipidsStrainIPCMIPCM(IP)2CWild type35\xa0±\xa0417\xa0±\xa0247\xa0±\xa04pdr10∆41\xa0±\xa0218\xa0±\xa0241\xa0±\xa01sur1∆61\xa0±\xa0313\xa0±\xa0126\xa0±\xa03ipt1∆46\xa0±\xa0348\xa0±\xa027\xa0±\xa01Note: Strains were W303-1A (wild type), NRY243 (pdr10∆), NRY409 (sur1∆), and NRY410 (ipt1∆). Sphingolipids were labeled with 32P-phosphate, extracted, and analyzed as described under Materials and Methods']}
|
Saccharomyces cerevisiae ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in
|
[
"Pdr10",
"ABC transporter",
"Sphingolipid",
"Lipid raft",
"Detergent-resistant membrane",
"Lateral segregation",
"Pdr12",
"Lem3",
"Dnf1",
"Dnf2"
] |
J Membr Biol
|
1242716400
|
Volatile organic compounds have been reported to serve some important roles in plant communication with other organisms, but little is known about the biological functions of most of these substances. To gain insight into this problem, we have compared differences in floral and vegetative volatiles between two closely related plant species with different life histories. The self-pollinating annual, Arabidopsis thaliana, and its relative, the outcrossing perennial, Arabidopsis lyrata, have markedly divergent life cycles and breeding systems. We show that these differences are in part reflected in the formation of distinct volatile mixtures in flowers and foliage. Volatiles emitted from flowers of a German A. lyrata ssp. petraea population are dominated by benzenoid compounds in contrast to the previously described sesquiterpene-dominated emissions of A. thaliana flowers. Flowers of A. lyrata ssp. petraea release benzenoid volatiles in a diurnal rhythm with highest emission rates at midday coinciding with observed visitations of pollinating insects. Insect feeding on leaves of A. lyrata ssp. petraea causes a variable release of the volatiles methyl salicylate, C11- and C16-homoterpenes, nerolidol, plus the sesquiterpene (E)-beta-caryophyllene, which in A. thaliana is emitted exclusively from flowers. An insect-induced gene (AlCarS) with high sequence similarity to the florally expressed (E)-beta-caryophyllene synthase (AtTPS21) from A. thaliana was identified from individuals of a German A. lyrata ssp. petraea population. Recombinant AlCarS converts the sesquiterpene precursor, farnesyl diphosphate, into (E)-beta-caryophyllene with alpha-humulene and alpha-copaene as minor products indicating its close functional relationship to the A. thaliana AtTPS21. Differential regulation of these genes in flowers and foliage is consistent with the different functions of volatiles in the two Arabidopsis species.
|
[
"Animals",
"Arabidopsis",
"Arabidopsis Proteins",
"Breeding",
"Circadian Rhythm",
"DNA, Complementary",
"Flowers",
"Gas Chromatography-Mass Spectrometry",
"Gene Expression Regulation, Enzymologic",
"Gene Expression Regulation, Plant",
"Host-Parasite Interactions",
"Larva",
"Mass Spectrometry",
"Molecular Sequence Data",
"Moths",
"Odorants",
"Plant Leaves",
"Polycyclic Sesquiterpenes",
"Recombinant Proteins",
"Reverse Transcriptase Polymerase Chain Reaction",
"Sequence Analysis, DNA",
"Sesquiterpenes",
"Species Specificity",
"Volatile Organic Compounds"
] |
other
|
PMC2687517
| null |
106
|
[
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J Membr Biol. 2009 May 19; 229(1):27-52
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NO-CC CODE
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ACE is present in MHC class II pathway and its catalytic activity is required for antigen processingOne hour after macrophage phagocytosis was induced with latex beads, ACE co-localized in the compartments of MHC class II (A), early endosome (B) and late endosome/lysosome (C). Stars indicate latex beads which were pinpointed under bright field. (A-C) Representative pictures of 3 independent experiments are shown. (D) WT macrophages were transfected with constructs encoding either wild-type ACE (ACE) or catalytic domain mutated ACE (mACE). Their presentation efficiency of OVA323–339 following OVA administration was assessed by IL-2 secretion of OT-II T cells. n=6. *P<0.05.
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nihms855384f5
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bac5b45e29b02f891e9653e20dc33e356c37bdcbdebb26c7a31a9bea0e72cf6e
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
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[{'image_id': 'nihms855384f6', 'image_file_name': 'nihms855384f6.jpg', 'image_path': '../data/media_files/PMC5493495/nihms855384f6.jpg', 'caption': 'MHC II antigen presentation is altered by ACE inhibition(A) Macrophages purified from WT and ACE10 mice were pre-treated with or without lisinopril for 2 hours and then co-incubated with OVA. OVA323–339 presentation was assessed through surface CD69 upregulation (top) or IL-2 secretion (bottom) by OT-II T cells. (B) WT and ACE10 mice were treated with lisinopril for 7 days followed by s.c. immunization of OVA-CFA. After another 9 days of lisinopril treatment, the spleens and the draining lymph nodes were harvested and the OVA323–339-specific T cell responses were evaluated for IFN-γ (top) and IL-17 (bottom) production. n=6–7. *P<0.05. **P<0.01, ***P<0.001, NS: Not significant.', 'hash': '70276875d50fa41e815123cd19dcce2936701476d4c566124e011fd5a2454b4f'}, {'image_id': 'nihms855384f1', 'image_file_name': 'nihms855384f1.jpg', 'image_path': '../data/media_files/PMC5493495/nihms855384f1.jpg', 'caption': 'The effects of ACE on ovalbumin MHC class II antigen presentation in vitroThe efficiency of ACE KO, WT and ACE10 macrophages in presenting OVA329–339, when fed with no antigen, OVA protein and OVA329–339 peptide was evaluated by upregulation of CD69 (A) and secretion of IL-2 (B) by OT-II T cells. n=6. *P<0.05, **P<0.01, ***P<0.001. This figure, and all figures, show the standard error of the mean (SEM).', 'hash': '8a90ab0716583c64856b0489ef5a70aab81102c0c77781664da2bfec7c016085'}, {'image_id': 'nihms855384f2', 'image_file_name': 'nihms855384f2.jpg', 'image_path': '../data/media_files/PMC5493495/nihms855384f2.jpg', 'caption': 'The effects of ACE on macrophage surface molecules and pinocytosis(A) Expression of MHC class II molecular I-Ab and co-stimulatory factors CD80 and CD86 on the surface of macrophages from ACE KO, WT and ACE10 mice. N=5. (B) Pinocytosis of fluorescein conjugated OVA by ACE KO, WT and ACE10 macrophages was measured by flow cytometric analysis of mean fluorescence intensity (MFI). n=5. *P<0.05, **P<0.01, ***P<0.001.', 'hash': '00aff1fbac513fadea668c90d7ed88651cbf323cf3c806d4d29f928c75da94b9'}, {'image_id': 'nihms855384f5', 'image_file_name': 'nihms855384f5.jpg', 'image_path': '../data/media_files/PMC5493495/nihms855384f5.jpg', 'caption': 'ACE is present in MHC class II pathway and its catalytic activity is required for antigen processingOne hour after macrophage phagocytosis was induced with latex beads, ACE co-localized in the compartments of MHC class II (A), early endosome (B) and late endosome/lysosome (C). Stars indicate latex beads which were pinpointed under bright field. (A-C) Representative pictures of 3 independent experiments are shown. (D) WT macrophages were transfected with constructs encoding either wild-type ACE (ACE) or catalytic domain mutated ACE (mACE). Their presentation efficiency of OVA323–339 following OVA administration was assessed by IL-2 secretion of OT-II T cells. n=6. *P<0.05.', 'hash': 'bac5b45e29b02f891e9653e20dc33e356c37bdcbdebb26c7a31a9bea0e72cf6e'}, {'image_id': 'nihms855384f4', 'image_file_name': 'nihms855384f4.jpg', 'image_path': '../data/media_files/PMC5493495/nihms855384f4.jpg', 'caption': 'ACE over-expression enhances OVA presentation to CD4+ T cells in vivoWT and ACE10 mice were immunized with OVA-CFA. The spleens and draining lymph nodes (LN) were harvested 9 days later. OVA329–339-specific CD4+ T cell activation was evaluated by production of the cytokines IFN-γ (A) and IL-17A (B). n=6–7. WT and ACE 10 mice were immunized with OVA-CFA (C) or OVA-Alum (D) followed by a boost of OVA alone 14 days later. Seven days after the boost, the titers of anti-OVA IgG subtypes were evaluated by ELISA. n=5. *P<0.05. **P<0.01, ***P<0.001.', 'hash': '0f1a1c97bd669d795d694ad4aa48e2a331715ce115df12ee0f72e792e6635723'}, {'image_id': 'nihms855384f3', 'image_file_name': 'nihms855384f3.jpg', 'image_path': '../data/media_files/PMC5493495/nihms855384f3.jpg', 'caption': 'The effects of ACE on HEL MHC class II antigen presentation in vitroThe efficiency of ACE KO, WT and ACE10 macrophages in presenting HEL-associated epitopes by I-Ab was measured by the secretion of IL-2 from their corresponding hybridomas. n=6. *P<0.05, **P<0.01, ***P<0.001.', 'hash': 'c84787a4dac9742dd738010639ab08afb0355e5e937ce4c2b290f5c7cbe7e894'}]
|
{'nihms855384f1': ['To test whether ACE functions in the processing and presentation of MHC class II peptides, we first examined the effects of ACE on the presentation of OVA323–339, the epitope of ovalbumin (OVA) presented by I-Ab. We used thioglycollate-induced peritoneal macrophages as the APCs since they are activated inflammatory macrophages and express relatively abundant MHC class II molecules. Macrophages derived from ACE knock-out (KO), wild type (WT) and ACE10, a mouse line with ACE over-expressed in macrophage lineage cells, were compared due to their expression of null, normal and increased level of ACE, respectively (12, 13) (Supplementary figure 1). After a 2-hour incubation with OVA protein, macrophages were fixed with paraformaldehyde and the ability to present OVA323–339 by MHC class II was measured by surface CD69 upregulation (Supplementary figure 2) and IL-2 secretion from OT-II T cells. For both CD69 expression and secretion of IL-2, there was a consistent pattern (<xref rid="nihms855384f1" ref-type="fig">Figure 1A, B</xref>). The ACE null cells gave the lowest values. Increasing ACE concentrations (WT vs. ACE10) resulted in increasing activation of the OT-II cells. In fact, there was an over 3-fold difference in both CD69 and IL-2 expression between ACE null and ACE over-expressing cells (p<0.001). If the background levels of expression are subtracted, the difference between the response to ACE null cells vs WT and ACE10 cells was about 3-fold and 8-fold (). The ACE null cells gave the lowest values. Increasing ACE concentrations (WT vs. ACE10) resulted in increasing activation of the OT-II cells. In fact, there was an over 3-fold difference in both CD69 and IL-2 expression between ACE null and ACE over-expressing cells (p<0.001). If the background levels of expression are subtracted, the difference between the response to ACE null cells vs WT and ACE10 cells was about 3-fold and 8-fold (p<0.001). To investigate if the difference is due to ACE changing the fundamental ability of macrophages to present antigens to T cells, we repeated the experiment by feeding the ACE KO, WT and ACE10 macrophages with the mature MHC class II peptide OVA323–339. No difference was found between the 3 groups of macrophages (<xref rid="nihms855384f1" ref-type="fig">Figure 1A, B</xref>). These data suggest that the basic ability of APCs to present MHC class II peptides is not affected by ACE levels. These data imply that ACE participates in the processing of MHC class II antigens.). These data suggest that the basic ability of APCs to present MHC class II peptides is not affected by ACE levels. These data imply that ACE participates in the processing of MHC class II antigens.'], 'nihms855384f2': ['To further investigate whether ACE expression affects the basic machinery of MHC class II antigen presentation, we evaluated the surface expression of MHC class II and co-stimulatory molecules CD80 and CD86 on ACE KO and ACE10 macrophages (<xref rid="nihms855384f2" ref-type="fig">Figure 2A</xref> and and Supplementary figure 3). The expression of these molecules does not differ when ACE expression changes. Further, the soluble antigen studied above was taken up by APC pinocytosis before going through antigen processing and presentation by MHC class II molecules, we studied whether protein uptake was affected by null or high levels of ACE. To investigate this, we incubated macrophages with Alexa Fluor 488-conjugated OVA for 1 hour. Cells were then studied by flow cytometry. No difference was observed between ACE KO, WT and ACE10 macrophages in taking up soluble antigen (<xref rid="nihms855384f2" ref-type="fig">Figure 2B</xref>).).'], 'nihms855384f3': ['To further understand the effects of ACE on MHC class II antigen presentation, we tested another model antigen, hen egg lysozyme (HEL), by feeding this protein to macrophages. The presentation of four MHC class II epitopes of HEL, i.e. HEL20–35, HEL30–53, HEL46–64 and HEL74–88, by I-Ab was evaluated using hybridomas Hb1.9, H30.44, H46.13 and BO4 (14) (<xref rid="nihms855384f3" ref-type="fig">Figure 3</xref>). Because three of these epitopes contain cysteine residues (). Because three of these epitopes contain cysteine residues (5), we added β-mercaptoethanol to the coincubation of macrophages and HEL to increase the efficiency of HEL presentation. Again, ACE affected the antigen presentation. With HEL, 3 of the 4 epitopes tested showed a direct or inverse relationship between APC expression of ACE and the efficiency of surface class II peptide expression. Specifically, higher ACE expression increased the presentation of HEL30–53, and decreased the presentation of epitopes HEL20–35 and HEL74–88. For HEL46–64, there was no difference in presentation between ACE KO, WT and ACE10 cells. For each of the three epitopes affected by ACE, there was a consistent pattern that epitope presentation by null cells and ACE 10 cells over expressing ACE showed the opposite effect. Thus, these data again indicate that ACE takes part in the processing of MHC class II peptides. Moreover, ACE has different effects on different peptide epitopes, as would be expected given substrate preferences of the enzyme.'], 'nihms855384f4': ['The previous in vitro studies suggest that ACE over-expression influences, and can even augment, the presentation of certain antigens to CD4+ T cells in vivo. Next, we used an immunization strategy to investigate this hypothesis. Given that ACE KO mice have hypotension and other physiologic defects (12, 15), we used WT and ACE10 mice to study the immune response to immunization. OVA protein was emulsified in complete Freund’s adjuvant (CFA) and injected s.c. into cohorts of WT and ACE10 mice. After 9 days, spleens and draining lymph nodes were collected and lymphocytes were restimulated with OVA323–339 peptide. The expression of IFN-γ and IL-17A by CD4+ T cells, which represents the activation of Th1 and Th17 cells respectively, was measured by ELISA (<xref rid="nihms855384f4" ref-type="fig">Figure 4A, B</xref> and and Supplementary figure 4A). The spleens and lymph nodes derived from ACE10 mice consistently produced a greater CD4+ T cell response compared to their counterparts derived from WT mice. For example, splenocytes from ACE10 mice produced 5.52-fold IFN-γ and 5.48-fold IL-17 as compared to WT cells (p<0.001). Data from cells from lymph nodes was similar, though more variable.', 'Antibody production by B cells requires help from CD4+ T cells. Thus, we also measured the production of anti-OVA antibodies. For all IgG subtypes evaluated, the ACE10 mice produced more anti-OVA antibodies compared to WT mice after CFA-OVA immunization and OVA enhancement (<xref rid="nihms855384f4" ref-type="fig">Figure 4C</xref>). In fact, for the most common IgG subtype, IgG1, ACE 10 mice averaged more than a 20-fold higher antibody titer (p<0.001). The adjuvant Alum exerts its effects through a different mechanism than CFA (). In fact, for the most common IgG subtype, IgG1, ACE 10 mice averaged more than a 20-fold higher antibody titer (p<0.001). The adjuvant Alum exerts its effects through a different mechanism than CFA (16). To exclude the possibility that the over-production of anti-OVA antibody by ACE10 mice is adjuvant-dependent, we followed the same protocol but used an Alum-OVA mixture for the initial immunization (<xref rid="nihms855384f4" ref-type="fig">Figure 4D</xref>). Again, the ACE10 mice produced more anti-OVA antibodies. Here, each class of IgG was significantly different between ACE10 and WT. To ensure this change, we also measured the total IgG titer in the plasma of unimmunized WT and ACE10 mice, and no baseline difference was noticed (). Again, the ACE10 mice produced more anti-OVA antibodies. Here, each class of IgG was significantly different between ACE10 and WT. To ensure this change, we also measured the total IgG titer in the plasma of unimmunized WT and ACE10 mice, and no baseline difference was noticed (Supplemental figure 4B). Thus, consistent with the in vitro antigen presentation studies, ACE over-expression can affect several of the CD4+ T cell responses to MHC class II-presented antigen in vivo.'], 'nihms855384f5': ['The discovery that ACE can participate in MHC class II antigen presentation prompted the question of where ACE encounters MHC class II antigens. ACE is typically exported to the cell membrane where it is bound by its C-terminal hydrophobic sequence. Thus, it is conceivable that the cell membrane is the place where ACE trims peptides presented by MHC class II molecules. It is also possible that in the formation of the endosome/phagosome, vacuolar ACE is internalized with its catalytic domains exposed to the lumen of the endosome/phagosome. To investigate the distribution of ACE in MHC class II compartments inside cells, we treated macrophages with 1 μm latex beads for 1 hour to induce phagocytosis. Cells were permeabilized and co-stained for ACE and the MHC class II molecule I-Ab. Upon phagocytosis, ACE accumulated around latex beads in some intracellular organelles or vacuoles where MHC class II molecules localize, which indicates that ACE is present in MHC class II compartments. (<xref rid="nihms855384f5" ref-type="fig">Figure 5A</xref>). With the same protocol, we also co-stained for ACE and phagosomal markers. As shown in ). With the same protocol, we also co-stained for ACE and phagosomal markers. As shown in <xref rid="nihms855384f5" ref-type="fig">Figures 5B</xref> and and <xref rid="nihms855384f5" ref-type="fig">5C</xref>, after phagocytosis, ACE exhibited partial co-localization with the early endosome marker EEA1 and late endosome/lysosome marker LAMP1 around latex beads. Therefore, ACE is present in the MHC class II antigen presentation pathway where it may be available to modify intracellular peptides, including peptides for the formation of mature MHC class II., after phagocytosis, ACE exhibited partial co-localization with the early endosome marker EEA1 and late endosome/lysosome marker LAMP1 around latex beads. Therefore, ACE is present in the MHC class II antigen presentation pathway where it may be available to modify intracellular peptides, including peptides for the formation of mature MHC class II.', 'To further investigate the relationship between ACE expression and MHC class II antigen presentation, we used nucleofection to transfect WT bone marrow-derived macrophages with an expression construct for catalytically active ACE or with a mutated ACE cDNA (mACE) encoding a protein in which the two catalytic domains of ACE were rendered inactive by point mutations (8). Twenty-four hours after transfection, live attached cells were primed with IFN-γ for 12 hr to upregulate the MHC class II machinery. Cells were then fed with OVA protein, and I-Ab presentation of OVA323–339 was measured by OT-II T cells. Transfection of the catalytically active ACE construct resulted in 2-fold more I-Ab-OVA323–339 presentation than that of cells expressing mACE (<xref rid="nihms855384f5" ref-type="fig">Figure 5D</xref>). These data strongly indicate that ACE catalytic activity participates in MHC class II antigen processing of at least some epitopes.). These data strongly indicate that ACE catalytic activity participates in MHC class II antigen processing of at least some epitopes.'], 'nihms855384f6': ['ACE inhibitors have been widely used in treating cardiovascular diseases. To understand whether pharmacological ACE inhibition would affect MHC class II antigen presentation, we treated WT and ACE10 peritoneal macrophages with the ACE inhibitor lisinopril for 2 hours before co-incubating them with OVA. Inhibiting ACE activity completely abrogated the enhanced ability of ACE10 cells in presenting OVA323–339, as measured by the upregulation of CD69 and secretion of IL-2 by OT-II cells (<xref rid="nihms855384f6" ref-type="fig">Figure 6A</xref>). Indeed, the conversion of ACE 10 cells from cells that over-present OVA). Indeed, the conversion of ACE 10 cells from cells that over-present OVA323–339 to cells equivalent to WT after short term inhibitor treatment implicates ACE catalytic activity as responsible for the ACE 10 phenotype.', 'To evaluate the effects of an ACE inhibitor in vivo, we treated WT and ACE10 mice with lisinopril for one week with a dose (6 mg/kg/day) which was sufficient to lower blood pressure by about 28 mmHg. This dose achieved complete inhibition of ACE and is higher than the typical dose in humans of about 0.5 mg/kg/day which decreases blood pressure by about 5 mmHg in normotensive subjects. The mice were then immunized s.c. with OVA-CFA. We continued ACE inhibition until 9 days after immunization. On harvesting, CD4+ T cells in the spleen and draining lymph nodes were evaluated in response to OVA323–339 challenge (<xref rid="nihms855384f6" ref-type="fig">Figure 6B</xref>). ACE inhibition significantly decreased CD4). ACE inhibition significantly decreased CD4+ T cell responses to OVA antigen in ACE10 mice and there was no difference between lisinopril-treated WT and lisinopril-treated ACE 10 mice. Of note, ACE inhibition also suppressed the presentation efficiency of WT cells. To investigate the possibility that the difference between the WT mice in the presence and the absence of ACE activity may be caused by T cell receptor (TCR) repertoire change, we assessed the spectrum of CD4+ TCR β-chain variable region (Vβ) in splenocytes isolated from WT and ACE KO mice. No difference was found in the use of CD4+ TCR Vβ in presence or absence of ACE (Supplemental Figure 5). Thus, both in vitro and in vivo studies consistently indicate that ACE affects the peripheral activation of CD4+ T cells by APCs.']}
|
Angiotensin converting enzyme affects the presentation of MHC class II antigens
|
[
"angiotensin converting enzyme",
"MHC class II",
"antigen presentation"
] |
Lab Invest
|
1499670000
|
In this paper we evaluate the effects of heavy alcohol consumption on sexual behavior, HIV acquisition, and antiretroviral treatment (ART) initiation in a longitudinal open cohort of 1929 serodiscordant couples in Lusaka, Zambia from 2002 to 2012. We evaluated factors associated with baseline heavy alcohol consumption and its association with condomless sex with the study partner, sex outside of the partnership, and ART initiation using multivariable logistic regression. We estimated the effect of alcohol consumption on HIV acquisition using multivariable Cox models. Baseline factors significantly associated with women's heavy drinking (drunk weekly or more in 12-months before enrollment) included woman's older age (adjusted prevalence odds ratio [aPOR] = 1.04), partner heavy drinking (aPOR = 3.93), and being HIV-infected (aPOR = 2.03). Heavy drinking among men was associated with less age disparity with partner (aPOR per year disparity = 0.97) and partner heavy drinking (aPOR = 1.63). Men's being drunk daily (aOR = 1.18), women's being drunk less than monthly (aOR = 1.39) vs. never drunk and being in a male HIV-negative and female HIV-positive union (aOR = 1.45) were associated with condomless sex. Heavy alcohol use was associated with having 1 or more outside sex partners among men (aOR drunk daily = 1.91, drunk weekly = 1.32, drunk monthly = 2.03 vs. never), and women (aOR drunk monthly = 2.75 vs. never). Being drunk weekly or more increased men's risk of HIV acquisition (adjusted hazard ratio [aHR] = 1.72). Men and women being drunk weekly or more was associated (p < 0.1) with women's seroconversion (aHR = 1.42 and aHR = 3.71 respectively). HIV-positive women who were drunk monthly or more had lower odds of initiating ART (aOR = 0.83; 95% CI = 0.70-0.99) adjusting for age, months since baseline and previous pregnancies. Individuals in HIV-serodiscordant couples who reported heavy drinking had more outside sex partnerships and condomless sex with their study partner and were more likely to acquire HIV. HIV-positive women had lower odds of initiating ART if they were heavy drinkers.
|
[
"Adult",
"Age Factors",
"Alcohol Drinking",
"Alcoholic Intoxication",
"Cohort Studies",
"Female",
"HIV Infections",
"Humans",
"Logistic Models",
"Male",
"Middle Aged",
"Odds Ratio",
"Prevalence",
"Proportional Hazards Models",
"Risk Factors",
"Sexual Behavior",
"Sexual Partners",
"Unsafe Sex",
"Young Adult",
"Zambia"
] |
other
|
PMC5493495
| null |
23
|
[
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] |
Lab Invest. 2017 Jul 10; 97(7):764-771
|
NO-CC CODE
|
|
Time-lapse and live in vivo imaging to visualize the engraftment process(a) Time-lapse in vivo imaging of tibia co-transplanted with 3×104 GFP+ SKL cells (white arrow) and 105 DsRed+ CD133+ hematopoietic progenitor cells (yellow arrowhead, n=3). (b) 5×106 DsRed+ Lin− cells were injected to Tie2-GFP mice to visualize the dynamic interaction of Tie2-GFP+ BM endothelium (EC) and engrafting cells (n=5). Images were simultaneously captured from Video S4 showing rolling/tethering (red arrow) and attaching/extravasating cells (white arrow) that migrate into BM cavity. A cell that is already crossed the endothelial barrier was also observed (blue arrow). Scale bar=200 μm. The observation time is same as appeared in Video S5. (c) Higher magnification images showing cells that tether and scan along the endothelium via millipede-like locomotion. Scale bar = 20μm. Time format = (mm:ss).
|
nihms828649f4
|
2
|
580c2c25b031160875ecc781ab3f62efcf7403aca3f6eee198bd9d8bb27a5e7d
|
nihms828649f4.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
704,
427
] |
[{'image_id': 'nihms828649f3', 'image_file_name': 'nihms828649f3.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f3.jpg', 'caption': 'Time-lapse imaging of the individual SKL cells showing endosteal engraftment(a) Blood flow (arrows) in the endosteal region. (b) Time lapse imaging of the area showing the engraftment of individual SKL cells. The white arrow indicates the main colony and blue arrows indicate the satellite colonies developed at later time points. Scale bar=100μm.', 'hash': '162388e636e1d4a481b76d8ead505ad54d80c0e29462c3a61f76648faef72b50'}, {'image_id': 'nihms828649f4', 'image_file_name': 'nihms828649f4.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f4.jpg', 'caption': 'Time-lapse and live in vivo imaging to visualize the engraftment process(a) Time-lapse in vivo imaging of tibia co-transplanted with 3×104 GFP+ SKL cells (white arrow) and 105 DsRed+ CD133+ hematopoietic progenitor cells (yellow arrowhead, n=3). (b) 5×106 DsRed+ Lin− cells were injected to Tie2-GFP mice to visualize the dynamic interaction of Tie2-GFP+ BM endothelium (EC) and engrafting cells (n=5). Images were simultaneously captured from Video S4 showing rolling/tethering (red arrow) and attaching/extravasating cells (white arrow) that migrate into BM cavity. A cell that is already crossed the endothelial barrier was also observed (blue arrow). Scale bar=200 μm. The observation time is same as appeared in Video S5. (c) Higher magnification images showing cells that tether and scan along the endothelium via millipede-like locomotion. Scale bar = 20μm. Time format = (mm:ss).', 'hash': '580c2c25b031160875ecc781ab3f62efcf7403aca3f6eee198bd9d8bb27a5e7d'}, {'image_id': 'nihms828649f5', 'image_file_name': 'nihms828649f5.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f5.jpg', 'caption': 'SKL cells tend to engraft on the specific niche with the limited number(a) Representative images of tibia window with different number of GFP+ SKL cell injection at day 4. Endosteum-engrafted colonies above 3500μm2 were marked with red arrowheads to speculate possible osteoblastic niche locations. (b) The average number of observed colonies in each group was shown in graph (n=5–10). Data analyzed by ANOVA and Tukey’s post test, *, P<0.05; **, P<0.01; n.s.= not significant (c) The same number of SKL cells from GFP and DsRed mice (3×104 cells) were injected into the same recipient to show the niche preference of SKL cells (n=3). Scale bars = 500μm for a, 300μm for c.', 'hash': 'ecde1cb001a98eadb1a635c010a8f2f2f1e31ce76c80fbf6b337ab34041b6619'}, {'image_id': 'nihms828649f2', 'image_file_name': 'nihms828649f2.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f2.jpg', 'caption': 'In vivo imaging of the same mouse over time demonstrates that SKL cells tend to engraft and proliferate near the endosteal region(a) Mosaic images of time-lapse in vivo imaging of a tibia window. 3×104 GFP+ SKL cells were injected at Day 0 and in vivo colony formation from SKL cells was monitored in the same animal over time (n=6). Scale bar=500μm (b) Higher magnification of single GFP+ cells engrafted on the endosteal surface developing into a colony (the boxed region at Day 4). Injected SKL cells homed to the marrow and developed into a colony over 4 days (Red arrowheads, the same area marked at the mosaic images). However, not every cell homed to the endosteal surface formed colonies (white arrows). Scale bar=200μm.', 'hash': '8296725723350510d348aba4affb01c67c5f768746430b9c21f279ae38af857c'}, {'image_id': 'nihms828649f1', 'image_file_name': 'nihms828649f1.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f1.jpg', 'caption': 'Time-lapse in vivo imaging of the tibia bone to visualize the engraftment of SKL cells(a) A diagram showing the processes of in vivo tibia imaging. Mice were lethally irradiated 2 days before cell injection. Each image was tiled into a mosaic to create the panoramic view of the tibia marrow. (b) Use of the RGB filter for real-time, true color video recording. Color-separated images from RGB channel demonstrate that individual GFP+ cells could be clearly visualized using 10× magnification (white arrows). Non-fluorescent SKL cells from C57BL6 mice did not generate any signal in the recipients (data not shown). (c) Formation of BM colonies from individual SKL cell in the RGB channel. The fate of a single GFP+ SKL cell was followed over 6 days. All scale bars = 200μm.', 'hash': '1fac9f19c16e7ef79c54a1ca8696b6880108e374ef24e41e10ba8fff83a5deaf'}, {'image_id': 'nihms828649f6', 'image_file_name': 'nihms828649f6.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f6.jpg', 'caption': 'Dye retaining cells are located on the endosteal region(a and b) Time-lapse in vivo tracking of dye retaining cells to visualize slow cycling cells (n=5). 4×103 GFP+ SKL cells stained with DiI membrane-dye (red) were injected into the femoral artery and monitored for engraftment. GFP+ DiI bright cells were located predominantly near the endosteum (white arrows). (c) The DiI dye that was initially observed in GFP+ SKL cells at the central marrow region at day 2 was rapidly diluted after cell proliferation after 24h. (d–f) FACS analysis of the dye retaining cells from the tibia at day 3 (n=5). Student’s t-test was done for statistical analysis. ***, P<0.001. All scale bars=200μm', 'hash': '2cfb37e88ea52e98d792f5348f6ed9e3e195233dc264330f2d37518438771c97'}, {'image_id': 'nihms828649f7', 'image_file_name': 'nihms828649f7.jpg', 'image_path': '../data/media_files/PMC5498248/nihms828649f7.jpg', 'caption': 'SKL cell engraftment in abnormal HSC niches(a) Time-lapse in vivo imaging of SKL cell engraftment in P-selectin knockout mice (n=5). Cells engrafted in the central marrow area engrafted and proliferated much slower (white arrow) than a cell that homed to the endosteal region (blue arrow). Higher magnification pictures of the boxed area on the right side show aberrant engraftment in the central marrow region (white arrow). 6×104 GFP+ SKL cells were injected at Day 0 after lethal irradiation. (b) In vivo imaging of SKL cell engraftment in OPN knockout mice showing rapid proliferation of SKL cells (n=4). Higher magnification pictures of the boxed area on the right side show clusters of SKL cells at the central marrow region disappearing at day 3 (red arrow). SKL cells in the endosteal regions showed rapid proliferation (asterisk). 3×104 GFP+ SKL cells were injected at Day 0 after lethal irradiation. (c) 3×103 GFP+ SLAM-SKL cells were injected at Day 0. Engraftment and proliferation of individual SLAM-SKL cells (red/blue arrows) were observed till Day 5.', 'hash': '44cceabc72ddca0bde58dc1af3d2e1e12e5ecd548bb644a6a89f536f277c2b10'}]
|
{'nihms828649f1': ['The goal of this study was to directly observe the key characteristics associated with HSC function in a living animal during the dynamic repopulation process following BMT. Using time-lapse intravital imaging of tibial long bone as described in <xref rid="nihms828649f1" ref-type="fig">Figure 1</xref>, we sought to directly visualize the functional abilities of homing, engraftment, clonal expansion and asymmetric cell fates that define HSC activity in lethally irradiated animals. We also wanted to determine if the tibia window technique could distinguish differing engraftment dynamics in transgenic mice with defective HSC niches, or with cell HSC enriched populations with altered engraftment patterns previously assessed via histology. To achieve this goal, we transplanted fluorescently-tagged, HSC enriched Sca-1, we sought to directly visualize the functional abilities of homing, engraftment, clonal expansion and asymmetric cell fates that define HSC activity in lethally irradiated animals. We also wanted to determine if the tibia window technique could distinguish differing engraftment dynamics in transgenic mice with defective HSC niches, or with cell HSC enriched populations with altered engraftment patterns previously assessed via histology. To achieve this goal, we transplanted fluorescently-tagged, HSC enriched Sca-1+, c-Kit+, Lineage− (SKL) cells or other test populations to directly observe their engraftment and repopulation dynamics in individual living recipients for up to six days post transplant.', 'Our initial aim was to selectively visualize key functional stem cell activities within the marrow of an individual living mouse over a period of days following BMT. We chose to use the classic Sca-1+, c-Kit+ Lin− (SKL) BM population isolated from the GFP+ donors. The SKL population is the most widely studied enriched HSC population, but even within SKL studies different laboratories often utilize disparate lineage antibody cocktails, FACS gates etc. We chose to use our SKL isolation protocol that we had previously used for successful single HSC transplants.(3) Our current success rate is 1:5 single cell engrafted recipient animals using this highly HSC enriched SKL population. The SKL population contains both enriched short and long term HSC activity as well as a variety of multipotent progenitors. Engraftment of SKL cells was visualized by surgically thinning one side of the tibia until the bone was sufficiently transparent to allow direct observation of the BM space (<xref rid="nihms828649f1" ref-type="fig">Figure 1</xref>).().(15, 16) Individual frames were tiled to produce mosaic images of the majority of tibia marrow space for unbiased observation (<xref rid="nihms828649f1" ref-type="fig">Figures 1</xref> and and Supplementary Figure 1). The use of an epifluorescence microscope coupled with triple bandpass RGB filter and 3CCD camera enabled rapid imaging the dynamic process of engraftment in true color and generate real time videos with the single cell level resolution (<xref rid="nihms828649f1" ref-type="fig">Figure 1b, c</xref> and all and all Supplementary Videos). Following the BMT, we noticed formation and expansion of engrafted GFP+ colonies mainly along the osteoblastic region of the bone (<xref rid="nihms828649f2" ref-type="fig">Figure 2</xref> and and Supplementary Video 1). Individual GFP+ SKL cells that homed to and engrafted near the endosteum were routinely observed by 12–48h post-transplant (<xref rid="nihms828649f2" ref-type="fig">Figure 2b</xref> and and Supplementary Figure 2a). Real time video of single GFP+ SKL cells rolling on the endothelium showed that some SKL cells could strongly tether themselves to the endosteal surface (Supplementary Video 2). By 52–60 hours, small but bright GFP+ colonies started to form predominantly along the endosteal region and then rapidly expanded over time to repopulate the marrow space (<xref rid="nihms828649f2" ref-type="fig">Figure 2</xref>). Time-lapse analysis of individual GFP). Time-lapse analysis of individual GFP+ SKL cells also showed that cells engrafted near the endosteal surface could actively expand and dynamically migrate to the surroundings to generate smaller satellite colonies (<xref rid="nihms828649f3" ref-type="fig">Figure 3</xref> and and Supplementary Video 3). 62.7±8.1% of colonies observed in the tibia window were bordered by the endosteum at day 4 (n=5). We observed that single or a few GFP+ SKL cells that initially engrafted near osterix positive cells (shown to help delineate the potential osteoblastic niche) could proliferate more quickly and form tighter, more populous colonies compared to the few scattered cells and colonies seen in the central marrow region of lethally irradiated recipients (Supplementary Figure 2), which is consistent with previous studies highlighting the importance of osterix in HSC niche formation.(18, 19) SKL enriched cell transplants showed the same overall patterns of engraftment as seen for GFP+ whole bone marrow and/or Lin− control transplants (Supplementary Figure 3 and data not shown).'], 'nihms828649f6': ['Animals were sacrificed from 24 hours to day 14 after the cell injection. BM cells were isolated as described above. For animals used in <xref rid="nihms828649f6" ref-type="fig">Figure 6</xref>, BM cells from the central and the endosteal marrow were separated differentially as described.(, BM cells from the central and the endosteal marrow were separated differentially as described.(17) Both cell populations were treated with ACK buffer on ice for 5–10 min to remove red cells. After the cell number determination, BM cells were re-suspended into FACS buffer and analyzed using BD FACS Canto II (BD bioscience).', 'HSC go through active differentiation in lethally irradiated mice to repopulate the hematopoietic system. HSC were shown to produce asymmetrical cell fates of both relative quiescence (self-renewal) and active proliferation/differentiation (reconstitution) in an ablative transplant environment.(1, 32, 33) One method allowing direct observation of differences in cell proliferation rates among progeny cells is differential cell membrane-dye retention.(34) GFP+ SKL cells were stained with red fluorescent DiI-membrane dye and used for BMT of recipient animals with tibia windows. We initially observed Dil-dye positive cells primarily engrafting in the endosteal area, but occasionally out in the central marrow region (<xref rid="nihms828649f6" ref-type="fig">Figures 6a and 6c</xref>). However, Dil-dye retaining cells after evidence of extensive GFP). However, Dil-dye retaining cells after evidence of extensive GFP+ proliferation/colony formation were only observed near the endosteal region. Dye retaining cells showing little or no proliferation also only persisted near the endosteum by 72 hours post-transplant (<xref rid="nihms828649f6" ref-type="fig">Figure 6b</xref>). The relative lack of Dil-dye retaining Sca-1). The relative lack of Dil-dye retaining Sca-1+, c-Kit+ cells/progeny outside of the endosteal region beyond 3 days post-transplant was confirmed by FACS analysis (<xref rid="nihms828649f6" ref-type="fig">Figure 6d–f</xref>) suggesting that the endosteal region selectively maintains slow cycling cells among rapidly dividing daughter cells.) suggesting that the endosteal region selectively maintains slow cycling cells among rapidly dividing daughter cells.'], 'nihms828649f4': ['We wanted to directly contrast the homing, engraftment and repopulation patterns of the HSC enriched SKL population versus a known multipotent hematopoietic progenitor population (MPP). We chose CD133+ BM cells from a DsRed transgenic donor as our test MPP population. CD133+ cells were shown to represent a short-term MPP population that can give rise to lymphoid, myeloid and endothelial lineages.(20–22) When both populations were injected, only GFP+ SKL cells were able to actively engraft and proliferate in the BM, preferentially in the endosteal region, which confirmed their homing and repopulation abilities (<xref rid="nihms828649f4" ref-type="fig">Figure 4a</xref>). A few DsRed). A few DsRed+ CD133+ progenitors homed to and engrafted in the marrow, but they failed to proliferate and form colonies. We also transplanted 106 Lin+ GFP+ BM cells in lethally irradiated recipients and followed them for 5 days to understand homing of differentiated cells (Supplementary Figure 4). A few individual Lin+ cells were observed transiently near the central marrow region, but no endosteal engraftment or colony formation was seen. Taken together, the results demonstrate that the SKL population can engraft and rapidly proliferate primarily near the endosteal region, an activity not observed from progenitors or mature cell populations.', 'HSC homing is initiated by multiple adhesion molecule cascade within BM endothelium and the niche. Selectins are one such adhesion molecule family that mediates tethering and rolling along the endothelium, followed by firm adherence and transendothelial migration.(28) We next aimed to understand the homing process by directly visualizing individual cells real time in tibia BM by injecting 5×106 Lin− donor-cells from DsRed mice to Tie2-GFP recipient mice where the vascular endothelium is GFP+. Among rapidly circulating cells, we could observe a few cells that roll and scan along the Tie2-GFP+ endothelium via millipede-like locomotion (<xref rid="nihms828649f4" ref-type="fig">Figure 4c</xref>). We also observed cells adhered to the endothelium that are about to exit and other cells that had already migrated to the BM cavity (white and blue arrows in ). We also observed cells adhered to the endothelium that are about to exit and other cells that had already migrated to the BM cavity (white and blue arrows in <xref rid="nihms828649f4" ref-type="fig">Figure 4b</xref> and and Supplementary Video 4). The adhesion and extravasation of hematopoietic stem/progenitor cells was only observed at a limited number of locations within the irradiated marrow vasculature.', 'First, using tibia windows we documented donor SKL populations homing to marrow via the circulation where they extravasate into the marrow space (<xref rid="nihms828649f4" ref-type="fig">Figure 4</xref>, , Supplementary Videos 2 and 4). Donor SKL engraft within the tibia long bone with a strong preference for a limited number of niches for both extravasation and engraftment within irradiated recipients (<xref rid="nihms828649f1" ref-type="fig">Figures 1</xref>––<xref rid="nihms828649f5" ref-type="fig">5</xref>, , Supplementary Videos 1 and 3). The SKL engraftment phenotype was indistinguishable from that observed with either whole bone marrow or mildly enriched Lin− donor populations (Supplementary Figure 3), suggesting that no major stem/progenitor activities were excluded by SKL enrichment. In contrast, Lin+ transplants failed to home to or engraft the marrow to any appreciable degree (Supplemental Figure 2). CD133+ MPP did occasionally engraft, but failed to significantly proliferate within the marrow. These data suggest that the SKL population contains the majority of homing/engraftment/expansion active cell types, or at least niche utilization phenotypes contained in whole marrow. Furthermore, significant marrow colony formation was only observed when populations known to contain HSC activity were transplanted. Thus, the ability to produce colony forming unit-BM (CFU-BM) during marrow repopulation likely represents HSC activity. However, the observable timeframe of the tibia window cannot distinguish short versus long-term HSC populations. Additional test populations containing known CFU and MPP activity without HSC function would be required to further support the CFU-BM as a measure of HSC activity. When comparing population studies, it is always possible that minor populations capable of engraftment and expansion excluded by the SKL enrichment process are not appreciated, especially if their numbers are low and their niche utilization phenotypes duplicate activities contained in the SKL population.', 'The predominant engraftment site observed with SKL, WBM or Lin− populations was located near the endosteum adjacent to Tie2+ vascular structures, with limited engraftment observed in the central marrow regions (<xref rid="nihms828649f4" ref-type="fig">Figure 4</xref> and and Supplementary Video 5). Occasional double occupancy in the niche, limiting-dilution and mixed green/red donor SKL experiments suggest that the number of engraftment sites located within the irradiated marrow is limited (<xref rid="nihms828649f2" ref-type="fig">Figure 2b</xref> and and <xref rid="nihms828649f5" ref-type="fig">Figure 5</xref>). Non-ablative transplants into NOG recipients showed a similar overall pattern of engraftment observed in irradiated transplants, although colony sizes were reduced. This supports the theory that HSC niches are restricted during normal homeostasis as well.). Non-ablative transplants into NOG recipients showed a similar overall pattern of engraftment observed in irradiated transplants, although colony sizes were reduced. This supports the theory that HSC niches are restricted during normal homeostasis as well.'], 'nihms828649f2': ['We observed that not every cell that homed to and engrafted at the endosteal surface developed into a colony (white arrows on <xref rid="nihms828649f2" ref-type="fig">Figure 2</xref> and and Supplementary Video 4). This indicates that many cells within the enriched SKL population may not be functional HSCs, or that otherwise functional HSC may engraft in areas of the marrow that do not support proliferation – perhaps areas that induce quiescence. (1, 27) However, it is also possible that there exists a limited number of distinctive HSC niches within the irradiated marrow capable of supporting functional HSC activity. To address this issue, we performed a series of limiting dilution transplantations with GFP+ SKL cells and demonstrated that when SKL cell numbers were limited, engraftment occurred mostly in the endosteal regions (<xref rid="nihms828649f5" ref-type="fig">Figure 5a</xref> and and Supplementary Video 3). The limited number of engraftment sites was easily saturated, and higher doses of SKL cells did not result in proportional increase of marrow colony number (<xref rid="nihms828649f5" ref-type="fig">Figure 5b</xref>). In addition, when a saturating dose of both GFP). In addition, when a saturating dose of both GFP+ SKL cells and DsRed+ SKL cells were injected in the lethally irradiated recipients, we observed that about 1/3 of developing colonies contained both green and red donor cells in the same endosteal regions (8 out of 25 colonies observed, <xref rid="nihms828649f5" ref-type="fig">Figure 5c</xref>).).', 'HSC undergo clonal expansion to repopulate both the HSC pool and peripheral blood following myeloablative transplant. The tibia window allowed direct observation of single cell engraftment within the marrow. (<xref rid="nihms828649f2" ref-type="fig">Figures 2</xref>, , <xref rid="nihms828649f3" ref-type="fig">3</xref> and and Supplementary Videos 1) The balance between proliferation and quiescence is critical for maintaining the appropriate number of HSCs.(43) Such asymmetric HSC fates were demonstrated by differential membrane dye retention within rapidly expanding GFP marrow colonies. The more quiescent cells were restricted to the endosteal region of the marrow with dye retaining cells being rapidly lost from the central marrow region (<xref rid="nihms828649f6" ref-type="fig">Figure 6</xref>). Our results suggest that there exist a limited number of specialized endosteal zones, possibly with a distinct blood vessel type for HSC maintenance, that play a critical role in preserving slow cycling stem cells among the rapidly proliferating daughter cells.(). Our results suggest that there exist a limited number of specialized endosteal zones, possibly with a distinct blood vessel type for HSC maintenance, that play a critical role in preserving slow cycling stem cells among the rapidly proliferating daughter cells.(44) Although recent studies have highlighted the important roles of BM endothelial cells to HSCs, stem cell activities are still found near the endosteal region of both normal and myeloablated conditions,(17, 44–48) which is in agreement to our observation. As suggested by several reviews, osteoblastic and perivascular/stromal niches may not be mutually exclusive to each other.(49, 50) But rather, they are likely to be physically and functionally involved together to facilitate engraftment of HSC. For instance, metaphyseal spongiosa in femur, where osteoblasts and endothelial/stromal cells are highly mixed, is active HSC engraftment sites. Although our method cannot fully visualize metaphyseal tibia, we also observed many colonies formed on the trabecules of woven tibia bone which supports this idea (<xref rid="nihms828649f3" ref-type="fig">Figure 3</xref>, , Supplementary Video 1 and 3).'], 'nihms828649f7': ['Next, we sought to determine if the tibia window model would facilitate direct observation of altered bone marrow engraftment and niche utilization phenotypes previously documented in the literature. Therefore, we visualized engraftment of GFP+ SKL cells in P-selectin knockout mice to observe HSC engraftment dynamics in the mutant bone marrow microenvironment. P-selectin is one of the major BM endothelial adhesion molecules involved in the initial rolling and extravasation steps on BMT. P-selectin null marrow would be predicted to have negative effects on WT-donor cell engraftment.(35) Indeed, we had to increase the injected cell number to 6×104 SKL cells to ensure survival in these mice as engraftment was very poor. Time-lapse imaging showed that there were small colonies developed in the central region of the P-selectin null marrow, showing that endosteal engraftment is severely compromised by the mutation (<xref rid="nihms828649f7" ref-type="fig">Figure 7a</xref>). While majority of the engrafted cells were scattered in the central marrow region, a few single SKL cells did engraft near the endosteum at 24–48h (). While majority of the engrafted cells were scattered in the central marrow region, a few single SKL cells did engraft near the endosteum at 24–48h (Supplementary Figure 7). These few cells formed clusters at later time points (blue arrows in <xref rid="nihms828649f7" ref-type="fig">Figure 7a</xref>), and eventually developed into much bigger colonies than others observed in the central marrow regions (white arrows in ), and eventually developed into much bigger colonies than others observed in the central marrow regions (white arrows in <xref rid="nihms828649f7" ref-type="fig">Figure 7a</xref>). Large colony formation was significantly delayed in P-selectin null recipients compared to normal.). Large colony formation was significantly delayed in P-selectin null recipients compared to normal.', 'Osteopontin (OPN) is a key component of the HSC niche that is secreted by osteoblasts. Studies have shown that it acts as a potent constraint on HSC proliferation as OPN-null microenvironment leads to increased HSC proliferation.(36, 37) We also observed rapid proliferation of GFP+ WT-SKL cells in these OPN-null recipient marrows in vivo. Rapidly proliferating colonies occurred mainly near the endosteal regions (<xref rid="nihms828649f7" ref-type="fig">Figure 7b</xref>). Rapidly proliferating colonies were also initially formed in the central marrow region of these mice, however, many of them dissipated quickly by day 4 (red arrows in ). Rapidly proliferating colonies were also initially formed in the central marrow region of these mice, however, many of them dissipated quickly by day 4 (red arrows in <xref rid="nihms828649f7" ref-type="fig">Figure 7b</xref>). While the observed GFP). While the observed GFP+ SKL cells were mainly located near the endosteum in C57BL6 mice at 18h after cell injection, the same population in P-selectin and OPN knockout mice was randomly distributed, showing that disruptions of vascular or endosteal HSC niche components result in aberrant homing and engraftment of SKL cells (Supplementary Figure 8).', 'The addition of signaling lymphocyte activation molecule (SLAM) markers CD150 and CD48 to the SKL enrichment scheme yields a highly purified myeloid biased HSC population that is reported to preferentially utilize the central vascular niche of bone marrow after transplant (4, 38). We isolated GFP+ CD150+, CD48− (SLAM)-SKL cells for in vivo imaging via a tibia window. The SLAM-SKL population showed a contrasting engraftment pattern to the parent SKL population (<xref rid="nihms828649f7" ref-type="fig">Figure 7c</xref>). While SKL cells predominantly expanded quickly within the endosteal niche, SLAM-SKL cells formed smaller colonies mostly in the central marrow or the perivascular/stromal niche during the observation period. Both populations provided subsequent long-term radioprotection to their recipients demonstrating HSC function (data not shown). The two engrafted HSC populations have distinct patterns of predominant niche utilization, with the SLAM-SKL population utilizing a subset of niches used by the parent SKL population and forming colonies within the marrow at a slower initial rate.). While SKL cells predominantly expanded quickly within the endosteal niche, SLAM-SKL cells formed smaller colonies mostly in the central marrow or the perivascular/stromal niche during the observation period. Both populations provided subsequent long-term radioprotection to their recipients demonstrating HSC function (data not shown). The two engrafted HSC populations have distinct patterns of predominant niche utilization, with the SLAM-SKL population utilizing a subset of niches used by the parent SKL population and forming colonies within the marrow at a slower initial rate.', 'The initial phases of homing, engraftment and marrow repopulation are very dynamic and variable processes. The utility of the tibia window model for assessing alterations to early bone marrow engraftment dynamics was demonstrated by the observed altered engraftment patterns in the abnormal HSC niches of P-selectin knockout (deleterious for homing) and OPN knockout (enhanced for proliferation) mice (<xref rid="nihms828649f7" ref-type="fig">Figure 7</xref>). The tibia window also allows direct observation of altered niche utilization among HSC populations as shown by engraftment of SLAM-SKL cells, which are highly enriched for a myeloid biased LT-HSC (). The tibia window also allows direct observation of altered niche utilization among HSC populations as shown by engraftment of SLAM-SKL cells, which are highly enriched for a myeloid biased LT-HSC (<xref rid="nihms828649f7" ref-type="fig">Figure 7c</xref>).().(38) The SLAM-SKL produced far fewer colonies located away from endosteum proving that the predominant HSC engraftment/repopulation activity of the parent SKL population along the endosteum is not due to HSCs from the SLAM-SKL subpopulation. This indicates each HSC population has different engraftment/proliferation kinetics and further experiments will be required to elucidate cellular dynamics of each population.']}
|
in vivo Extended time-lapse imaging of tibia bone marrow to visualize dynamic hematopoietic stem cell engraftment
| null |
Leukemia
|
1501225200
|
In many contexts, the problem arises of determining which of many candidate mutations is the most likely to be causative for some phenotype. It is desirable to have a way to evaluate this probability that relies as little as possible on previous knowledge, to avoid bias against discovering new genes or functions. We have isolated mutants with blocked cell cycle progression in and determined mutant genome sequences. Due to the intensity of UV mutagenesis required for efficient mutant collection, the mutants contain multiple mutations altering coding sequence. To provide a quantitative estimate of probability that each individual mutation in a given mutant is the causative one, we developed a Bayesian approach. The approach employs four independent indicators: sequence conservation of the mutated coding sequence with ; severity of the mutation relative to wild-type based on Blosum62 scores; meiotic mapping information for location of the causative mutation relative to known molecular markers; and, for a subset of mutants, the transcriptional profile of the candidate wild-type genes through the mitotic cell cycle. These indicators are statistically independent, and so can be combined quantitatively into a single probability calculation. We validate this calculation: recently isolated mutations that were not in the training set for developing the indicators, with high calculated probability of causality, are confirmed in every case by additional genetic data to indeed be causative. Analysis of "best reciprocal BLAST" (BRB) relationships among and other eukaryotes indicate that the temperature sensitive-lethal (Ts-lethal) mutants that our procedure recovers are highly enriched for fundamental cell-essential functions conserved broadly across plants and other eukaryotes, accounting for the high information content of sequence alignment to .
|
[
"Arabidopsis",
"Bayes Theorem",
"Chlamydomonas",
"Genome, Plant",
"Meiosis",
"Models, Genetic",
"Mutation"
] |
other
|
PMC5498248
| null |
19
|
[
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"{'Citation': 'Remm M., Storm C. E., Sonnhammer E. L., 2001. \\u2003Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J. Mol. Biol. 314(5): 1041–1052.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '11743721'}}}",
"{'Citation': 'Rogozin I. B., Basu M. K., Csürös M., Koonin E. V., 2009. \\u2003Analysis of rare genomic changes does not support the unikont-bikont phylogeny and suggests cyanobacterial symbiosis as the point of primary radiation of eukaryotes. Genome Biol. Evol. 1: 99–113.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC2817406'}, {'@IdType': 'pubmed', '#text': '20333181'}]}}",
"{'Citation': 'Tulin F., Cross F. R., 2014. \\u2003A microbial avenue to cell cycle control in the plant superkingdom. Plant Cell 26(10): 4019–4038.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4247570'}, {'@IdType': 'pubmed', '#text': '25336509'}]}}",
"{'Citation': 'Tulin F., Cross F. R., 2015. \\u2003Cyclin-dependent kinase regulation of diurnal transcription in Chlamydomonas. Plant Cell 27(10): 2727–2742.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4682320'}, {'@IdType': 'pubmed', '#text': '26475866'}]}}",
"{'Citation': 'Tulin F., Cross F. R., 2016. \\u2003Patching holes in the Chlamydomonas genome. G3 (Bethesda) 6: 1899–1910.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4938644'}, {'@IdType': 'pubmed', '#text': '27175017'}]}}",
"{'Citation': 'Yampolsky L. Y., Stoltzfus A., 2005. \\u2003The exchangeability of amino acids in proteins. Genetics 170: 1459–1472.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC1449787'}, {'@IdType': 'pubmed', '#text': '15944362'}]}}",
"{'Citation': 'Yoon H. S., Hackett J. D., Ciniglia C., Pinto G., Bhattacharya D. A., 2004. \\u2003Molecular timeline for the origin of photosynthetic eukaryotes. Mol. Biol. Evol. 21(5): 809–18.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '14963099'}}}",
"{'Citation': 'Zones J. M., Blaby I. K., Merchant S. S., Umen J. G., 2015. \\u2003High-resolution profiling of a synchronized diurnal transcriptome from Chlamydomonas reinhardtii reveals continuous cell and metabolic differentiation. Plant Cell 27(10): 2743–2769.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4682324'}, {'@IdType': 'pubmed', '#text': '26432862'}]}}"
] |
Leukemia. 2017 Jul 28; 31(7):1582-1592
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NO-CC CODE
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Phagocytosis, hemocyte recruitment to wounds, and wound sealing are not impaired in leukemic lines. (A and B) Ex-vivo phagocytosis of Texas Red–conjugated E. coli (K-12 strain). A representative image of the HFP/w and HRS-leukemia lines is shown after phagocytosis. The red arrow indicates phagocytosed E. coli. (C) Quantification of the phagocytosis index. All leukemic lines show wild-type levels of phagocytic capacity. (D–F) Hemocyte migration to the wound edge in leukemic situation is comparable to wild type. Dashed line in phase-contrast and the GFP channel encircle the wound edges. (E and F) The increased number of hemocytes in the leukemic lines leads to a more blurry appearance in both GFP and merge channels. (G) Wounds in leukemic lines are sealed equally efficiently as in wild type. Bar in (A) and (B) is 10 μm, and in (D–F) is 50 μm. Data represent means of SD; Student’s t-test on the normalized data was performed in (C). ANOVA followed by Tukey’s multiple comparison test was performed in (G). n.s., not significant.
|
2139f3
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2
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6accb70dfa2fb118c72c8d43b8dc4fd11a4c092f6c1a8f23f7d68405bd7300cc
|
2139f3.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
762,
1382
] |
[{'image_id': '2139f3', 'image_file_name': '2139f3.jpg', 'image_path': '../data/media_files/PMC5499123/2139f3.jpg', 'caption': 'Phagocytosis, hemocyte recruitment to wounds, and wound sealing are not impaired in leukemic lines. (A and B) Ex-vivo phagocytosis of Texas Red–conjugated E. coli (K-12 strain). A representative image of the HFP/w and HRS-leukemia lines is shown after phagocytosis. The red arrow indicates phagocytosed E. coli. (C) Quantification of the phagocytosis index. All leukemic lines show wild-type levels of phagocytic capacity. (D–F) Hemocyte migration to the wound edge in leukemic situation is comparable to wild type. Dashed line in phase-contrast and the GFP channel encircle the wound edges. (E and F) The increased number of hemocytes in the leukemic lines leads to a more blurry appearance in both GFP and merge channels. (G) Wounds in leukemic lines are sealed equally efficiently as in wild type. Bar in (A) and (B) is 10 μm, and in (D–F) is 50 μm. Data represent means of SD; Student’s t-test on the normalized data was performed in (C). ANOVA followed by Tukey’s multiple comparison test was performed in (G). n.s., not significant.', 'hash': '6accb70dfa2fb118c72c8d43b8dc4fd11a4c092f6c1a8f23f7d68405bd7300cc'}, {'image_id': '2139f4', 'image_file_name': '2139f4.jpg', 'image_path': '../data/media_files/PMC5499123/2139f4.jpg', 'caption': 'Toll and Imd signaling are regulated in opposite ways in leukemic larvae. Compared to control (HFP/w), expression (A) is found upregulated while Dpt A and Cec A1 (B and C) mRNA levels are downregulated in leukemic larvae compared to untreated larvae (normalized to Rpl32 after RT-qPCR, all levels are shown relative to nontreated controls, which are set to 1). Upon wounding, Drs is also upregulated in control larvae to levels similar to nonwounded leukemic lines [HFP/w in (A)]. However, no further Drs induction is observed after wounding leukemic larvae. In contrast, Dpt A and Cec A1 levels are induced after wounding in all lines. Data represent means of SD; Student’s t-test: ** P < 0.01, * P < 0.05. n.s., not significant.', 'hash': '2181e12d6d04a6f8ae0aafbb5bf3e80d776c7c27244cbb091eba23190fdc0190'}, {'image_id': '2139f5', 'image_file_name': '2139f5.jpg', 'image_path': '../data/media_files/PMC5499123/2139f5.jpg', 'caption': 'Survival of septic wounded leukemic larvae is similar to wild type. (A) Aseptic wounding: control wild-type larvae show a slight increased survival compared to leukemic larvae; however, this is not significant. (B–D) Septic wounding with both Gram-negative (E. coli and Erwinia carotovora carotovora) and Gram-positive bacteria (S. aureus). None of the septic wounds caused significant increases in mortality for the leukemic lines compared with controls. Survival was monitored until eclosion. Log-rank test on GraphPad 6.0 was performed to determine statistical significance for the survival curves. (A) P = 0.1182, (B) P = 0.2074, (C) P = 0.3682, and (D) P = 0.3491.', 'hash': '11343b8ea273d019e1666d5528c69b1ef24d4d530d7559b7c540da330edc5805'}, {'image_id': '2139f2', 'image_file_name': '2139f2.jpg', 'image_path': '../data/media_files/PMC5499123/2139f2.jpg', 'caption': 'Leukemic lines at higher temperatures die and contain hemocyte aggregates at the pupal stage. (A) At 29°, HR failed to eclose and larval/pupal mortality was observed for HRS and HRL. (B) Control HFP/w pupae. Opening of pupal cage at 46 hr after puparium formation (APF) at 29° showed fewer hemocytes and formation of adult structures (outlined in phase contrast). (C–E) Opening of pupae from leukemic lines showed clusters of hemocytes and an absence of adult structures. The dashed line in (C) outlines the vacuole-like structure (perhaps due to drying out). Green arrows point toward GFP-positive hemocytes. Scale bar, 20 μm.', 'hash': 'f348ea78c78455960981a74044a2e87d4c5afab4b6777ecaefbb176a251eecc9'}, {'image_id': '2139f1', 'image_file_name': '2139f1.jpg', 'image_path': '../data/media_files/PMC5499123/2139f1.jpg', 'caption': 'Establishing Drosophila leukemia by expressing RasV12 alone and in combination with knockdown of tumor suppressors in hemocytes. (A) Larval hemocytes from control crosses expressing eGFP (w; hml (Δ)-GAL4 UAS-eGFP × w1118, which is abbreviated to HFP/w). (B–D) The number of hemocytes increases drastically when a dominant active form of Ras (UAS-RasV12) alone (HR) or in combination with RNAi lines of tumor suppressors scribble (scrib, HRS) or lethal giant larvae [(l(2)gl, HRL] is expressed using the same driver as in (A). (A–D) show phase contrast exposures while (A’–D’) show the corresponding images under fluorescence, revealing eGFP-expressing hemocytes. (E) Quantification of late third-instar larval hemocytes for the genotypes shown in (A–D). (F) Leukemia lines are viable in the prepupal stage (12 hr of metamorphosis) at 25°. No sign of larval lethality is seen in the leukemic lines. White arrow indicates the normal prepupa. (G) Quantification of eclosion rates at 25° for all lines. Expression of RasV12 alone in hemocytes (HR) does not interfere with eclosion rates at 25°; however, reduced eclosion is observed with HRS and HRL. ANOVA followed by Tukey’s multiple comparison test was performed. The same and different letters above columns on the graphs indicate nonsignificance [a to a for instance, see (G)] and significance [a to b for example, see (E) and (G)], respectively. Data represent the means with SD; P < 0.0001 and P < 0.01 was found for (E) and (G), respectively. Bar in (A–D), 50 μm.', 'hash': '751cb8ce82be4860886b81a80d2cdaeb06dad9898edfc1a70a2b96bc6493c54c'}, {'image_id': '2139f6', 'image_file_name': '2139f6.jpg', 'image_path': '../data/media_files/PMC5499123/2139f6.jpg', 'caption': 'Leukemic lines combined with tumor suppressor knockdowns are more susceptible to nematode infection. (A) Schematic diagrams of the expression pattern of the two different GAL4 drivers used. Bx-GAL4 expression is in the salivary glands and wing discs, whereas hml (Δ)-GAL4 expression is in the larval lymph glands and in both circulating and sessile hemocytes. (B) Upon infection with entomopathogenic Heterorhabditis bacteriophora, the combination of Ras85DV12 expression and knockdown of tumor suppressors in hemocytes showed increased mortality compared to larvae expressing either construct alone (UAS- Ras85DV12 and UAS-RNAi of tumor suppressors) or in salivary glands and wing discs. Comparisons were made with respective controls (Bx-GAL4/w and HFP/w). Mortality was normalized to controls which were set to 1. Data represent means of SD; Student’s t-test: ** P < 0.01, * P < 0.05. (C) Knocking down of different RNAi lines using two GAL4 driver lines (Bx-GAL4 and HFP) and expression of Ras85DV12 alone with HFP only. When compared to respective controls, none of them showed significant mortality upon nematode infection. L, lymph gland; n.s., not significantm, S, salivary gland; W, wing disc.', 'hash': 'c5a156421419d86390e73d7dca88d3a0f4f79aa524790a5682dd4b39c527960e'}, {'image_id': '2139f7', 'image_file_name': '2139f7.jpg', 'image_path': '../data/media_files/PMC5499123/2139f7.jpg', 'caption': 'Larval locomotion is impaired in HRS compared to control (HFP/w) in the presence of nematodes. (A) In the absence of nematodes no differences were observed in the distance covered by leukemic lines and control crosses whereas in the presence of nematodes HRS lines showed reduced mobility. (B) Bending frequencies do not differ in the absence of nematodes whereas in the presence of nematodes HRS larvae show reduced bending compared to controls. Each dot represents the mean value for a replicate and the middle line represents the mean of the replicates. Error bar represents SEM; sample size was at least 115 larvae. Fisher’s LSD test: ** P < 0.01, * P < 0.05.', 'hash': 'be84d4e364cdb7c73f76027de35560f4014f50e4dd015da72db221bbd7e0d9a1'}]
|
{'2139f1': ['ANOVA followed by Tukey’s multiple comparison test, Student’s t-test (unpaired, two sided), Fisher’s LSD test, and Log-rank test on GraphPad 6.0 (survival analysis) were used to determine statistical significance. Data represent means of SD (<xref ref-type="fig" rid="2139f1">Figure 1</xref>, , <xref ref-type="fig" rid="2139f2">Figure 2</xref>, , <xref ref-type="fig" rid="2139f3">Figure 3</xref>, , <xref ref-type="fig" rid="2139f4">Figure 4</xref>, , <xref ref-type="fig" rid="2139f5">Figure 5</xref>, and , and <xref ref-type="fig" rid="2139f6">Figure 6</xref>) and SEM () and SEM (<xref ref-type="fig" rid="2139f7">Figure 7</xref>); ** ); ** P < 0.01, * P < 0.05.', 'To induce leukemic states in Drosophila hemocytes, we expressed a dominant-active form of Ras (RasV12) in hemocytes. To increase hemocyte-specific expression, we relied on the hemolectin driver (HmlΔ-GAL4) instead of the previously used collagen IV driver (Cg-GAL4) (Asha et al. 2003). To further increase the tumorigenic potential, we combined RasV12 with knockdowns of l(2)gl and scrib, which had previously been used in combination with RasV12 to successfully induce tumors in tissues other than hemocytes (Brumby and Richardson 2003; Pagliarini and Xu 2003). We observed a substantial increase in hemocyte counts both on expression of RasV12 alone and when coexpressed with one of the RNAi constructs for tumor suppressors (<xref ref-type="fig" rid="2139f1">Figure 1, A–E</xref>). The increase was ∼30-fold for ). The increase was ∼30-fold for RasV12-overexpression (HR in <xref ref-type="fig" rid="2139f1">Figure 1</xref>) compared with the control crosses and even more pronounced upon knockdown of tumor suppressors. ) compared with the control crosses and even more pronounced upon knockdown of tumor suppressors. L(2)gl RNAi; Ras85DV12 –larvae (HRL) showed the strongest effect with a ∼170-fold increase in hemocyte numbers while scribRNAi; Ras85DV12 (HRS) larvae contained ∼90 times more hemocytes when compared to control (HFP/w). At 25°, all combinations were viable up to the pupal stage and migrated along the wall of culture vials to prepare for pupation (<xref ref-type="fig" rid="2139f1">Figure 1F</xref>), but only ), but only RasV12-expressing pupae show eclosion rates comparable to wild type. In contrast, only a fraction of the combinations with tumor suppressors managed to eclose (<xref ref-type="fig" rid="2139f1">Figure 1G</xref>). When the temperature was raised to 29° to allow for a higher Gal4 activity, all three tumor lines died either at the larval or pupal stage (). When the temperature was raised to 29° to allow for a higher Gal4 activity, all three tumor lines died either at the larval or pupal stage (<xref ref-type="fig" rid="2139f2">Figure 2A</xref>). While upon bleeding, control pupae were shown to contain precursors for adult structures (). While upon bleeding, control pupae were shown to contain precursors for adult structures (<xref ref-type="fig" rid="2139f2">Figure 2B</xref>), most likely partially unfolded imaginal discs, larvae from all tumor lines were desiccated (), most likely partially unfolded imaginal discs, larvae from all tumor lines were desiccated (<xref ref-type="fig" rid="2139f2">Figure 2C</xref>) and the remaining hemolymph contained aggregates of GFP-expressing hemocytes () and the remaining hemolymph contained aggregates of GFP-expressing hemocytes (<xref ref-type="fig" rid="2139f2">Figure 2, C–E</xref>). Moreover, adult tissue formation was not observed in leukemic pupae (). Moreover, adult tissue formation was not observed in leukemic pupae (<xref ref-type="fig" rid="2139f2">Figure 2, C–E</xref>).).', 'We show that by expressing dominant-active RasV12 in hemocytes, a substantial increase in hemocyte numbers is induced, confirming earlier results using a different driver (Asha et al. 2003). Hemocyte overproliferation is further exacerbated when tumor suppressors are knocked down (<xref ref-type="fig" rid="2139f1">Figure 1</xref>). This establishes a set of three ). This establishes a set of three Drosophila leukemia models which display a graded increase in hemocyte numbers. All models show developmental defects at higher temperature but no such defects at the last larval instar at 25° (<xref ref-type="fig" rid="2139f2">Figure 2</xref>). The severity of the developmental phenotype positively correlates with the increase in hemocyte numbers and may therefore be at least partially due to a redistribution of resources upon extensive hemocyte overproliferation. This is in line with the cachexia-like phenotype, which eventually leads to pupal death (). The severity of the developmental phenotype positively correlates with the increase in hemocyte numbers and may therefore be at least partially due to a redistribution of resources upon extensive hemocyte overproliferation. This is in line with the cachexia-like phenotype, which eventually leads to pupal death (<xref ref-type="fig" rid="2139f2">Figure 2, C–E</xref>).).'], '2139f3': ['To address the physiology and immune status of leukemic larvae we first assessed their phagocytic capacity by counting phagocytosed Texas red–conjugated bacteria and found that none of the leukemic lines displayed a reduction in phagocytosis (<xref ref-type="fig" rid="2139f3">Figure 3, A–C</xref>). Similarly, upon wounding, hemocytes were attracted to wound sites in all lines and larvae survived wounding equally well, although there was a trend for HRS larvae to survive slightly worse than the other lines (). Similarly, upon wounding, hemocytes were attracted to wound sites in all lines and larvae survived wounding equally well, although there was a trend for HRS larvae to survive slightly worse than the other lines (<xref ref-type="fig" rid="2139f3">Figure 3, D–G</xref>). To check whether the proliferation of hemocytes had any proinflammatory effects, we analyzed the expression of antimicrobial peptide gene reporters for two major ). To check whether the proliferation of hemocytes had any proinflammatory effects, we analyzed the expression of antimicrobial peptide gene reporters for two major Drosophila immune pathways, including Drs (a proxy for the Toll pathway, <xref ref-type="fig" rid="2139f4">Figure 4A</xref>), Diptericin A (), Diptericin A (Dpt A, an Imd reporter, <xref ref-type="fig" rid="2139f4">Figure 4B</xref>), and Cecropin A1 (), and Cecropin A1 (Cec A1, which receives input from both pathways, <xref ref-type="fig" rid="2139f4">Figure 4C</xref>). Compared to untreated control larvae, ). Compared to untreated control larvae, Drs expression was activated in leukemic larvae and both Dpt A and Cec A1 reduced, although to a lesser extent. Wounding nonleukemic larvae induced Drs to the same degree as the leukemic stage (<xref ref-type="fig" rid="2139f4">Figure 4A</xref>). ). Dpt A and Cec A1, which are more dependent on Imd signaling, were induced to a similar extent upon wounding all leukemic and control lines (<xref ref-type="fig" rid="2139f4">Figure 4B</xref>). Taken together, this indicates that several aspects of cellular immunity appear normal in leukemic lines but there is a shift in the immune status, which mimics some aspects of wounding.). Taken together, this indicates that several aspects of cellular immunity appear normal in leukemic lines but there is a shift in the immune status, which mimics some aspects of wounding.'], '2139f5': ['To assess the immune competence of leukemic lines, we infected larvae with either Gram-positive or Gram-negative bacteria (<xref ref-type="fig" rid="2139f5">Figure 5</xref>). Leukemic lines survived equally well as controls upon infection with ). Leukemic lines survived equally well as controls upon infection with Staphylococcus aureus, which is primarily cleared via phagocytosis (Defaye et al. 2009). This is in line with our previous observation that phagocytic ability is not affected in leukemic hemocytes (<xref ref-type="fig" rid="2139f3">Figure 3, A–C</xref>). Similarly, infection with ). Similarly, infection with E. coli and the natural pathogen Erwinia carotovora carotovora failed to induce higher mortality in leukemic larvae.'], '2139f6': ['To analyze the immune status of leukemic lines in a more natural setting, we infected larvae with the EPN H. bacteriophora and its associated bacterium Photorhabdus luminescens. Heterorhabditis infects insect hosts by entering the hemocoel via the cuticle or the gut and subsequently releases Photorhabdus into the hemolymph. All three leukemic lines were infected using EPNs as well as control crosses and single knockdown lines for the tumor suppressors. We also included crosses where all combinations were expressed under control of the Beadex driver (Bx-GAL4) in the wing discs and the salivary glands [<xref ref-type="fig" rid="2139f6">Figure 6A</xref>, see also , see also Hauling et al. (2014)]. Although some lines with only one construct showed a trend toward increased susceptibility, none of these were significant (<xref ref-type="fig" rid="2139f6">Figure 6C</xref>). Only the leukemic lines that expressed ). Only the leukemic lines that expressed RasV12 in combination with tumor suppressor knockdowns were consistently more sensitive to nematode infections (<xref ref-type="fig" rid="2139f6">Figure 6B</xref>). Of note, even crosses with the Beadex driver showed similar mortality when compared to their respective control, although immune activation had been observed upon expression of both ). Of note, even crosses with the Beadex driver showed similar mortality when compared to their respective control, although immune activation had been observed upon expression of both RasV12 alone and in combination with tumor suppressor knockdowns (Hauling et al. 2014 and Krautz, R. Khalili, D. Hauling, T. and Theopold, U., unpublished data).'], '2139f7': ['Since we did not detect any gross developmental defects or deficiencies in the cellular response of leukemic larvae, we considered alternative explanations for the increased susceptibility of leukemic lines toward EPNs such as subtle differences in their behavior. To analyze larval locomotion, we used frustrated total internal reflection of infrared light (Risse et al. 2013), which we recently adapted for use in infection studies (Kunc et al. 2017). When exposed to nematodes, HRS larvae covered shorter distances than larvae from control crosses or the two other leukemic lines. However, no such difference was observed in the absence of nematodes (<xref ref-type="fig" rid="2139f7">Figure 7A</xref>). The results from measuring the frequency of bending, which is part of larval avoidance behavior, were less clear but they showed that in the presence of nematodes, the bending frequency differed between HR and HRS larvae compared to controls (). The results from measuring the frequency of bending, which is part of larval avoidance behavior, were less clear but they showed that in the presence of nematodes, the bending frequency differed between HR and HRS larvae compared to controls (<xref ref-type="fig" rid="2139f7">Figure 7B</xref>). This suggests that in the presence of nematodes, leukemic larvae are at least by some measures less mobile, which will decrease their chances of escaping infection.). This suggests that in the presence of nematodes, leukemic larvae are at least by some measures less mobile, which will decrease their chances of escaping infection.', 'When larvae were infected with EPNs, those expressing RasV12 in combination with either knockdown for tumor suppressors were more susceptible. This was most significant for the scrib knockdown. As expected for a complex model such as EPN infections, our data do not fully explain why leukemic lines are more susceptible but the dysregulation of the two major immune pathways provides a partial explanation: precocious activation of Toll-dependent genes may induce a proinflammatory state which hampers the response toward EPNs. Alternatively, or in addition, the repression of Imd targets may delay the response against the symbiotic bacteria that are released by EPNs after their entry into the hemocoel (Hallem et al. 2007; Kucerova et al. 2015; Arefin et al. 2014; Castillo et al. 2015). We also observed behavioral changes in the leukemic lines, some of which can be linked to the presence of nematodes (<xref ref-type="fig" rid="2139f7">Figure 7</xref>) and a trend for decreased survival after injury () and a trend for decreased survival after injury (<xref ref-type="fig" rid="2139f5">Figure 5A</xref>), which together may exacerbate the susceptibility toward EPNs. Taken together our data indicate that ), which together may exacerbate the susceptibility toward EPNs. Taken together our data indicate that Drosophila leukemia models, which combine overexpression of RasV12 with knockdown of tumor suppressors, are more susceptible toward a naturally relevant infection despite a lack of developmental, behavioral, and immune defects in standard assays. This mimics the specific defects observed in some leukemias (Forconi and Moss 2015) and provides useful tools to study subtle interactions between tumor progression, inflammation, and immunity. Further screens for modifiers will permit a detailed genetic dissection of the genes and pathways involved. These will include genetic screens as well as screens for potential chemical modifiers (McCubrey et al. 2008) and will address candidate pathways involved in cachexia, stress, and sensors for cellular integrity (Sonoshita and Cagan 2017).']}
|
Drosophila The Immune Phenotype of Three Leukemia Models
|
[
"Ras",
"oncogene",
"nematodes",
"insect immunity",
"hemocyte",
"Genetics of Immunity"
] |
None
|
1493967600
|
Cells fine-tune their metabolic programs according to nutrient availability in order to maintain homeostasis. This is achieved largely through integrating signaling pathways and the gene expression program, allowing cells to adapt to nutritional change. Dbp2, a member of the DEAD-box RNA helicase family in , has been proposed to integrate gene expression with cellular metabolism. Prior work from our laboratory has reported the necessity of in proper gene expression, particularly for genes involved in glucose-dependent regulation. Here, by comparing differentially expressed genes in ∆ to those of 700 other deletion strains from other studies, we find that and , which form a complex and inhibit transcription of numerous genes, corepress a common set of genes with Gene ontology (GO) annotations reveal that these corepressed genes are related to cellular metabolism, including respiration, gluconeogenesis, and alternative carbon-source utilization genes. Consistent with a direct role in metabolic gene regulation, loss of either or results in increased cellular respiration rates. Furthermore, we find that corepressed genes have a propensity to be associated with overlapping long noncoding RNAs and that upregulation of these genes in the absence of correlates with decreased binding of Cyc8 to these gene promoters. Taken together, this suggests that Dbp2 integrates nutrient availability with energy homeostasis by maintaining repression of glucose-repressed, Cyc8-targeted genes across the genome.
|
[
"Adaptation, Physiological",
"DEAD-box RNA Helicases",
"Energy Metabolism",
"Gene Deletion",
"Gene Expression Regulation, Fungal",
"Nuclear Proteins",
"Repressor Proteins",
"Response Elements",
"Saccharomyces cerevisiae",
"Saccharomyces cerevisiae Proteins"
] |
other
|
PMC5499123
| null |
75
|
[
"{'Citation': 'Akita O., Nishimori C., Shimamoto T., Fujii T., Iefuji H., 2000. \\u2003Transport of pyruvate in Saccharomyces cerevisiae and cloning of the gene encoded pyruvate permease. Biosci. Biotechnol. Biochem. 64: 980–984.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '10879467'}}}",
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"{'Citation': 'Beck Z. T., Cloutier S. C., Schipma M. J., Petell C. J., Ma W. K., et al. , 2014. \\u2003Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae. Genetics 198: 1001–1014.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4224148'}, {'@IdType': 'pubmed', '#text': '25164881'}]}}",
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] |
G3 (Bethesda). 2017 May 5; 7(7):2139-2149
|
NO-CC CODE
|
|
Chronic exposure to PMMTM induces
neoplastic transformation
of human bronchial epithelial cells. (A) Schematic representation
of chronic exposure model. BEAS-2B cells were continuously exposed
to noncytotoxic concentration (1 μg/mL) of PMCON,
PMMTM, Si, and Mo for 3 months and designated as B-PMCON, B-PMMTM, B-Si, and B-Mo cells, respectively.
BEAS-2B cells maintained in culture without particle exposure (B-NTX)
served as passage control cells. (B, C) Cells were seeded on 0.5%
agar plates, and after 2 weeks they were visualized under a phase
contrast microscope. (C) Quantification of large colonies (>50
μm
in diameter). Data are mean ± SD (n = 4). *P < 0.05 (power >95%) vs passage control B-NTX cells.
**P < 0.05 (power >80%) vs B-PMCON cells.
|
es-2014-04263u_0002
|
2
|
53f31e3d385b1a702a64d25764c66164096e022b1e960ac3601b1021461b3a68
|
es-2014-04263u_0002.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
666,
819
] |
[{'image_id': 'es-2014-04263u_0007', 'image_file_name': 'es-2014-04263u_0007.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0007.jpg', 'caption': 'Analysis of ex vivo tumors of human lung cancer H460 cells.\nAt\n3 weeks postinjection, SC tumors were dissected from mice bearing\nH460 and B-PMCON, B-PMMTM, B-Si, or B-Mo cells.\n(A) Representative bioluminescence images of mice and SC tumors. (B)\nQuantitative analysis of bioluminescence signals from SC tumors. Data\nare mean ± SD (n = 4). *P <\n0.05 (power >60%) vs H460 and B-PMCON coinjection. (C)\nThe weight of dissected SC tumors. (D) Quantitative analysis of bioluminescence\nsignals from the back of mice.', 'hash': '4ec75014bea74a1c4a274bc509dd12f3021e53ab6c816b433e477e0ded1afc44'}, {'image_id': 'es-2014-04263u_0008', 'image_file_name': 'es-2014-04263u_0008.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0008.jpg', 'caption': 'No caption found', 'hash': '7177c4a1dee10c3c0686039ec2f3debc6cde37d3c91644f061a0c5ab2a02e64e'}, {'image_id': 'es-2014-04263u_0006', 'image_file_name': 'es-2014-04263u_0006.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0006.jpg', 'caption': 'Chronic PMMTM exposed cells\npromote tumor formation\nof human nonsmall cell lung cancer H460 cells in mice. (A) Growth\nkinetics of H460 or transformed B-PMCON, B-PMMTM, B-Si, and B-Mo cells (1 × 106 cells) when SC injected\ninto the NSG mice alone. E indicates the end of experiment. (B) Transformed\ncells at the dose of 6 × 105 cells were coinjected\nwith luciferase-labeled H460 cells at the dose of 3 × 105 cells (2:1 ratio) into the left and right flanks of NSG mice.\nTumor formation was monitored weekly by IVIS bioluminescence imaging.\nRepresentative IVIS images of mice at day 1 and week 2 are shown.\n(C, D) Normalization of tumor bioluminescence signals at 1 (C) and\n2 (D) weeks postinjection to their initial signal at day 1. Data are\nmean ± SD (n = 4). *P <\n0.05 (power >60%) vs H460 and B-PMCON coinjection.', 'hash': 'a2b780f0678f461b16fd4af8d5e622f02bad711f9b6679c15388860a56607137'}, {'image_id': 'es-2014-04263u_0001', 'image_file_name': 'es-2014-04263u_0001.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0001.jpg', 'caption': 'Effect of acute PMMTM exposure on cytotoxicity of human\nbronchial epithelial cells. (A) Subconfluent monolayers of BEAS-2B\ncells were left untreated (nontreatment, NTX) or treated with various\nconcentrations (0.1–10 μg/mL) of PMMTM and\nPMCON for 48 h and analyzed for cell viability using MTT\nassay. (B) Cells were treated with Si or Mo at the same concentration\nrange and analyzed for cell viability after 48 h by MTT assay. Data\nare mean ± SD (n = 4).', 'hash': '201e58fe6ec23d31bc7277af57a28a4d07667c9425640c55965dcd40cea92b11'}, {'image_id': 'es-2014-04263u_0002', 'image_file_name': 'es-2014-04263u_0002.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0002.jpg', 'caption': 'Chronic exposure to PMMTM induces\nneoplastic transformation\nof human bronchial epithelial cells. (A) Schematic representation\nof chronic exposure model. BEAS-2B cells were continuously exposed\nto noncytotoxic concentration (1 μg/mL) of PMCON,\nPMMTM, Si, and Mo for 3 months and designated as B-PMCON, B-PMMTM, B-Si, and B-Mo cells, respectively.\nBEAS-2B cells maintained in culture without particle exposure (B-NTX)\nserved as passage control cells. (B, C) Cells were seeded on 0.5%\nagar plates, and after 2 weeks they were visualized under a phase\ncontrast microscope. (C) Quantification of large colonies (>50\nμm\nin diameter). Data are mean ± SD (n = 4). *P < 0.05 (power >95%) vs passage control B-NTX cells.\n**P < 0.05 (power >80%) vs B-PMCON cells.', 'hash': '53f31e3d385b1a702a64d25764c66164096e022b1e960ac3601b1021461b3a68'}, {'image_id': 'es-2014-04263u_0005', 'image_file_name': 'es-2014-04263u_0005.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0005.jpg', 'caption': 'Chronic PMMTM exposure enhances migration of human bronchial\nepithelial cells. (A) Confluent monolayers of B-NTX, B-PMCON, B-PMMTM, B-Si, and B-Mo cells were wounded, and the\ncells were allowed to migrate for 24 h. Wound space was visualized\nunder a phase contrast microscope and analyzed by comparing the change\nin wound space as a percentage of wound closure. Data are mean ±\nSD (n = 3). *P < 0.05 (power\n>80%) vs passage control B-NTX cells. **P <\n0.05\n(power >80%) vs B-PMCON cells. (B) Representative micrographs\nfrom three independent experiments were shown.', 'hash': '2255418a314089c565ae32acda185ef70a45431e31b0998abe40c4e4b5c54258'}, {'image_id': 'es-2014-04263u_0004', 'image_file_name': 'es-2014-04263u_0004.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0004.jpg', 'caption': 'Chronic PMMTM exposure alters cell\ncycle of human bronchial\nepithelial cells. (A) B-NTX, B-PMCON, B-PMMTM, B-Si, and B-Mo cells were serum starved for 12 h to synchronize\ntheir cell cycle. They were then cultured in a complete medium for\n8 h and analyzed for their cell cycle by flow cytometry. Representative\nhistograms from three independent experiments were shown. (B) Plots\nare percentages of total cells (50\u2009000 events) in each phase\nof the cell cycle (G1, S, and G2/M).', 'hash': '82829ee56450f286fe8ab07104d89819a47a1d5c8ddfae526e6d66959da10f22'}, {'image_id': 'es-2014-04263u_0003', 'image_file_name': 'es-2014-04263u_0003.jpg', 'image_path': '../data/media_files/PMC4224494/es-2014-04263u_0003.jpg', 'caption': 'Chronic PMMTM exposure accelerates proliferation of\nhuman bronchial epithelial cells. (A) B-PMCON, B-PMMTM, B-Si, B-Mo, and B-NTX cells were plated in 24-well plates\nat the density of 3 × 104 cells in growth medium.\nAfter 2 and 5 days, the cells were counted using an automated cell\ncounter. Data are mean ± SD (n = 3). *P < 0.05 (power >95%) vs passage control B-NTX cells.\n(B) Cells were labeled with membrane dye CellVue Claret. After 4 days\nof culture, cellular fluorescence intensity was determined by flow\ncytometry, and proliferative index was calculated. (C) Representative\nflow cytometric histograms from three independent experiments showing\nbrightly stained parental cells and weakly stained daughter cells.', 'hash': '46ec20a79de660e0fd61df455637eac596366f7fc2b47017e07e8233cb8a1852'}]
|
{'es-2014-04263u_0001': ['The purpose\nof this study was to establish an experimental human lung cell model\nfor PMMTM carcinogenesis studies that would allow further\nmolecular and cellular mechanistic studies underlying cancer-like\nphenotypes. We first characterized the acute cytotoxic effect of PMs\nto determine their noncytotoxic concentrations for subsequent long-term\nexposure studies. Human bronchial epithelial cells were exposed to\nvarious concentrations (0.1–10 μg/mL) of PMCON and PMMTM for 48 h, and cell viability was determined\nby MTT assay. The results showed that none of the PMCON and PMMTM treatments caused a significant effect on cell\nviability as compared to nontreated (NTX) control (Figure <xref rid="es-2014-04263u_0001" ref-type="fig">1</xref>A). We similarly tested the dose effect of inorganic\nchemical elements of PMA). We similarly tested the dose effect of inorganic\nchemical elements of PMMTM (Si and Mo) on cell viability.\nThe results similarly showed the noncytotoxic effect of Si and Mo\nat the treatment doses of 0.1–10 μg/mL (Figure <xref rid="es-2014-04263u_0001" ref-type="fig">1</xref>B). As we observed a slight increase in cell viability\n(proliferation), albeit not significant, at the high dose of PMB). As we observed a slight increase in cell viability\n(proliferation), albeit not significant, at the high dose of PMMTM (10 μg/mL), we used a lower dose (1 μg/mL)\nin our subsequent chronic exposure studies.'], 'es-2014-04263u_0002': ['To mimic the long-term carcinogenic process, cells were chronically\nexposed to a noncytotoxic concentration of PMs at 1 μg/mL (0.1\nμg/cm2 surface area dose) and passaged biweekly.\nThis surface area dose mimics the in vivo dose in rodents of 0.5 mg\nor approximately 8.5 years of human inhalation exposure as described\nin Materials and Methods. In this study, the\ncells were exposed to PMMTM, PMCON, or left\nuntreated for 3 months (Figure <xref rid="es-2014-04263u_0002" ref-type="fig">2</xref>A), after which\nthey were grown in complete medium (without treatment) for at least\n10 passages and examined for anchorage-independent growth by soft-agar\ncolony formation assay, which is one of the most stringent indicators\nof neoplastic transformation.A), after which\nthey were grown in complete medium (without treatment) for at least\n10 passages and examined for anchorage-independent growth by soft-agar\ncolony formation assay, which is one of the most stringent indicators\nof neoplastic transformation.23 To determine\nthe key inorganic constituents of PMMTM that may contribute\nto its pathological effect, cells were similarly exposed to Si and\nMo, and analyzed for cell transformation. Figure <xref rid="es-2014-04263u_0002" ref-type="fig">2</xref>B,C show that as compared to particle-control B-PMB,C show that as compared to particle-control B-PMCON cells, the B-PMMTM and B-Mo cells formed larger and greater\nnumbers of colony, whereas the B-Si cells exhibited a similar colony\nforming activity. These results indicate the neoplastic transformation\nof B-PMMTM and B-Mo cells.', 'Anchorage-independent growth\nhas been well correlated with the\ntumorigenicity and invasiveness of several cancer cell types.24 Colony formation under soft agar assay, the\ngold standard test to evaluate the ability of cells to undergo anchorage-independent\ngrowth, is therefore the most stringent indicator for neoplastic transformation.\nWe showed that chronic PMMTM-exposed B-PMMTM cells induced larger number and size of colonies as compared to\nchronic PMCON-exposed B-PMCON cells and passage-matched\ncontrol B-NTX cells (Figure <xref rid="es-2014-04263u_0002" ref-type="fig">2</xref>). It has previously\nbeen reported that BEAS-2B cells might undergo squamous differentiation\nin the presence of serum.). It has previously\nbeen reported that BEAS-2B cells might undergo squamous differentiation\nin the presence of serum.33,34 However, because B-PMCON and B-NTX cells showed no phenotypic changes or neoplastic\nbehavior under the culture condition, it can be concluded that the\nB-PMMTM cells, not B-PMCON and B-NTX cells,\nhave undergone differentiation and show altered phenotype due to continued\nexposure. In order to delineate the chemical effects of PMMTM inorganic elements, bronchial epithelial cells were similarly exposed\nto Si and Mo and neoplastic transformation was observed in the Mo-exposed\nB-Mo cells, but not Si-exposed B-Si cells. Given that the magnitude\nof Mo effect was similar to the PMMTM, despite its higher\nconcentration than those presented in PMMTM, it is likely\nthat (i) some other elements could be involved in the PMMTM effect and that (ii) such an effect arose from the synergistic effect\nof more than one component (e.g., Si and Mo).'], 'es-2014-04263u_0003': ['Excessive cell\ngrowth is one of the carcinogenic properties of\nmalignant cells.24,25 To determine whether chronic\nPM exposure affects cell growth characteristic, the exposed cells\nwere analyzed for cell proliferation by direct cell counting and dye-based\nassays. Figure <xref rid="es-2014-04263u_0003" ref-type="fig">3</xref>A shows that the B-PMA shows that the B-PMMTM and B-Mo cells exhibited a significantly higher proliferation rate\nthan the B-PMCON cells, which grew at a similar rate as\nthe B-NTX and B-Si cells. To confirm this result which was based on\ndirect cell counting assay, the cells were stained with membrane dye\nCellVue Claret, and their proliferative index was determined by flow\ncytometry. This dye-based assay measures cell proliferation based\non the principle of dye dilution upon cell division. Consistent with\nthe direct cell counting result, the dye-based assay indicated a higher\nproliferative index of B-PMMTM and B-Mo cells compared\nto B-PMCON and B-Si cells (Figure <xref rid="es-2014-04263u_0003" ref-type="fig">3</xref>B). Analysis of cellular fluorescence intensity further indicated\nthe division of parental cells with the observed seventh generation\nof daughter cells only in the B-PMB). Analysis of cellular fluorescence intensity further indicated\nthe division of parental cells with the observed seventh generation\nof daughter cells only in the B-PMMTM and B-Mo cells (Figure <xref rid="es-2014-04263u_0003" ref-type="fig">3</xref>C), thus substantiating the above finding.C), thus substantiating the above finding.', 'Various carcinogenic\nproperties representing the hallmarks of malignant\ncells were further assessed in this study. The transformed B-PMMTM and B-Mo cells demonstrated excessive cell growth and altered\ncell cycle (Figures <xref rid="es-2014-04263u_0003" ref-type="fig">3</xref> and and <xref rid="es-2014-04263u_0004" ref-type="fig">4</xref>). To our knowledge, this is the first demonstration of the\ninduction of cell proliferation by chronic low-dose PM exposure, although\nthe inhibition of cell proliferation). To our knowledge, this is the first demonstration of the\ninduction of cell proliferation by chronic low-dose PM exposure, although\nthe inhibition of cell proliferation35,36 and induction\nof cytotoxicity37,38 by acute high-dose PMs from urban\nand industrial areas have previously been demonstrated. Interestingly,\nB-PMMTM and B-Mo cells promoted cell cycle at different\nphases, possibly due to (i) the low content of Mo present in the PMMTM that might not be sufficient to drive the cells to G2/M\nphase, and (ii) the PMMTM proliferative effect was probably\nthe result of components other than Mo or a synergistic effect of\nmore than one component. Cell motility was also shown to increase\nsignificantly in the B-PMMTM and B-Mo cells as compared\nto B-NTX cells (Figure <xref rid="es-2014-04263u_0005" ref-type="fig">5</xref>), thus indicating\ntheir aggressive behaviors, which could be important in tumor progression.), thus indicating\ntheir aggressive behaviors, which could be important in tumor progression.'], 'es-2014-04263u_0004': ['To delineate the mechanism of\nPMMTM-induced cell proliferation,\nwe investigated the cell cycle progression of synchronized B-PMMTM, B-PMCON, B-Si, and B-Mo cells by flow cytometry\nusing propidium iodide (PI) DNA staining assay. As depicted in Figure <xref rid="es-2014-04263u_0004" ref-type="fig">4</xref>A, a higher percentage of B-PMA, a higher percentage of B-PMMTM cells\ncompared to B-PMCON cells entered the S phase (∼80%\nvs 50%) and reached the G2/M transition phase (∼5% vs 1%),\nwhereas the B-Mo cells had a significant portion in the G2/M phase\n(∼10%). These results indicated the promotion of S phase entry\nby chronic PMMTM exposure and the transition to G2/M phase\nby chronic Mo exposure.'], 'es-2014-04263u_0005': ['The aggressive behavior of PMMTM-exposed cells was examined\nby assessing their migratory activity, which is a key determinant\nof tumor invasion and progression.26,27 Cell migration\nwas determined by scratch or wound healing assay. At 24 h after the\nscratch, B-PMMTM and B-Mo cells showed a significantly\nhigher motility rate toward the wound compared to B-NTX, B-PMCON and B-Si cells, as judged by their greater wound closure\n(Figure <xref rid="es-2014-04263u_0005" ref-type="fig">5</xref>A,B). These results indicate the induction\nof aggressive cell behavior by chronic exposure to PMA,B). These results indicate the induction\nof aggressive cell behavior by chronic exposure to PMMTM and Mo.'], 'es-2014-04263u_0006': ['Carcinogenesis is a multistep sequential process consisting of\nthree major stages, namely, initiation, promotion, and progression.12,28,29 Certain carcinogens can act in\none or all of these stages, which results in neoplastic transformation\nand tumor development.30 Having demonstrated\nthe neoplastic transformation of B-PMMTM cells, we next\nassessed their tumorigenic potential in vivo. The B-PMMTM cells and their control B-PMCON cells as well as B-NTX,\nB-Mo, and B-Si cells (1 × 106) were injected into\nNSG mice subcutaneously and tumor formation was determined over time.\nNo tumor formation was observed with any of the above treatments including\nthose injected with the neoplastic B-PMMTM and B-Mo cells\n(Figure <xref rid="es-2014-04263u_0006" ref-type="fig">6</xref>A), indicating their inherent nontumorigenicity.\nTo test whether these cells might possess tumor-promoting activity,\nwe coinjected the B-PMA), indicating their inherent nontumorigenicity.\nTo test whether these cells might possess tumor-promoting activity,\nwe coinjected the B-PMMTM, B-PMCON, B-Si, or\nB-Mo cells (6 × 105) with tumorigenic human lung cancer\nH460 cells (3 × 105), which have been modified to\nexpress luciferase to aid quantitation of tumor formation in mice\nby bioluminescence imaging (Figure <xref rid="es-2014-04263u_0006" ref-type="fig">6</xref>B). Tumor\nluminescence signals were quantified over time and normalized to their\ninitial signal at the time of inoculation (day 1). At 1 week postinjection,\ntumor luminescence was higher in mice bearing the H460 cells with\nB-PMB). Tumor\nluminescence signals were quantified over time and normalized to their\ninitial signal at the time of inoculation (day 1). At 1 week postinjection,\ntumor luminescence was higher in mice bearing the H460 cells with\nB-PMMTM, B-Mo, or B-Si cells as compared to the mice bearing\nthe H460 with B-PMCON cells (Figure <xref rid="es-2014-04263u_0006" ref-type="fig">6</xref>C). At 2 weeks postinjection, the tumor luminescence intensity was\nhigh only in the mice injected with H460 cells and B-PMC). At 2 weeks postinjection, the tumor luminescence intensity was\nhigh only in the mice injected with H460 cells and B-PMMTM or B-Mo cells, but not B-PMCON or B-Si cells (Figure <xref rid="es-2014-04263u_0006" ref-type="fig">6</xref>D). These results indicate the tumor-promoting activity\nof B-PMD). These results indicate the tumor-promoting activity\nof B-PMMTM and B-Mo cells.', 'The potential role of PMMTM in lung carcinogenesis was\nfurther evaluated in vivo using a mouse xenograph model. Our results\ndemonstrated that B-PMMTM cells, although not directly\ntumor-inducing in mice, promoted tumor formation and metastasis of\nhuman lung cancer H460 cells (Figures <xref rid="es-2014-04263u_0006" ref-type="fig">6</xref> and and <xref rid="es-2014-04263u_0007" ref-type="fig">7</xref>). The limitation of our in vivo study is the relatively\nsmall number of animals per group, and given the high individual biological\nvariation, we could not obtain statistical power >80%. Despite\nthe\nlow statistical power, we were still able to achieve statistical significance\n(). The limitation of our in vivo study is the relatively\nsmall number of animals per group, and given the high individual biological\nvariation, we could not obtain statistical power >80%. Despite\nthe\nlow statistical power, we were still able to achieve statistical significance\n(P < 0.05). These results however are in good\nagreement with previous reports showing the hypermethylation of tumor\nsuppressor p16 by PMs from urban areas, which could lead to cancer\ndevelopment39 and the induction of lung\ncarcinoma by Mo inhalation exposure in mice.40'], 'es-2014-04263u_0007': ['At the end of the experiments\n(week 3), SC tumors were dissected\nand their bioluminescence were determined and compared between groups\n(Figure <xref rid="es-2014-04263u_0007" ref-type="fig">7</xref>A). Figure A). Figure <xref rid="es-2014-04263u_0007" ref-type="fig">7</xref>B shows a stronger bioluminescence signal in tumors from H460 and\nB-PMB shows a stronger bioluminescence signal in tumors from H460 and\nB-PMMTM or H460 and B-Mo cells, compared to those from\nH460 and B-PMCON or H460 and B-Si cells. Analysis of tumor\nweight of the samples further supported the tumor-promoting role of\nB-PMMTM and B-Mo cells (Figure <xref rid="es-2014-04263u_0007" ref-type="fig">7</xref>C). Interestingly, we observed notable tumor bioluminescence signals\nin mice bearing H460 and B-PMC). Interestingly, we observed notable tumor bioluminescence signals\nin mice bearing H460 and B-PMMTM, B-Si or B-Mo cells after\nthe dissection of SC tumors (Figure <xref rid="es-2014-04263u_0007" ref-type="fig">7</xref>A,D),\nsuggesting metastasis of tumor cells to neighboring tissues and strengthening\nthe important role of chronic PMA,D),\nsuggesting metastasis of tumor cells to neighboring tissues and strengthening\nthe important role of chronic PMMTM and Mo exposure in\ntumor promotion.']}
|
Appalachian
Mountaintop Mining Particulate Matter
Induces Neoplastic Transformation of Human Bronchial Epithelial Cells
and Promotes Tumor Formation
| null |
Environ Sci Technol
|
1415088000
|
Epidemiological studies suggest that living near mountaintop coal mining (MTM) activities is one of the contributing factors for high lung cancer incidence. The purpose of this study was to investigate the long-term carcinogenic potential of MTM particulate matter (PMMTM) exposure on human bronchial epithelial cells. Our results show that chronic exposure (3 months) to noncytotoxic, physiological relevant concentration (1 μg/mL) of PMMTM, but not control particle PMCON, induced neoplastic transformation, accelerated cell proliferation, and enhanced cell migration of the exposed lung cells. Xenograft transplantation of the PMMTM-exposed cells in mice caused no apparent tumor formation, but promoted tumor growth of human lung carcinoma H460 cells, suggesting the tumor-promoting effect of PMMTM. Chronic exposure to the main inorganic chemical constituent of PMMTM, molybdenum but not silica, similarly induced cell transformation and tumor promotion, suggesting the contribution of molybdenum, at least in part, in the PMMTM effects. These results provide new evidence for the carcinogenic potential of PMMTM and support further risk assessment and implementation of exposure control for PMMTM.
|
[
"Animals",
"Appalachian Region",
"Carcinogens",
"Cell Line, Tumor",
"Cell Movement",
"Cell Proliferation",
"Cell Transformation, Neoplastic",
"Coal Mining",
"Epithelial Cells",
"Humans",
"Lung",
"Lung Neoplasms",
"Mice",
"Mice, SCID",
"Molybdenum",
"Particulate Matter",
"Silicon Dioxide",
"Toxicity Tests, Chronic",
"Xenograft Model Antitumor Assays"
] |
other
|
PMC4224494
| null |
41
|
[
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Environ Sci Technol. 2014 Nov 4; 48(21):12912-12919
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Effect of Siah1a on mGluR surface expression and subcellular distribution. A, phase contrast image, GFP fluorescence, and surface cy3-conjugated anti-myc labeling of mGluR5a-myc from a cell expressing mGluR5a-myc alone (upper) and a cell coexpressing Siah1a (lower). Cy3-conjugated primary anti-myc antibody was applied to live cells before fixation to ensure labeling of only extracellular surface epitopes (see methods). B, Fluorescence measurments for cells expressing mGluR5a-myc (black bar) and mGluR5a-myc + Siah1a (open bar), as in A. The number of cells analyzed for each group is shown in parentheses. Units are arbitrary fluorescence units derived from 8 bit images (see methods). No significant difference was detected between the two groups (T-Test, p > 0.3). C, Phase contrast image and GFP fluorescence from a cell expressing mGluR1a-GFP alone (upper) and mGluR1a-GFP with Siah1a (lower).
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[{'image_id': '1471-2202-2-15-2', 'image_file_name': '1471-2202-2-15-2.jpg', 'image_path': '../data/media_files/PMC58838/1471-2202-2-15-2.jpg', 'caption': 'Siah1a effect is specific for group I mGluRs. Bar graph illustrating average (+SEM) calcium current inhibition via the indicated receptor in the absence (filled bars) and presence (open bars) of Siah1a. The number of cells from each group is indicated in parentheses. * indicates a significant difference from control (p < 0.05, ANOVA).', 'hash': '2c9cc63c6db18c4cda5eac40f7815602ec3680b120a742ee9e630a84680fdf46'}, {'image_id': '1471-2202-2-15-5', 'image_file_name': '1471-2202-2-15-5.jpg', 'image_path': '../data/media_files/PMC58838/1471-2202-2-15-5.jpg', 'caption': 'Summary of GFP-Siah1a-CT effect. A, Image showing GFP flourescence from a cell expressing GFP-Siah1a-CT. B, Bar graph illustrating average (+SEM) calcium current via the indicated receptor in the absence (filled bar) and presence (open bar) of GFP-Siah1a-CT. The number of cells from each group is indicated in parentheses. * indicates a significant difference from control (p < 0.05, ANOVA).', 'hash': '7b3fe1dc4dc571703f22774650126a39abf2360f2102c843b426746871581683'}, {'image_id': '1471-2202-2-15-4', 'image_file_name': '1471-2202-2-15-4.jpg', 'image_path': '../data/media_files/PMC58838/1471-2202-2-15-4.jpg', 'caption': 'Effect of GFP-Siah1a-CT on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). Voltage protocol was as in figure 1. The scale bars indicate 20 msec and 0.5 nA (A) or 1 nA (C). Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10mV).', 'hash': '4a0220c12e99e9875877654c860ed800f29e8e95b65e5541c05da8d3287e553a'}, {'image_id': '1471-2202-2-15-3', 'image_file_name': '1471-2202-2-15-3.jpg', 'image_path': '../data/media_files/PMC58838/1471-2202-2-15-3.jpg', 'caption': 'Effect of Siah1a on mGluR surface expression and subcellular distribution. A, phase contrast image, GFP fluorescence, and surface cy3-conjugated anti-myc labeling of mGluR5a-myc from a cell expressing mGluR5a-myc alone (upper) and a cell coexpressing Siah1a (lower). Cy3-conjugated primary anti-myc antibody was applied to live cells before fixation to ensure labeling of only extracellular surface epitopes (see methods). B, Fluorescence measurments for cells expressing mGluR5a-myc (black bar) and mGluR5a-myc + Siah1a (open bar), as in A. The number of cells analyzed for each group is shown in parentheses. Units are arbitrary fluorescence units derived from 8 bit images (see methods). No significant difference was detected between the two groups (T-Test, p > 0.3). C, Phase contrast image and GFP fluorescence from a cell expressing mGluR1a-GFP alone (upper) and mGluR1a-GFP with Siah1a (lower).', 'hash': 'a26c0537b20c6385c4b40bd00c442d89b41b4981123b781a9ff019c2b9de60d4'}, {'image_id': '1471-2202-2-15-6', 'image_file_name': '1471-2202-2-15-6.jpg', 'image_path': '../data/media_files/PMC58838/1471-2202-2-15-6.jpg', 'caption': 'CaM reverses the effect of Siah1a on group I mGluR-mediated calcium current inhibition. Bar graph illustrating average (+SEM) calcium current via the indicated receptor in cells expressing mGluR5b alone ("mGluR5b"), mGluR5b plus Siah1a ("+Siah1a") and mGluR5b + Siah1a and CaM ("+CaM"). The number of cells from each group is indicated in parentheses. * indicates a significant difference from control (p < 0.05, ANOVA). ** indicates a significant difference from "+Siah1a".', 'hash': 'ef455c32b03fdf77f77c9dda4eb8da2fa3f6514b821e4812f702d44ad72b74e3'}, {'image_id': '1471-2202-2-15-1', 'image_file_name': '1471-2202-2-15-1.jpg', 'image_path': '../data/media_files/PMC58838/1471-2202-2-15-1.jpg', 'caption': 'Effect of Siah1a expression on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). A triple pulse voltage protocol was used wherein cells were sustained at a holding potential of-80 mV and stepped to an initial test pulse to +10 mV for 25 msec, to +80 mV for 50 msec, then following a 10 msec step to -80 mV a second test pulse to +10 mV was applied. The scale bars indicate 20 msec and 0.4 nA. Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) and norepinephrine (NE) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10 mV).', 'hash': '2da7b6aa9f41735146a8a8a27655d373f6f0ae59da939fd03bed50b76e8ae877'}]
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{'1471-2202-2-15-1': ['To examine the role of Siah1a expression on mGluR function, SCG neurons were injected with cDNA encoding several group I mGluRs (mGluR1, 5 and their splice variants) with or without Siah1a expression. Calcium current modulation was then examined upon application of extracellular 100 μM glutamate. The triple pulse voltage protocol [39] was used as the standard test pulse to assess calcium current magnitude and voltage dependence of inhibition [38,40]. Figure <xref ref-type="fig" rid="1471-2202-2-15-1">1A</xref> illustrates sample current traces from before ("con") and during inhibition by glutamate ("Glu"), in an mGluR5b-expressing cell. Consistent with previous reports [ illustrates sample current traces from before ("con") and during inhibition by glutamate ("Glu"), in an mGluR5b-expressing cell. Consistent with previous reports [24,38], calcium current inhibition via mGluR5b appeared partially voltage dependent. The time course of inhibition by glutamate and 10 μM norepinephrine (NE), acting through endogenous α2 adrenergic receptors [41], in this cell (figure <xref ref-type="fig" rid="1471-2202-2-15-1">1B</xref>) illustrates that inhibition was rapid and reversible.) illustrates that inhibition was rapid and reversible.', 'In contrast to that in cells expressing mGluR5b alone, calcium current inhibition by glutamate in cells coexpressing mGluR5b and Siah1a was quite small (figure <xref ref-type="fig" rid="1471-2202-2-15-1">1C</xref>). Average inhibition of calcium currents by glutamate in mGluR5b-expressing cells was 33 ± 5%, (n = 7). Inhibition was significantly reduced to 10 ± 6% (n = 5; p < 0.05) in cells coexpressing mGluR5b and Siah1a (figure ). Average inhibition of calcium currents by glutamate in mGluR5b-expressing cells was 33 ± 5%, (n = 7). Inhibition was significantly reduced to 10 ± 6% (n = 5; p < 0.05) in cells coexpressing mGluR5b and Siah1a (figure <xref ref-type="fig" rid="1471-2202-2-15-2">2</xref>). This reduction of modulation by glutamate appeared to be specific for mGluRs, as NE-mediated calcium current inhibition was unaltered (figure ID). Calcium current inhibition resulting from NE application in mGluR-expressing cells was 58 ± 6% (n = 12). In cells coexpressing Siah1a, NE produced an inhibition of 54 ± 6% (n = 11; figure ). This reduction of modulation by glutamate appeared to be specific for mGluRs, as NE-mediated calcium current inhibition was unaltered (figure ID). Calcium current inhibition resulting from NE application in mGluR-expressing cells was 58 ± 6% (n = 12). In cells coexpressing Siah1a, NE produced an inhibition of 54 ± 6% (n = 11; figure <xref ref-type="fig" rid="1471-2202-2-15-2">2</xref>).).', 'Effect of GFP-Siah1a-CT on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). Voltage protocol was as in figure <xref ref-type="fig" rid="1471-2202-2-15-1">1</xref>. The scale bars indicate 20 msec and 0.5 nA (A) or 1 nA (C). Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10mV).. The scale bars indicate 20 msec and 0.5 nA (A) or 1 nA (C). Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10mV).'], '1471-2202-2-15-2': ['The strong reduction in calcium current modulation by glutamate in the presence of Siah1a was observed with every group I mGluR tested. Cells expressing an extracellularly myc-tagged mGluR5a (mGluR5a-myc) exhibited calcium current inhibition of 42 ± 4% (n = 6) upon glutamate application. This inhibition was significantly reduced to 22 ± 5% (n = 6) in cells coexpressing Siah1a (p < 0.05; figure <xref ref-type="fig" rid="1471-2202-2-15-2">2</xref>). Similarly, calcium current inhibition in cells expressing a C-terminally GFP-tagged mGluR1a (mGluR1a-GFP) and mGluR1a-GFP + Siah1a was 46 ± 5% (n = 5) and 23 ± 6% (n = 6), respectively. In addition to group I mGluRs, mGluR2 (a group II mGluR) was also examined. However, Siah1a had no detectable effect on mGluR2-mediated calcium current inhibition, consistent with reports demonstrating that Siah1a does not bind to group II mGluRs [). Similarly, calcium current inhibition in cells expressing a C-terminally GFP-tagged mGluR1a (mGluR1a-GFP) and mGluR1a-GFP + Siah1a was 46 ± 5% (n = 5) and 23 ± 6% (n = 6), respectively. In addition to group I mGluRs, mGluR2 (a group II mGluR) was also examined. However, Siah1a had no detectable effect on mGluR2-mediated calcium current inhibition, consistent with reports demonstrating that Siah1a does not bind to group II mGluRs [36]. Cells expressing mGluR2 alone exhibited 57 ± 5% inhibition of calcium current in response to glutamate (n = 6), while those coexpressing Siah1a were inhibited 61 ± 2% (n = 6; figure <xref ref-type="fig" rid="1471-2202-2-15-2">2</xref>). Together, these data demonstrate that in the presence of Siah1a, the function of group I mGluRs is selectively inhibited. These data also suggest that Siah1a is acting at the level of the receptor, since much of the calcium current modulation through group I mGluRs proceeds through a Gβγ-mediated pathway similar to that used by α). Together, these data demonstrate that in the presence of Siah1a, the function of group I mGluRs is selectively inhibited. These data also suggest that Siah1a is acting at the level of the receptor, since much of the calcium current modulation through group I mGluRs proceeds through a Gβγ-mediated pathway similar to that used by α2 adrenergic receptors and mGluR2 [38,40].'], '1471-2202-2-15-3': ['Figure <xref ref-type="fig" rid="1471-2202-2-15-3">3A</xref> ( (upper) illustrates a cell injected with cDNA encoding mGluR5a-myc alone (and coexpressing pEGFP as a positive marker for expression). Column 1 ("Phase") and column 2 ("GFP") show a phase contrast image and GFP fluorescence of such a cell. Column 3 ("anti-myc cy3") shows surface labeling of the extracullular myc epitope on mGluR5a-myc using a cy3 fluorescently-conjugated primary antibody. Live cells were exposed to antibody prior to fixation to ensure that only receptors expressed on the cell surface would be detected. Uninjected cells displayed no detectable cy3 labeling (data not shown). In similarly treated cells coexpressing Siah1a, surface expression, measured as total cell fluorescence (figure <xref ref-type="fig" rid="1471-2202-2-15-3">3B</xref>), was not significantly reduced (p = 0.33) and no receptor clustering was observed (figure ), was not significantly reduced (p = 0.33) and no receptor clustering was observed (figure <xref ref-type="fig" rid="1471-2202-2-15-3">3A</xref>, , lower). Similar experiments using a GFP-tagged group I mGluR, GFP-mGluR1a, indicated no detectable changes in receptor distribution when coexpressed with Siah1a (figure <xref ref-type="fig" rid="1471-2202-2-15-3">3C</xref>). These data suggest that neither a reduction in receptor levels nor a change in receptor association with scaffolding proteins appears to be responsible for the diminished group I mGluR-mediated calcium current modulation observed in the presence of Siah1a.). These data suggest that neither a reduction in receptor levels nor a change in receptor association with scaffolding proteins appears to be responsible for the diminished group I mGluR-mediated calcium current modulation observed in the presence of Siah1a.', 'Siah1a has been shown to be a member of the ubiquitin family of proteins involved in protein degradation through the proteasome [30,31,33]. Therefore, group I mGluR degradation was considered a potential mechanism for the decrease in mGluR-mediated calcium current modulation observed in SCG neurons. Although an obvious decrease in receptor levels in the presence of Siah1a was not detected (figure <xref ref-type="fig" rid="1471-2202-2-15-3">3</xref>), it is possible that introduction of high levels of Siah1a changed the rate of mGluR degradation such that a new equilibrium was reached with lower levels of active receptor than in the absence of Siah1a. Such a change may have been difficult to detect with the fluorescence techniques used here (figure ), it is possible that introduction of high levels of Siah1a changed the rate of mGluR degradation such that a new equilibrium was reached with lower levels of active receptor than in the absence of Siah1a. Such a change may have been difficult to detect with the fluorescence techniques used here (figure <xref ref-type="fig" rid="1471-2202-2-15-3">3</xref>). Therefore, a deletion mutant of Siah1a was constructed, GFP-Siah1a-CT, with the N-terminal RING-fmger domain deleted. This RING-fmger domain has been shown to be necessary for the ubiquitination function of Siah1a [). Therefore, a deletion mutant of Siah1a was constructed, GFP-Siah1a-CT, with the N-terminal RING-fmger domain deleted. This RING-fmger domain has been shown to be necessary for the ubiquitination function of Siah1a [46]. As a positive control for expression, this construct was fused to the C-terminus of GFP (figure <xref ref-type="fig" rid="1471-2202-2-15-5">5A</xref>). Figure ). Figure <xref ref-type="fig" rid="1471-2202-2-15-4">4A</xref> illustrates sample currents from a cell expressing mGluR5a alone. As with mGluR5b, mGluR5a produced a rapid and reversible calcium current inhibition when 100 μM glutamate was applied to the bath (figure illustrates sample currents from a cell expressing mGluR5a alone. As with mGluR5b, mGluR5a produced a rapid and reversible calcium current inhibition when 100 μM glutamate was applied to the bath (figure <xref ref-type="fig" rid="1471-2202-2-15-4">4B</xref>). Coexpression of GFP-Siah1a-CT caused a significant reduction in the magnitude of calcium current modulation via mGluR5a (figure ). Coexpression of GFP-Siah1a-CT caused a significant reduction in the magnitude of calcium current modulation via mGluR5a (figure <xref ref-type="fig" rid="1471-2202-2-15-4">4C,D</xref>). Glutamate-mediated calcium current inhibition in cells expressing mGluR5a alone was 33 ± 4% (n = 7). In cells expressing mGluR5a and GFP-Siah1a-CT, inhibition was reduced to 14 ± 3 (n = 6; p < 0.05; figure ). Glutamate-mediated calcium current inhibition in cells expressing mGluR5a alone was 33 ± 4% (n = 7). In cells expressing mGluR5a and GFP-Siah1a-CT, inhibition was reduced to 14 ± 3 (n = 6; p < 0.05; figure <xref ref-type="fig" rid="1471-2202-2-15-5">5B</xref>). Therefore, a ubiquitin-deficient Siah1a construct that can bind mGluRs is capable of producing a similar reduction in mGluR-mediated calcium current modulation as the full length Siah1a. This suggests that the effect of Siah1a on group I mGluRs is a result of its physical association with the receptor, rather than a change in receptor levels.). Therefore, a ubiquitin-deficient Siah1a construct that can bind mGluRs is capable of producing a similar reduction in mGluR-mediated calcium current modulation as the full length Siah1a. This suggests that the effect of Siah1a on group I mGluRs is a result of its physical association with the receptor, rather than a change in receptor levels.', 'To detect surface expression of the mGluR5a-myc construct, a cy3-conjugated, mouse anti-myc antibody (Sigma) was applied to live cells at room temperature for 20 minutes at a 1:150 dilution. Cells were subsequently fixed in a 4% formaldehyde (Fisher) and 4% sucrose (Fisher) solution in phosphate buffered saline (PBS) for 20 minutes, washed with PBS and imaged. Images were acquired using a Spot RT Monochrome, cooled CCD camera (Diagnostic Instruments, Inc.) mounted onto a Nikon Eclipse TE300 inverted microscope with a 40X objective (Nikon). Images were obtained using Spot software (Diagnostic Instruments) and figures were constructed using Adobe Photoshop and Canvas (Deneba Systems) software. Image analysis was performed using NIH Image software. All images analyzed in figure <xref ref-type="fig" rid="1471-2202-2-15-3">3B</xref> were taken using a 3 second exposure, chosen to put the majority of cells\' fluorescence near the center of the camera\'s dynamic range. Images of 3 cells (out of 38) were discarded for having large areas of saturation. Using 8 bit images, total fluorescence of a small area encompassing the entire cell was measured and the fluorescence of an identically sized area of background was subtracted to yield a measure of total fluorescence for each cell. Since there was no detectable change in cell size accompanying Siah1a expression measured electrophysiologically (data not shown), the fluorescence measurments were not normalized to total area. were taken using a 3 second exposure, chosen to put the majority of cells\' fluorescence near the center of the camera\'s dynamic range. Images of 3 cells (out of 38) were discarded for having large areas of saturation. Using 8 bit images, total fluorescence of a small area encompassing the entire cell was measured and the fluorescence of an identically sized area of background was subtracted to yield a measure of total fluorescence for each cell. Since there was no detectable change in cell size accompanying Siah1a expression measured electrophysiologically (data not shown), the fluorescence measurments were not normalized to total area.'], '1471-2202-2-15-6': ['Calcium current inhibition in cells expressing the group I mGluR, mGluR5b was 29 ± 2% (figure <xref ref-type="fig" rid="1471-2202-2-15-6">6</xref>; n = 21), similar to that through other group I mGluRs tested (see figure ; n = 21), similar to that through other group I mGluRs tested (see figure <xref ref-type="fig" rid="1471-2202-2-15-2">2</xref>). Coexpression of Siah1a caused a reduction in calcium current modulation to 12 ± 3%, as observed previously (n = 14). Next, when CaM was coexpressed with mGluR5b and Siah1a, calcium current modulation was significantly increased (over that in cells expressing mGluR5b and Siah1a) to 25 ± 5% (n = 12; figure ). Coexpression of Siah1a caused a reduction in calcium current modulation to 12 ± 3%, as observed previously (n = 14). Next, when CaM was coexpressed with mGluR5b and Siah1a, calcium current modulation was significantly increased (over that in cells expressing mGluR5b and Siah1a) to 25 ± 5% (n = 12; figure <xref ref-type="fig" rid="1471-2202-2-15-6">6</xref>). Finally, CaM binding to the mGluRs did not appear to be Ca). Finally, CaM binding to the mGluRs did not appear to be Ca2+-dependent. When results obtained in the presence of 11 mM EGTA, a relatively high level of Ca2+ buffering, were compared to those in low Ca2+ buffering (with 0.1 mM intracellular BAPTA), no difference was detected. Under low Ca2+-buffering conditions, calcium current inhibition in control (mGluR5b alone) was 25 ± 4% (n = 9). With Siah1a and Siah1a + CaM inhibition was 7 ± 6% (n = 3) and 30 ± 18% (n = 3), respectively. Since the results were indistinguishable with high or low Ca2+ buffering, the data from both sets were pooled in the final results (figure <xref ref-type="fig" rid="1471-2202-2-15-6">6</xref>). It should also be noted that coexpression of CaM with mGluR5a in the absence of Siah1a yields no changes in calcium current modulation (data not shown). These results indicate that the presence of high levels of CaM can reverse the effect of Siah1a on calcium current modulation, presumably by competing for binding on the C-terminal tail of the receptor.). It should also be noted that coexpression of CaM with mGluR5a in the absence of Siah1a yields no changes in calcium current modulation (data not shown). These results indicate that the presence of high levels of CaM can reverse the effect of Siah1a on calcium current modulation, presumably by competing for binding on the C-terminal tail of the receptor.']}
|
A role for Seven in Absentia Homolog (Siah1a) in metabotropic glutamate receptor signaling
| null |
BMC Neurosci
|
1002524400
|
[{'@Label': 'BACKGROUND', '@NlmCategory': 'BACKGROUND', '#text': 'Diarrhoea caused by Escherichia coli is an important cause of infant morbidity and mortality in developing countries. Enteropathogenic Escherichia coli (EPEC) is considered one of the major causes of diarrhoea in children living in developing countries. The ability of diarrhoeagenic strains of E. coli to adhere to and colonize the intestine is the first step towards developing the disease. EPEC strains adhere to enterocytes and HeLa cells in a characteristic pattern known as localized adherence. Many epidemiological studies of diarrhoea have shown that breast-feeding protects infants from intestinal infections. Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to the protection from diarrhoeal agents.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'The effects of human milk and its protein components on the localized adherence of EPEC were investigated. Non-immunoglobulin components of human milk responsible for the inhibition of EPEC adhesion to HeLa cells were isolated by chromatographic fractionation of human whey proteins. Besides secretory immunoglobulin A, which has been previously reported to affect the adhesion of EPEC, free secretory component (fSC) and lactoferrin (Lf) were isolated. Even in concentrations lower than those usually found in whole milk, fSC and Lf were able to inhibit the adhesion of EPEC. alpha-lactalbumin was also isolated, but showed no activity on EPEC adhesion.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'This study demonstrated that the immunoglobulin fraction, the free secretory component and lactoferrin of human milk inhibit EPEC adhesion to HeLa cells. These results indicate that fSC and Lf may be important non-specific defence factors against EPEC infections.'}]
|
[
"Bacterial Adhesion",
"Caseins",
"Chromatography",
"Escherichia coli",
"HeLa Cells",
"Humans",
"Lactalbumin",
"Lactoferrin",
"Milk, Human"
] |
other
|
PMC58838
| null |
41
|
[
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] |
BMC Neurosci. 2001 Oct 8; 2:15
|
NO-CC CODE
|
|
Selinexor inhibits nuclear export and forces nuclear accumulation of ACE-2 in vitro. Vero E6 cells were incubated with 500 nM selinexor or DMSO for 24 h. a) Cells were fixed with 3% paraformaldehyde, incubated with anti-ACE-2 antibody (Invitrogen, #MA5-32307) followed by a rabbit secondary antibody, Alexa Fluor 488, green-fluorescent dye (Thermo Fisher, #A11008), then visualized with the Echo Revolve fluorescent microscope (ECHO) at 60× magnification. Membrane-bound ACE-2 receptors are present in DMSO-treated cells only (white arrows). b) Sub-cellular fractionation was performed post-treatment then analyzed by immunoblots and c) quantified by densitometric analysis. d) Vero E6 and HEK 293 cells were treated with selinexor (550 nM, 24 h) and whole protein lysates were analyzed on immunoblots.
|
gr1_lrg
|
2
|
840f870bae1196466fa0abe00bb456d8a68946b0549fd6f1fe68bf2c3336fd5e
|
gr1_lrg.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
789,
558
] |
[{'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr3_lrg.jpg', 'caption': 'Selinexor decreased SARS-CoV-2 viral load in ferret lung tissue. Levels of viral RNA in the lungs were measured post-mortem on Day 4 by qPCR. Lower limit of detection range is indicated by dashed lines. Data are expressed as mean\xa0±\xa0SEM.', 'hash': 'b23c1aa50b9c96d31895e5ab477f6939ca8f2c2410e576cee80f6f27c18dbe63'}, {'image_id': 'gr7_lrg', 'image_file_name': 'gr7_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr7_lrg.jpg', 'caption': 'Selinexor diminishes inflammatory cytokine levels associated with SARS-CoV-2 infection in human cells. Human peripheral blood mononuclear cells were incubated with LPS (1000\xa0ng/ml) and increasing concentrations of selinexor for 6, 16, 24, and 40\xa0h. Supernatant was collected and analyzed by individual ELISAs. Data are expressed as mean\xa0±\xa0SEM.', 'hash': '706ea4396c1656b72d78fd3c148b3b61c0d790d301833e2a038a36b0cd3fe85c'}, {'image_id': 'ga1_lrg', 'image_file_name': 'ga1_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/ga1_lrg.jpg', 'caption': 'No caption found', 'hash': '18e4e918053c00d10987fa9ff94606d383301d032f947243d4d391b1fa0f88f8'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr2_lrg.jpg', 'caption': 'Selinexor inhibits SARS-CoV-2 viral replication and shedding in vitro. a) Vero E6 cells were pre-treated with selinexor before SARS-CoV-2 infection (left – Prophylactic) or treated with selinexor at the time of infection (right – Therapeutic). Both were incubated with selinexor during infection. Viral load was assessed by plaque assay after 4 days of cell incubation with overlay (Selinexor CC50 of Vero E6 Cells at 96\xa0h\xa0=\xa0434\xa0nM). b) Vero E6 cells were infected with SARS-CoV-2 and viral load was assessed in conditioned media collected after 4 days of incubation. c) Vero E6 cells were infected with SARS-CoV-2 at a MOI of 0.01 for 1\xa0h then selinexor was added to overlay media at 0, 24, 36 and 48 h post infection. Viral load was assessed by plaque assay after 4 days of incubation.', 'hash': 'dda9abc647e7b5047108071100fb5bd3859b4a943812480e4fb9b346f1377a11'}, {'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr6_lrg.jpg', 'caption': 'Quantitative assessment of selinexor effects on alveolitis and bronchitis after SARS-CoV-2 lung infection. Histopathological analysis was performed on formalin-fixed lungs. Tissues were analyzed for the a) extent and b) severity of alveolitis, and severity of c) bronchitis. Quantification based on 5 sections from 6 animals. Data are expressed as mean\xa0±\xa0SEM.', 'hash': 'ebd6a167171a883f13f40d26cc6902882b51af4d4d8a8ff678f24930eefdc101'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr4_lrg.jpg', 'caption': 'Selinexor protects from pathological changes associated with SARS-CoV-2 infection in nasal turbinates. Histopathological analysis in formalin-fixed tissue demonstrates inflammation of the nasal turbinates (rhinitis) in infected ferrets treated with placebo (a), but not in infected ferrets treated with selinexor (b) or uninfected animals (c, d). Inflammation is seen as infiltrates of inflammatory cells (leukocytes) in the lamina propria/submucosa (epithelium) of the nasal turbinates with associated edema and prominent (dilated) blood vessels. e) Quantification based on 5 sections from 6 animals to determine the severity of rhinitis (score details described in methods). Data are expressed as mean\xa0±\xa0SEM.', 'hash': '4661e0246f01ba2beb91dead69f4c16e2dd33315a4d80400a2f2d5c61a7f7228'}, {'image_id': 'gr8_lrg', 'image_file_name': 'gr8_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr8_lrg.jpg', 'caption': 'Blocking nuclear export inhibits SARS-CoV-2 infection, replication and viral propagation. a) Selinexor treatment blocks nuclear export and induces nuclear localization of the ACE-2 protein. Our results demonstrate that selinexor induces significant retention of ACE-2 in the cell nucleus while a small portion remained on the cell surface as seen in Fig. 1a and b. The reduction in ACE-2 on the cell surface confers protection from SARS-CoV-2 infection. b) A schematic model demonstrates how inhibition of nuclear export protects cells from SARS-CoV-2 infection by: reducing membranal presentation of ACE-2, blocking the cytoplasmic shuttling of the host protein GLTSCR2, (Wang et al., 2016), and sequestering the viral proteins ORF3b (Freundt et al., 2009), ORF9b (Moshynskyy et al., 2007; Sharma et al., 2011), and the nucleocapsid protein (Timani et al., 2005; You et al., 2007) in the nucleus. This allows for the activation of the innate immune response and the production of the type I interferons.', 'hash': 'd3a25152628498a1ce9fd75165c4f7065b16ba4954e5ee736e4c1e1b0c4d9724'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr1_lrg.jpg', 'caption': 'Selinexor inhibits nuclear export and forces nuclear accumulation of ACE-2 in vitro. Vero E6 cells were incubated with 500\xa0nM selinexor or DMSO for 24\xa0h. a) Cells were fixed with 3% paraformaldehyde, incubated with anti-ACE-2 antibody (Invitrogen, #MA5-32307) followed by a rabbit secondary antibody, Alexa Fluor 488, green-fluorescent dye (Thermo Fisher, #A11008), then visualized with the Echo Revolve fluorescent microscope (ECHO) at 60× magnification. Membrane-bound ACE-2 receptors are present in DMSO-treated cells only (white arrows). b) Sub-cellular fractionation was performed post-treatment then analyzed by immunoblots and c) quantified by densitometric analysis. d) Vero E6 and HEK 293\xa0cells were treated with selinexor (550\xa0nM, 24\xa0h) and whole protein lysates were analyzed on immunoblots.', 'hash': '840f870bae1196466fa0abe00bb456d8a68946b0549fd6f1fe68bf2c3336fd5e'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC8213878/gr5_lrg.jpg', 'caption': 'Selinexor protects from pathological changes associated with SARS-CoV-2 lung infection. Histopathological analysis in formalin-fixed tissue demonstrates inflammation of the bronchial tubes (bronchitis) in infected ferrets treated with placebo (a), but not in infected ferrets treated with selinexor (b) or uninfected animals (c, d). Insets show higher magnifications of indicated fields.', 'hash': 'ee4d206cd3bbbb591004ad85bcca1362b6ab748f38b0d16cc19b02de45b12a05'}]
|
{'gr1_lrg': ['We postulated that treatment with selinexor would lead to ACE-2 accumulation within the nucleus, thus protecting the cells from SARS-CoV-2 infection. In order to test if XPO1 inhibition by selinexor affects the subcellular distribution of ACE-2 in vitro, Vero E6 cells were treated with selinexor for 24\xa0h and ACE-2 localization was analyzed by immunofluorescence and sub-cellular fractionation. Selinexor treatment induced the nuclear retention of the ACE-2 receptor, while DMSO-treated cells showed membrane bound ACE-2 (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>\nA). Immunoblots of sub-cellular fractions showed that XPO1 inhibition by selinexor induced nuclear accumulation of ACE-2, with decreased ACE-2 in the membrane/cytoplasmic fraction, and nearly a 5-fold increase in nuclear protein levels (\nA). Immunoblots of sub-cellular fractions showed that XPO1 inhibition by selinexor induced nuclear accumulation of ACE-2, with decreased ACE-2 in the membrane/cytoplasmic fraction, and nearly a 5-fold increase in nuclear protein levels (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B and C). Selinexor treatment also led to nuclear accumulation of the well-established XPO1 cargo p53 tumor suppressor protein (B and C). Selinexor treatment also led to nuclear accumulation of the well-established XPO1 cargo p53 tumor suppressor protein (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B and C). Assessment of total protein levels showed that cells treated with selinexor had an overall reduction in GLTSCR2 and an expected decrease of XPO1 protein (B and C). Assessment of total protein levels showed that cells treated with selinexor had an overall reduction in GLTSCR2 and an expected decrease of XPO1 protein (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>D) (D) (Kashyap et al., 2016; Lee et al., 2020). Importantly, these effects were observed at concentrations that do not affect cell viability as indicated by levels of full-length caspase 3 (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>D). These results suggest that selinexor could potentially protect host cells from SARS-CoV-2 infection by sequestering ACE-2 in the nucleus to reduce the expression of membrane ACE-2 receptors.D). These results suggest that selinexor could potentially protect host cells from SARS-CoV-2 infection by sequestering ACE-2 in the nucleus to reduce the expression of membrane ACE-2 receptors.Fig. 1Selinexor inhibits nuclear export and forces nuclear accumulation of ACE-2 in vitro. Vero E6 cells were incubated with 500\xa0nM selinexor or DMSO for 24\xa0h. a) Cells were fixed with 3% paraformaldehyde, incubated with anti-ACE-2 antibody (Invitrogen, #MA5-32307) followed by a rabbit secondary antibody, Alexa Fluor 488, green-fluorescent dye (Thermo Fisher, #A11008), then visualized with the Echo Revolve fluorescent microscope (ECHO) at 60× magnification. Membrane-bound ACE-2 receptors are present in DMSO-treated cells only (white arrows). b) Sub-cellular fractionation was performed post-treatment then analyzed by immunoblots and c) quantified by densitometric analysis. d) Vero E6 and HEK 293\xa0cells were treated with selinexor (550\xa0nM, 24\xa0h) and whole protein lysates were analyzed on immunoblots.Fig. 1', 'Our results demonstrated that at least four of the SARS-CoV-2 proteins (nucleocapsid, ORF3b, ORF9b and the spike protein) contain predicted NESs, and three of these have been experimentally validated by others to be XPO1 cargos (Table 1). Similarly, we found that five of the human host proteins we tested contain NESs, including GLTSCR2 and Iκb, which have been previously experimentally validated (Table 1). ACE-2, the functional receptor for the SARS-CoV-2 spike glycoprotein contains two predicted NESs (Table 1). Our studies confirmed that XPO1 inhibition by selinexor induced nuclear retention of ACE-2 and reduced its cell surface localization (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>), providing experimental evidence to support ACE-2 as an XPO1 cargo protein. Reduction of ACE-2 membranal expression diminishes SARS-CoV-2 viral infection, as was demonstrated in ACE-2 knockout mice (), providing experimental evidence to support ACE-2 as an XPO1 cargo protein. Reduction of ACE-2 membranal expression diminishes SARS-CoV-2 viral infection, as was demonstrated in ACE-2 knockout mice (Kuba et al., 2005). Therefore, our findings that selinexor inhibited SARS-CoV-2 viral replication and shedding in vitro (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>) are consistent with a mechanism by which selinexor-mediated XPO1-inhibition leads to a reduction in the cellular membrane fraction of ACE-2, which helps to protect from viral infection. We showed that selinexor inhibited viral infection when given prophylactically, at the time of viral infection and also up to 24\xa0h following cell infection () are consistent with a mechanism by which selinexor-mediated XPO1-inhibition leads to a reduction in the cellular membrane fraction of ACE-2, which helps to protect from viral infection. We showed that selinexor inhibited viral infection when given prophylactically, at the time of viral infection and also up to 24\xa0h following cell infection (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>). These results suggest that selinexor can protect healthy tissue from infection in an infected individual. Importantly, selinexor levels required to block viral replication are not cytotoxic, with a therapeutic index of 35 (). These results suggest that selinexor can protect healthy tissue from infection in an infected individual. Importantly, selinexor levels required to block viral replication are not cytotoxic, with a therapeutic index of 35 (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>).).', 'The ACE-2 receptor is ubiquitously expressed in many organs including lungs, heart, kidney, testis, gut, and brain (Saponaro et al., 2020). One of its roles is to catalyze the formation of angiotensin-(1–7) through either degradation of angiotensin II or conversion of angiotensin I into angiotensin-(1–9), which in turn, is converted to angiotensin-(1–7) (Verdecchia et al., 2020). Through this process, ACE-2 receptors protect tissue from vasoconstriction, fibrosis, enhanced inflammation, pulmonary damage, and thrombosis that occurs when AT1 receptors bind angiotensin II (Verdecchia et al., 2020). As we have provided evidence that ACE-2 is an XPO1 cargo, it is worth discussing the potential clinical outcome of the membranal reduction of ACE-2 in selinexor treated patients. In vivo studies show mild acute lung injury in ACE-2 knockout mice (Kuba et al., 2005). Unlike the ACE-2 knockout, the effects of selinexor on the membranal expression of ACE-2 are milder and transient. Selinexor treatment reduced membranal and cytoplasmic ACE-2 without completely depleting it from the cell membrane (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>). In addition, preclinical and clinical use of selinexor demonstrated maximal activity in the first 48\xa0h after drug dosing, and human selinexor treatment for a number of non-viral indications demonstrated tolerability with no hyperactivation of AT). In addition, preclinical and clinical use of selinexor demonstrated maximal activity in the first 48\xa0h after drug dosing, and human selinexor treatment for a number of non-viral indications demonstrated tolerability with no hyperactivation of AT1 receptors (Abdul Razak et al., 2016; Grosicki et al., 2020; Kalakonda et al., 2020).'], 'gr2_lrg': ['To test the anti-viral effects of selinexor on SARS-CoV-2 infection in vitro, Vero E6 cells were incubated with varying concentrations of selinexor starting either 6\xa0h prior (prophylactic) or at the time of viral infection (therapeutic) (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\nA). Both experiments demonstrated that selinexor has potent anti-SARS-CoV-2 activity \nA). Both experiments demonstrated that selinexor has potent anti-SARS-CoV-2 activity in vitro, with 50% inhibition (EC50) of SARS-CoV-2 replication at 10\xa0nM and EC90 at 100\xa0nM in the Vero E6 cells. To test the cytotoxic effects of selinexor on Vero E6 cells, the cells were incubated with increasing concentrations of selinexor and the CC50 was found to be 434\xa0nM. These results demonstrated that the therapeutic index ([TI]\xa0=\xa0CC50/EC50) of selinexor against SARS-CoV-2 infection in Vero E6 cells is 35.Fig. 2Selinexor inhibits SARS-CoV-2 viral replication and shedding in vitro. a) Vero E6 cells were pre-treated with selinexor before SARS-CoV-2 infection (left – Prophylactic) or treated with selinexor at the time of infection (right – Therapeutic). Both were incubated with selinexor during infection. Viral load was assessed by plaque assay after 4 days of cell incubation with overlay (Selinexor CC50 of Vero E6 Cells at 96\xa0h\xa0=\xa0434\xa0nM). b) Vero E6 cells were infected with SARS-CoV-2 and viral load was assessed in conditioned media collected after 4 days of incubation. c) Vero E6 cells were infected with SARS-CoV-2 at a MOI of 0.01 for 1\xa0h then selinexor was added to overlay media at 0, 24, 36 and 48 h post infection. Viral load was assessed by plaque assay after 4 days of incubation.Fig. 2', 'To test the anti-viral activity of selinexor on uninfected neighboring cells, Vero E6 cells were incubated prophylactically with different concentrations of selinexor starting 6\xa0h prior to infection with SARS-CoV-2 and continuing for the duration of the experiment. This allowed viral shedding of SARS-CoV-2 virus from infected cells into the conditioned media and the infection of other cultured cells in the plate. Four days after the initial infection, the viral load was calculated as plaque forming units (PFU)/ml of conditioned media. The results show that selinexor inhibited SARS-CoV-2 viral shedding and therefore protected the infection of neighboring cells with an IC90\xa0<\xa010\xa0nM (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B)B).\n', 'To test how long after SARS-CoV-2 viral infection selinexor remains effective inhibiting viral propagation, Vero E6 cells infected with SARS-CoV-2 were treated with selinexor at 0, 24, 36, and 48 h post infection. Assessment of viral load demonstrated that selinexor inhibited SARS-CoV-2 viral propagation in vitro even when added up to 48 h after infection (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>C).C).'], 'gr3_lrg': ['To assess the in vivo therapeutic efficacy of selinexor against SARS-CoV-2 infection, we used a ferret model of viral challenge where animals were infected intranasally with SARS-CoV-2, then treated with either selinexor (5\xa0mg/kg) or placebo (vehicle only) for three days starting 4\xa0h after infection. On day 4 post-SARS-CoV-2 infection, placebo-treated animals had a mean viral RNA of 4.6 log10 viral copies/gram (vc/g) of lung tissue, whereas selinexor-treated animals had a mean of 3.8 vc/g (p\xa0=\xa00.0335), with most animals measuring in lower limit of detection of the assay (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\n).\n).Fig. 3Selinexor decreased SARS-CoV-2 viral load in ferret lung tissue. Levels of viral RNA in the lungs were measured post-mortem on Day 4 by qPCR. Lower limit of detection range is indicated by dashed lines. Data are expressed as mean\xa0±\xa0SEM.Fig. 3'], 'gr4_lrg': ['Histopathological analysis of formalin-fixed tissue from the respiratory tracts of animals that had been infected with SARS-CoV-2 showed severe neutrophilic rhinitis with lamina propria necrosis and inflammation in the lamina propria when treated with placebo. Animals treated with selinexor had mild to moderate neutrophilic rhinitis with mild and focal epithelial necrosis that was significantly less severe (p\xa0=\xa00.0001) (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\nA–E). The lungs demonstrated inflammation of the bronchial tubes (bronchitis) in infected ferrets treated with placebo, but not in infected ferrets treated with selinexor (\nA–E). The lungs demonstrated inflammation of the bronchial tubes (bronchitis) in infected ferrets treated with placebo, but not in infected ferrets treated with selinexor (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>\nA–D). Quantification of pathological changes in the lungs demonstrated that the extent of alveolitis was significantly decreased in selinexor-treated animals with a score of 1.5 out of 3 compared to 2.04 in the vehicle placebo-treated animals (p\xa0=\xa00.045) (\nA–D). Quantification of pathological changes in the lungs demonstrated that the extent of alveolitis was significantly decreased in selinexor-treated animals with a score of 1.5 out of 3 compared to 2.04 in the vehicle placebo-treated animals (p\xa0=\xa00.045) (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>\nA). The average severity of alveolitis and bronchitis was also numerically lower in the selinexor-treated animals, but the difference did not reach statistical significance (\nA). The average severity of alveolitis and bronchitis was also numerically lower in the selinexor-treated animals, but the difference did not reach statistical significance (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>B and C).B and C).Fig. 4Selinexor protects from pathological changes associated with SARS-CoV-2 infection in nasal turbinates. Histopathological analysis in formalin-fixed tissue demonstrates inflammation of the nasal turbinates (rhinitis) in infected ferrets treated with placebo (a), but not in infected ferrets treated with selinexor (b) or uninfected animals (c, d). Inflammation is seen as infiltrates of inflammatory cells (leukocytes) in the lamina propria/submucosa (epithelium) of the nasal turbinates with associated edema and prominent (dilated) blood vessels. e) Quantification based on 5 sections from 6 animals to determine the severity of rhinitis (score details described in methods). Data are expressed as mean\xa0±\xa0SEM.Fig. 4Fig. 5Selinexor protects from pathological changes associated with SARS-CoV-2 lung infection. Histopathological analysis in formalin-fixed tissue demonstrates inflammation of the bronchial tubes (bronchitis) in infected ferrets treated with placebo (a), but not in infected ferrets treated with selinexor (b) or uninfected animals (c, d). Insets show higher magnifications of indicated fields.Fig. 5Fig. 6Quantitative assessment of selinexor effects on alveolitis and bronchitis after SARS-CoV-2 lung infection. Histopathological analysis was performed on formalin-fixed lungs. Tissues were analyzed for the a) extent and b) severity of alveolitis, and severity of c) bronchitis. Quantification based on 5 sections from 6 animals. Data are expressed as mean\xa0±\xa0SEM.Fig. 6'], 'gr7_lrg': ['As described above, selinexor as well as other SINE compounds force the nuclear retention and functional activation of IκB and other anti-inflammatory proteins, leading to attenuation of NF-κB and other pro-inflammatory transcription factors (Widman et al., 2018; Wu et al., 2018). To further evaluate the anti-inflammatory effects of selinexor, PBMCs from human volunteers were isolated, stimulated with LPS, and incubated with increasing concentrations of selinexor. Cell free supernatants were collected at 6, 16, 24, and 40 h post-incubation and assayed by ELISA for selected pro- and anti-inflammatory cytokines. Compared with vehicle, selinexor inhibited production of IL-1β, IL-6, TNF-α, IL-10, and IFN-γ at each time point. In addition, production of GM-CSF and IL-8 showed moderate decreases with the highest dose of selinexor at later timepoints (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>\n).\n).Fig. 7Selinexor diminishes inflammatory cytokine levels associated with SARS-CoV-2 infection in human cells. Human peripheral blood mononuclear cells were incubated with LPS (1000\xa0ng/ml) and increasing concentrations of selinexor for 6, 16, 24, and 40\xa0h. Supernatant was collected and analyzed by individual ELISAs. Data are expressed as mean\xa0±\xa0SEM.Fig. 7'], 'gr8_lrg': ['Taken together, XPO1 inhibition, including the reduction of membranal ACE-2 receptor (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>A), exerts therapeutic effects due to the inhibition of viral infection, activation of the type I interferon response, and anti-inflammatory activity (A), exerts therapeutic effects due to the inhibition of viral infection, activation of the type I interferon response, and anti-inflammatory activity (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>B), blocking viral infection, replication, and propagation of SARS-CoV-2. Future studies will directly examine the role of additional host and viral proteins in SARS-CoV-2 infection and the effects of selinexor on their functions in order to identify combinations with other drugs that may synergize and enhance selinexor activity for the potential use of SINE compounds, including selinexor, in the clinic.B), blocking viral infection, replication, and propagation of SARS-CoV-2. Future studies will directly examine the role of additional host and viral proteins in SARS-CoV-2 infection and the effects of selinexor on their functions in order to identify combinations with other drugs that may synergize and enhance selinexor activity for the potential use of SINE compounds, including selinexor, in the clinic.Fig. 8Blocking nuclear export inhibits SARS-CoV-2 infection, replication and viral propagation. a) Selinexor treatment blocks nuclear export and induces nuclear localization of the ACE-2 protein. Our results demonstrate that selinexor induces significant retention of ACE-2 in the cell nucleus while a small portion remained on the cell surface as seen in <xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>a and b. The reduction in ACE-2 on the cell surface confers protection from SARS-CoV-2 infection. a and b. The reduction in ACE-2 on the cell surface confers protection from SARS-CoV-2 infection. b) A schematic model demonstrates how inhibition of nuclear export protects cells from SARS-CoV-2 infection by: reducing membranal presentation of ACE-2, blocking the cytoplasmic shuttling of the host protein GLTSCR2, (Wang et al., 2016), and sequestering the viral proteins ORF3b (Freundt et al., 2009), ORF9b (Moshynskyy et al., 2007; Sharma et al., 2011), and the nucleocapsid protein (Timani et al., 2005; You et al., 2007) in the nucleus. This allows for the activation of the innate immune response and the production of the type I interferons.Fig. 8']}
|
in vivo Selinexor, a novel selective inhibitor of nuclear export, reduces SARS-CoV-2 infection and protects the respiratory system
|
[
"Exportin-1",
"XPO1",
"CRM1",
"SARS-CoV-2",
"COVID19",
"Selinexor",
"SINE compound"
] |
Antiviral Res
|
1629356400
|
The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1β, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option.
|
[
"Active Transport, Cell Nucleus",
"Angiotensin-Converting Enzyme 2",
"Animals",
"Antiviral Agents",
"COVID-19",
"Chlorocebus aethiops",
"Cytokines",
"Ferrets",
"Humans",
"Hydrazines",
"Karyopherins",
"Receptors, Cytoplasmic and Nuclear",
"Respiratory System",
"SARS-CoV-2",
"Triazoles",
"Tumor Suppressor Proteins",
"Vero Cells",
"Virus Replication",
"COVID-19 Drug Treatment",
"Exportin 1 Protein"
] |
other
|
PMC8213878
| null |
51
|
[
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] |
Antiviral Res. 2021 Aug 19; 192:105115
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NO-CC CODE
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Protein network tethers rDNA to the nuclear envelopea, Representative 3D reconstructions of cells from double immunofluorescence analysis reveal the relative organization of Net1-GFP (green) and Heh1-Myc13 (red). Signal boundaries are shown with clipping planes for Heh1-Myc13. Percentages of green relative to full red volumes are shown (mean ± s.d.; n = 5). b, Nocodazole-arrested (G2/M) cells were subjected to FISH to visualize rDNA and DAPI staining (pseudocoloured red) to visualize bulk nuclear DNA. Cells with different rDNA morphologies are indicated by double arrowheads (spooled lines), arrows (amorphous), or triangles (two bodies) with quantifications in Supplementary Fig. 6b. Scale bar, 3 μm. c, Live cells with TetI-RFP-marked rDNA and expressing Rad52-YFP and nucleolar Nop1-CFP were imaged. Images depicting most observed phenotypes are shown. More images and quantifications are in Supplementary Fig. 6d-f. Scale bars are 1 μm (white) and 3 μm (black). d, e, Relative fold enrichment of indicated TAP-tagged proteins are shown. rDNA organization schematics are shown on graphs. Gels for d and e are shown in Supplementary Figs 7a and 7d, respectively. Detailed IGS1 ChIP is shown in Supplementary Fig. 7b, c.
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nihms-71410-f0003
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2
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abb65c2fd00730db77a7add45e4440fa8e170b0fb8d4fd63421c4854a9c02dad
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nihms-71410-f0003.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
686,
647
] |
[{'image_id': 'nihms-71410-f0001', 'image_file_name': 'nihms-71410-f0001.jpg', 'image_path': '../data/media_files/PMC2596277/nihms-71410-f0001.jpg', 'caption': 'Protein network extending from rDNA to the nuclear envelopea, rDNA repeats on Chr. XII. Each unit yields a Pol I-transcribed 35S precursor rRNA (processed into 25S, 18S, and 5.8S) and a Pol III-transcribed 5S rRNA. CEN, centromere; TEL, telomere; IGS, intergenic spacer; RE, recombination enhancer;, replication fork block; TIR, Pol I transcription initiation region; O, DNA replication origin; Mbp, megabase pairs. Vertical arrowheads indicate insertion sites of mURA3 reporter genes used in this study. b, Nuclear organization at the G2/M cell cycle stage. No1., nucleolus; ONM, outer nuclear membrane. c-f, Purification of native complexes. c, e, Protein detection in silver-stained gels. TAP cleavage during protein purification leaves a calmodulin binding protein (CBP) fragment. d, f, LC-MS/MS analysis. Number of unique peptides followed by percent coverage of the protein sequence is shown. SI contains full protein lists (Supplementary Table 1, part A) and spectral counts (Supplementary Tables 2 and 3).', 'hash': 'ceb7304da108b5a7399724c337ce8ae6d613a09703a6646ecf406c06566094f6'}, {'image_id': 'nihms-71410-f0002', 'image_file_name': 'nihms-71410-f0002.jpg', 'image_path': '../data/media_files/PMC2596277/nihms-71410-f0002.jpg', 'caption': 'Role of perinuclear protein network at rDNA repeatsa, b, Rates of ADE2 marker loss (mean ± s.d.) relative to wild-type (WT) (a) and representative colonies (b) are shown. c, CHEF analysis of rDNA stability. Chromosomes resolved by CHEF were probed with IGS1 rDNA (Chr. XII, top). Ethidium bromide (EtBr) staining of Chr. IV and smaller chromosomes shows quality of the preparation (bottom). Values corresponding to rDNA copy-number averages and smear edges are indicated. Chr. XII size for sir2Δ was too heterogeneous to estimate copy-number. d, Unlike Sir2 and Cohibin, CLIP is dispensable for rDNA silencing. Ten-fold serial dilutions of cells with the mURA3 reporter gene inserted at IGS1/IGS2 (see Fig. 1a for locations) or outside rDNA at the LEU2 locus are shown.', 'hash': '31f00f5c2f9758e9fbbc6592babbd7c2ee52f345a1f66d617170edb240e9a9fd'}, {'image_id': 'nihms-71410-f0004', 'image_file_name': 'nihms-71410-f0004.jpg', 'image_path': '../data/media_files/PMC2596277/nihms-71410-f0004.jpg', 'caption': "Targeted perinuclear tethering promotes rDNA repeat stabilitya, Fusion of Heh1 to Sir2 in lrs4Δ cells. Heh1 is shown embedded in the INM. b, FISH reveals that fusion of Heh1 and Sir2 restores the separation of rDNA signal from DAPI staining in nocodazole-arrested cells lacking Lrs4. Scale bar, 3 μm. c, Relative rates of ADE2 marker loss (mean ± s.d.). Asterisk, P < 0.0001 for Student's t test. d, CHEF analysis of rDNA stability. Chromosomes resolved by CHEF were probed with IGS1 rDNA (Chr. XII, top). EtBr staining of Chr. IV and smaller chromosomes shows quality of the preparation (bottom). e, Functional organization of the perinuclear molecular network tethering rDNA to the nuclear periphery. Repeat instability results from the loss of either the INM CLIP proteins or rDNA silencing complexes.", 'hash': '1d7762e1f2037b7a2fb9f35dc6ba6fc36c8caae79358dfcabed31146e60edc5c'}, {'image_id': 'nihms-71410-f0003', 'image_file_name': 'nihms-71410-f0003.jpg', 'image_path': '../data/media_files/PMC2596277/nihms-71410-f0003.jpg', 'caption': 'Protein network tethers rDNA to the nuclear envelopea, Representative 3D reconstructions of cells from double immunofluorescence analysis reveal the relative organization of Net1-GFP (green) and Heh1-Myc13 (red). Signal boundaries are shown with clipping planes for Heh1-Myc13. Percentages of green relative to full red volumes are shown (mean ± s.d.; n = 5). b, Nocodazole-arrested (G2/M) cells were subjected to FISH to visualize rDNA and DAPI staining (pseudocoloured red) to visualize bulk nuclear DNA. Cells with different rDNA morphologies are indicated by double arrowheads (spooled lines), arrows (amorphous), or triangles (two bodies) with quantifications in Supplementary Fig. 6b. Scale bar, 3 μm. c, Live cells with TetI-RFP-marked rDNA and expressing Rad52-YFP and nucleolar Nop1-CFP were imaged. Images depicting most observed phenotypes are shown. More images and quantifications are in Supplementary Fig. 6d-f. Scale bars are 1 μm (white) and 3 μm (black). d, e, Relative fold enrichment of indicated TAP-tagged proteins are shown. rDNA organization schematics are shown on graphs. Gels for d and e are shown in Supplementary Figs 7a and 7d, respectively. Detailed IGS1 ChIP is shown in Supplementary Fig. 7b, c.', 'hash': 'abb65c2fd00730db77a7add45e4440fa8e170b0fb8d4fd63421c4854a9c02dad'}]
|
{'nihms-71410-f0001': ['Eukaryotic rDNA is tandemly repeated anywhere from ∼100 to over 10,000 times8. rDNA repeats provide the foundation for at least one ribosome-manufacturing compartment, the nucleolus. Budding yeast Saccharomyces cerevisiae has 100-200 rDNA units tandemly arranged on chromosome XII (Chr. XII) and forming one nucleolus (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1a, b</xref>))8. In addition to harboring rRNA-coding DNA sequences, each unit contains intergenic spacers (IGS1 and 2) that promote repeat integrity (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1a</xref>))9-11. Recruitment of nucleolar protein complexes RENT (regulator of nucleolar silencing and telophase exit; composed of Cdc14, Net1/Cfi1, and Sir2) and Cohibin (mitotic monopolin proteins Lrs4 and Csm1) to IGS1 suppresses unequal recombination at the repeats3,12-16. This suppression is seemingly linked to the ability of these complexes to induce rDNA silencing, which involves chromatin changes preventing RNA Polymerase II-driven transcription within IGSs of rDNA4,5,16-19.', 'Purification of Cohibin suggested an association with INM proteins of unknown function16. To gain insight into the possible role of this association in nucleolar organization, we purified native Cohibin and INM proteins using tandem affinity purification (TAP). The TAP-tagged proteins are functional in vivo (Ref.16 and below). We detected purified complexes by silver staining and total protein mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Negative controls were untagged cells. Purification of Lrs4 and Csm1 yielded peptides of INM proteins Heh1 (helix extension helix 1, also called Src1) and Nur1 (nuclear rim 1, Ydl089w) (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1c, d</xref>; ; Supplementary Table 1, part A)16. Heh1, the orthologue of human Man1, is a member of a family of INM proteins containing a highly conserved LAP-Emerin-Man1 domain (LEM, also called HEH; Supplementary Fig. 2)20-22. LEM-domain proteins are linked to multiple clinical conditions via emerging roles in fundamental cellular processes, including gene expression and chromatin organization6,7,20,21,23,24. Little is known about Heh1 and Nur120, which we define here as chromosome linkage INM proteins (CLIP). Purification of either INM protein yielded peptides for both Heh1 and Nur1 (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1e, f</xref>; Supplementary Text, section A). Purification of Heh2, an Heh1 homologue (; Supplementary Text, section A). Purification of Heh2, an Heh1 homologue (Supplementary Fig. 2)20, did not yield peptides for CLIP or Cohibin proteins (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1e, f</xref>; ; Supplementary Fig. 3c; Supplementary Table 1). Moreover, TAP-tagged Heh1, Lrs4, and Csm1 coimmunoprecipitated with Myc13-tagged Lrs4, Heh1, and Nur1, respectively (Supplementary Fig. 3d). Migration of Heh1 to 115 kDa, instead of the predicted 95 kDa, led us to identify multiple post-translational modifications of the protein and fluctuation of Heh1 levels over the cell cycle with peaks at interphase and mitosis (Supplementary Figs 3a, e, f, and 4). These findings physically link rDNA-associated complexes to INM proteins.', 'How rDNA is separated from the bulk of nuclear DNA is unknown (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1b</xref>). We tested if the perinuclear network studied here affects this subnuclear separation. Nocodazole-arrested cells were analyzed by fluorescence in-situ hybridization (FISH) to visualize rDNA and 4\',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining to visualize bulk nuclear DNA. Wild-type cells exhibited line-shaped rDNA spooling away from the DNA bulk towards the nuclear periphery (). We tested if the perinuclear network studied here affects this subnuclear separation. Nocodazole-arrested cells were analyzed by fluorescence in-situ hybridization (FISH) to visualize rDNA and 4\',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining to visualize bulk nuclear DNA. Wild-type cells exhibited line-shaped rDNA spooling away from the DNA bulk towards the nuclear periphery (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3b</xref> and quantification in and quantification in Supplementary Fig. 6b)27. Deletion of Lrs4, Csm1, or Heh1, but not Heh2, caused rDNA to adopt amorphous distributions often overlapping DAPI signal and a small percentage of cells exhibited two separable rDNA bodies (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3b</xref>; ; Supplementary Fig. 6b), which may reflect severe loss of interactions between rDNA repeats on Chr. XII sister chromatids. Nur1 deletion caused smaller changes in rDNA morphology, which appeared less condensed (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3b</xref>; 65 ± 6% of cells, mean ± s.d.). Disorganization of rDNA was also observed in asynchronous cells (; 65 ± 6% of cells, mean ± s.d.). Disorganization of rDNA was also observed in asynchronous cells (Supplementary Fig. 6c).', 'a, b, Rates of ADE2 marker loss (mean ± s.d.) relative to wild-type (WT) (a) and representative colonies (b) are shown. c, CHEF analysis of rDNA stability. Chromosomes resolved by CHEF were probed with IGS1 rDNA (Chr. XII, top). Ethidium bromide (EtBr) staining of Chr. IV and smaller chromosomes shows quality of the preparation (bottom). Values corresponding to rDNA copy-number averages and smear edges are indicated. Chr. XII size for sir2Δ was too heterogeneous to estimate copy-number. d, Unlike Sir2 and Cohibin, CLIP is dispensable for rDNA silencing. Ten-fold serial dilutions of cells with the mURA3 reporter gene inserted at IGS1/IGS2 (see <xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1a</xref> for locations) or outside rDNA at the for locations) or outside rDNA at the LEU2 locus are shown.'], 'nihms-71410-f0002': ['Peripheral association of genes is linked to silent chromatin assembly, which seemingly stabilizes repeats by limiting access to recombination proteins2,7. Thus, CLIP may assemble at IGS1 to cooperate with RENT and Cohibin to silence transcription and inhibit unequal rDNA recombination. Therefore, we monitored unequal sister chromatid exchange (USCE) by measuring the rate of loss of an ADE2 marker gene from rDNA repeats. Deletion of Sir2, Lrs4, or Csm1 increased USCE, as expected (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2a, b</xref>; ; Supplementary Table 4)16,25. USCE also increased following deletion of Heh1 or Nur1, but not Heh2 (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2a, b</xref>; ; Supplementary Table 4). heh1Δ nur1Δ cells displayed additive USCE defects compared to single mutants, suggesting that INM proteins play partially overlapping roles at rDNA. Moreover, deletion of Heh1, Lrs4, or Csm1 exacerbated the effect of loosing Sir2 (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2a</xref>; ; Supplementary Table 4)16 suggesting that Sir2 stabilizes rDNA via CLIP/Cohibin-dependent and -independent processes. Since increases in USCE affect rDNA copy-number on Chr. XII, we analyzed its size using contour-clamped homogeneous electric field (CHEF). Chr. XII measured ∼2.83 Mbp in wild-type cells (∼190 rDNA units) and chromosome smearing in sir2Δ cells was indicative of severe changes in rDNA copy-number (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2c</xref>; ; Supplementary Fig. 5a), as expected4,17. Interestingly, deletion of Lrs4, Csm1, Heh1, or Nur1 resulted in marked changes in rDNA copy-number averages and chromosome smearing patterns (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2c</xref>; described in Supplementary Text, section B). Together, these data suggest that the perinuclear protein network studied here is required for rDNA repeat stability.; described in Supplementary Text, section B). Together, these data suggest that the perinuclear protein network studied here is required for rDNA repeat stability.', 'We next studied the ability of cells to silence an RNA Pol II-transcribed mURA3 reporter gene positioned within IGS1 or IGS2 by assessing cellular growth on synthetic complete (SC) medium that is either lacking uracil (-Ura, silencing disrupts growth) or supplemented with 5-fluoro-orotic acid (+5FOA, silencing allows growth). Deletion of Sir2, Lrs4, or Csm1 disrupted IGS1 silencing (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2d</xref>), as expected), as expected16. Surprisingly, in contrast to rDNA repeat stability, Heh1 and Nur1 were dispensable for silencing (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2d</xref>; ; Supplementary Fig. 5b), suggesting that silencing is insufficient for proper repeat size regulation.'], 'nihms-71410-f0003': ['We next asked whether tethering rDNA repeats to INM proteins via Cohibin limited recombination independently of silencing. Using immunofluorescence, we visualized the functional green fluorescent protein (GFP)-tagged Net1 and Myc13-tagged Heh1 (Supplementary Table 4)3,16. Net1 associates with rDNA in the nucleolus throughout the cell cycle and recruits Sir2 to IGS13. However, enrichment of Sir2 at rDNA in chromatin immunoprecipitation (ChIP) experiments is unaffected by deletion of Cohibin (Supplementary Figs 1 and 8c). To measure the limit of Net1-GFP internalization, the nucleus, delineated by peripheral Heh1-Myc13 signal, was divided in three concentric zones of equal area, zone I being most peripheral26. Cells were categorized according to whether the centre of the least peripheral Net1-GFP focus localizes to zone I, II, or III. Most wild-type cells displayed peripheral Net1-GFP localization (zone I, 74%) while few contained central Net1-GFP staining (zone III, 2%) (Supplementary Fig. 6a). Lrs4 or Csm1 deletion drastically shifted Net1-GFP to zone II or III (Supplementary Fig. 6a) and expanded the volume occupied by Net1-GFP within nuclear space in 3D (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3a</xref>), suggesting that optimal perinuclear localization of the rDNA-associated Net1 requires Cohibin.), suggesting that optimal perinuclear localization of the rDNA-associated Net1 requires Cohibin.', 'We next studied the localization of a specific site within rDNA repeats in live cells harboring a tetO array at rDNA repeats and expressing tetO-binding TetI-RFP (TetI fused to red fluorescent protein) and the nucleolar Nop1-CFP (Nop1 protein fused to cyan fluorescent protein)28. TetI-RFP localized inside or at the periphery of the nucleolus in most wild-type cells (93%; <xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3c</xref>; ; Supplementary Fig. 6d, e). Deletion of Lrs4 or Heh1 shifted TetI-RFP outside of the nucleolus or to its periphery (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3c</xref>; ; Supplementary Fig. 6d, e). sir2Δ cells exhibited less severe mislocalization of TetI-RFP (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3c</xref>; ; Supplementary Fig. 6d, e). Deletion of Lrs4, Heh1, or Sir2 also induced the formation of extranucleolar DNA repair centers, as marked by clustering of the yellow fluorescent protein-tagged Rad52 recombination protein, Rad52-YFP (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3c</xref>; ; Supplementary Fig. 6d, f). Fewer sir2Δ cells exhibited Rad52 foci compared to heh1Δ or lrs4Δ cells (Supplementary Fig. 6f). This is in contrast to USCE in sir2Δ cells, which is higher than that of heh1Δ or lrs4Δ cells (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2a</xref>), suggesting that more recombinations in ), suggesting that more recombinations in sir2Δ cells are unequal crossovers. Alternatively, a higher incidence of Rad52-YFP foci in cells lacking Heh1 or Lrs4 might suggest that these proteins stabilize several genetic loci. Most Rad52-YFP foci (61%-65%) did not overlap TetI-RFP signal in lrs4Δ, heh1Δ, or sir2Δ cells (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3c</xref>; ; Supplementary Fig. 6d, f), likely reflecting the occurrence of one or few repair events per rDNA array and their distance from tetO sequences. While we cannot exclude the possibility that the tetO array contributes to rDNA mislocalization in lrs4Δ, heh1Δ, or sir2Δ cells, disruption of rDNA organization in lrs4Δ and heh1Δ cells lacking tetO sites, as revealed by FISH and immunofluorescence (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3a, b</xref>; ; Supplementary Fig. 6a-c), argues against this possibility and suggests that the perinuclear complexes studied here help stabilize wild-type rDNA repeats. Together, these results suggest that Heh1 and Lrs4, and to a lesser extent Sir2, are required for sequestration of rDNA in the peripherally located nucleolus, and show that loss of sequestration correlates with increased repeat instability (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2</xref>) and Rad52 recombination foci.) and Rad52 recombination foci.', 'To further analyze CLIP-Cohibin links, we performed ChIP using a combination of dimethyl adipimidate and formaldehyde crosslinkers. We observed 2.85±0.37 and 2.35±0.12 fold enrichments for IGS1 sequences in Lrs4-TAP and Heh1-TAP immunoprecipitations, respectively (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3d, e</xref>; ; Supplementary Fig. 7). We did not detect an enrichment using Nur1-TAP, likely due to its low abundance or weaker association with Cohibin (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3d</xref>; Supplementary Figs ; Supplementary Figs 3a and 7a-c). More importantly, deletion of Lrs4 abolished the IGS1 enrichment of Heh1-TAP without affecting its levels (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3e</xref>; ; Supplementary Fig. 7d-f). In contrast, no enrichment was detected for Heh2-TAP (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3e</xref>; ; Supplementary Fig. 7d), an INM protein that neither interacts with Cohibin (<xref ref-type="fig" rid="nihms-71410-f0001">Fig. 1</xref>) nor affects rDNA stability () nor affects rDNA stability (<xref ref-type="fig" rid="nihms-71410-f0002">Fig. 2</xref>), although expressed to similar levels as Heh1 (data not shown)), although expressed to similar levels as Heh1 (data not shown)29. Together, these data indicate that CLIP/Cohibin-mediated tethering of rDNA repeats to the INM is required for repeat stability.', 'Standard ChIP (Supplementary Fig. 8) was conducted as described14. Modified ChIP (<xref ref-type="fig" rid="nihms-71410-f0003">Fig. 3</xref>; ; Supplementary Fig. 7) was conducted as described14,16 with protein-protein crosslinkers added34. Modifications are as follows: Yeast cultures (50 ml) were grown to OD600 ∼0.8. Cells were centrifuged, washed with ice-cold phosphate buffer saline (PBS), suspended in 10 ml ice-cold/fresh protein-protein crosslinking solution (10 mM DMA and 0.25% dimethyl sulfoxide in PBS) and nutated at room temperature (RT, 45 min). PBS-washed cells were resuspended in 50 ml of 1% formaldehyde in PBS (11 h), then Glycine was added to 125 mM. PBS-washed cells were resuspended in 400 μl of lysis buffer, subjected to bead-beating, and procedures were continued as described14,16 except that RNase was added prior to proteinase K. Dilutions for IP and input DNA were 1:2 and 1:20,000, respectively. [α-32P]-dCTP-labeled and EtBr-stained products were quantified with Molecular Imager/QuantityOne (Bio-Rad) and Image ReaderLAS-3000/ImageGauge (Fuji), respectively.'], 'nihms-71410-f0004': ['To determine if perinuclear tethering suppresses recombination in the absence of Cohibin proteins, which are required for rDNA silencing and suppression of recombination, we created a strain in which rDNA was linked to Heh1 via Sir2. We fused HEH1 and SIR2 genes in lrs4Δ cells creating a hybrid HEH1-SIR2 gene (<xref ref-type="fig" rid="nihms-71410-f0004">Fig. 4a</xref>). This yielded a fusion protein of expected size (∼175 kDa) detectable in anti-Sir2 immunoblotting (). This yielded a fusion protein of expected size (∼175 kDa) detectable in anti-Sir2 immunoblotting (Supplementary Fig. 8a). Fusion of Heh1 and Sir2 restored the separation of rDNA from bulk nuclear DNA in lrs4Δ cells (<xref ref-type="fig" rid="nihms-71410-f0004">Fig. 4b</xref>), reduced unequal recombination (), reduced unequal recombination (<xref ref-type="fig" rid="nihms-71410-f0004">Fig. 4c</xref>; ; Supplementary Table 4), and increased homogeneity in the size of Chr. XII in cell populations (<xref ref-type="fig" rid="nihms-71410-f0004">Fig. 4d</xref>; ; Supplementary Fig. 8b). Furthermore, ChIP revealed that Heh1-Sir2 associated with rDNA to similar levels as Sir2 (Supplementary Fig. 8c). Moreover, Heh1-Sir2 did not rescue IGS1-specific increases in histone H3 acetylation, a marker for loss of silencing, caused by Lrs4 deletion (Supplementary Fig. 8c). The inability of Heh1-Sir2 to fully restore rDNA stability may be due to other Lrs4 functions, such as silencing or perhaps chromosome condensation, which suppress recombination at repeats. Attempts to fuse Heh1 with other perinuclear proteins, such as Tof2 or Ku70, did not yield viable cells (data not shown). Thus, tethering rDNA to the INM can promote repeat stability at least partially independently of silencing.', 'Our results suggest that Sir2-dependent silencing alone cannot inhibit recombination within the repetitive rDNA locus and that INM-mediated perinuclear chromosome tethering ensures repeat stability (<xref ref-type="fig" rid="nihms-71410-f0004">Fig. 4e</xref>; ; Supplementary Fig. 1). Extranucleolar Rad52 focus formation in lrs4Δ, heh1Δ, or sir2Δ cells concurs with suggestions that while early rDNA recombination steps occur inside the nucleolus, Rad52 sumoylation and a high local concentration of the Smc5-Smc6 complex preclude Rad52 focus formation within nucleolar space28. Thus, our findings suggest that rDNA repeats unleashed from the INM accumulate lesions that can better access the nucleoplasm where high concentrations of functional Rad52 promote DNA repair by homologous recombination. Therefore, perinuclear tethering likely sequesters repeats away from recombination factors and may be required for Cohibin and RENT to stably align rDNA sister chromatids during replication to prevent unequal crossovers (<xref ref-type="fig" rid="nihms-71410-f0004">Fig. 4e</xref>). Recombination between homologous repeats dispersed in the genome often instigates catastrophic chromosomal rearrangements. We anticipate that proteins studied here are members of perinuclear networks that control recombination at multiple loci to maintain genome stability.). Recombination between homologous repeats dispersed in the genome often instigates catastrophic chromosomal rearrangements. We anticipate that proteins studied here are members of perinuclear networks that control recombination at multiple loci to maintain genome stability.']}
|
Role for perinuclear chromosome tethering in maintenance of genome stability
|
[
"Nucleolus",
"rDNA",
"ribosomal RNA genes",
"copy number",
"unequal recombination",
"silencing",
"heterochromatin",
"chromosome",
"Heh1",
"Man1",
"Nur1",
"Ydl089w",
"CLIP",
"LEM domain",
"HEH fold",
"Emerin",
"Sir2",
"SIRT1",
"RENT",
"Net1",
"Cdc14",
"nuclear envelope",
"perinuclear",
"nuclear periphery"
] |
Nature
|
1228377600
|
This discussion focuses on the dynamic nature of three chronic congenital infections of man, namely those caused by cytomegaloviruses, rubella virus, and Toxoplasma gondii. The spectra of the fetal infection are described and the risks associated with the acute and chronic phases of the fetal infections are contrasted. Because of their frequency and subtlety, emphasis is placed on the late-appearing sequelae which are associated with the persistent and/or recurring aspects of these infections.
|
[
"Central Nervous System Diseases",
"Chorioretinitis",
"Chronic Disease",
"Congenital Abnormalities",
"Cytomegalovirus Infections",
"Deafness",
"Gestational Age",
"Heart Defects, Congenital",
"Humans",
"Infant, Newborn",
"Rubella",
"Toxoplasmosis, Congenital"
] |
other
|
PMC2596277
| null |
12
|
[
"{'Citation': 'Am J Dis Child. 1969 May;117(5):522-39', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '4181125'}}}",
"{'Citation': 'N Engl J Med. 1970 Oct 8;283(15):771-8', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '5456233'}}}",
"{'Citation': 'N Engl J Med. 1971 Jul 29;285(5):267-74', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '4326257'}}}",
"{'Citation': 'J Infect Dis. 1975 Nov;132(5):582-6', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '171324'}}}",
"{'Citation': 'N Engl J Med. 1976 Aug 26;295(9):468-70', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '181675'}}}",
"{'Citation': 'N Engl J Med. 1977 Jun 2;296(22):1254-8', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '193004'}}}",
"{'Citation': 'Pediatrics. 1977 May;59(5):669-78', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '193086'}}}",
"{'Citation': 'J Clin Invest. 1977 Oct;60(4):838-45', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '197126'}}}",
"{'Citation': 'Scand J Infect Dis. 1980;12(1):1-5', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '6768125'}}}",
"{'Citation': 'Pediatrics. 1980 Nov;66(5):758-62', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '6159568'}}}",
"{'Citation': 'Pediatrics. 1980 Nov;66(5):767-74', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '7432882'}}}",
"{'Citation': 'N Engl J Med. 1982 Apr 22;306(16):945-9', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '6278309'}}}"
] |
Nature. 2008 Dec 4; 456(7222):667-670
|
NO-CC CODE
|
|
Activation of heterochromatin in the Zscan4+ cells. (A) ES cells were co-immunostained for Zscan4 (not shown) and euchromatin markers—H3K4me3, H3K9ac, H3K14ac, and H3K27ac (green). DNA was counterstained with DAPI (red). Arrows indicate DNA-dense heterochromatin foci. Scale bars, 5 µm. (B) Fluorescence intensities of euchromatin markers in the Zscan4+ cells compared with those in the Zscan4− cells. n = 15 for each group. Error bars, S.D. *P < 0.01, **P < 0.001. (C) Immunoblot analyses of Zscan4, H3K9ac, H3K14ac, H3K18ac, H3K27ac, H3K4me2, H3K4me3, H3K9me3, HP1α, and pan-H3 marker. The MC1-ZE7 cells were FACS-sorted into cells with Em+ (i.e. Zscan4+ cells) and cells with Em− (i.e. Zscan4− cells) and analysed by the immunoblotting. See also Supplementary Fig. S2.
|
dsv01302
|
2
|
86e17cee66d139f859cf65816c53798c635430809df1002892ff7ad4b791afb2
|
dsv01302.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
790,
329
] |
[{'image_id': 'dsv01304', 'image_file_name': 'dsv01304.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01304.jpg', 'caption': 'DNA demethylation coupled with histone hyperacetylation in heterochromatin in Zscan4+ cells. (A) Proportion of H3K27ac peaks with the high levels of DNA methylation (>50% methylated CpGs per total CpGs by the bisulfite sequencing),35 the average log-enrichment in binding of Lamin B1,36 the average log-enrichment of H3K9me2, and the log-enrichment of H3K27ac, in a sliding window of 300 H3K27ac peaks, which were sorted by the difference in the H3K27ac between Em+ and Em− cells. (B) Comparison of DNA methylation levels with the changes in H3K27ac levels between Em+ and Em− cells. H3K27ac peaks were sorted by the decreasing log ratio of H3K27ac in the Em+ versus Em− cells (x-axis), and then the proportion of peaks with DNA methylation (y-axis) was estimated in a sliding window of 300 peaks. (C) Representative examples of genes with H3K27 hyperacetylation (H3K27ac ChIP-seq, this study) and DNA demethylation (by the HELP assay, this study) in the Em+ cells compared with the Em− cells. (D) Bisulfite sequencing analyses of Tmem92 and Tdpoz4 regions (blue bars in Fig.\xa04C) were performed on Em+ and Em− cells. Open and filled circles indicate unmethylated or methylated CpG sites, respectively. The percentage of methylated CpGs per total CpGs is presented below each data set. See also Supplementary Fig. S4.', 'hash': '4a788707167b477c161850677dc17714a409a8b1705ba09e9463c68a5d699b00'}, {'image_id': 'dsv01303', 'image_file_name': 'dsv01303.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01303.jpg', 'caption': 'Correlation between histone hyperacetylations and gene expression in the Zscan4+ cells. (A) MC1-ZE7 cells were FACS-sorted into Em+ and Em− cells and analysed by the ChIP-seq using an anti-H3K27ac antibody. (B) Fractions (%) of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ or Em− cells. Error bars, S.E. (C) A scatterplot showing the comparison of H3K27ac peaks between the Em+ and Em− cells. Red dots (1,429), named ‘zH3K27ac peaks’, indicate H3K27ac peaks with significantly more (>3-fold) sequence reads in the Em+ cells compared with the Em− cells. (D) A plot showing the correlation between the gene expression differences in Em+ versus Em− cells (x-axis) and the proportion of genes with at least one zH3K27ac peak within 100 Kb from TSS, identified with a sliding window of 500 genes (y-axis). (E) Localizations of ZAGs (purple) and zH3K27ac peaks (blue) on mouse genomes. See also Supplementary Fig. S3.', 'hash': '13d7302e0b8c559f941d5b73aa98c8d78c6b15477214c0f9a59a71cf24c877ba'}, {'image_id': 'dsv01302', 'image_file_name': 'dsv01302.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01302.jpg', 'caption': 'Activation of heterochromatin in the Zscan4+ cells. (A) ES cells were co-immunostained for Zscan4 (not shown) and euchromatin markers—H3K4me3, H3K9ac, H3K14ac, and H3K27ac (green). DNA was counterstained with DAPI (red). Arrows indicate DNA-dense heterochromatin foci. Scale bars, 5 µm. (B) Fluorescence intensities of euchromatin markers in the Zscan4+ cells compared with those in the Zscan4− cells. n = 15 for each group. Error bars, S.D. *P < 0.01, **P < 0.001. (C) Immunoblot analyses of Zscan4, H3K9ac, H3K14ac, H3K18ac, H3K27ac, H3K4me2, H3K4me3, H3K9me3, HP1α, and pan-H3 marker. The MC1-ZE7 cells were FACS-sorted into cells with Em+ (i.e. Zscan4+ cells) and cells with Em− (i.e. Zscan4− cells) and analysed by the immunoblotting. See also Supplementary Fig. S2.', 'hash': '86e17cee66d139f859cf65816c53798c635430809df1002892ff7ad4b791afb2'}, {'image_id': 'dsv01305', 'image_file_name': 'dsv01305.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01305.jpg', 'caption': 'Both activating and repressing chromatin remodelling complexes localize in heterochromatin in the Zscan4+ cells. (A) Immunoblot analyses of Flag-Zscan4, Lsd1/Kdm1a, Mta1, Brg1, Hdac1, and Kap1/Trim28 proteins after immunoprecipitating the nuclear extracts of tet-Zscan4 ES cells by antibodies against Flag-tag, Lsd1/Kdm1a, Mta1, and Brg1. The tet-Zscan4 ES cells were cultured in the Dox+ (without Zscan4 overexpression) and Dox− (with Zscan4 overexpression) for 3 days. *, possible cross-reactive polypeptides. **, non-specific bands. (B) Triple immunostaining analyses of ES cells with the Zscan4 antibody (red), the CREST antibody (white, an anti-centromere protein), and various antibodies indicated (green). Blue, DAPI. Arrows indicate the clustered centromeres. Scale bars, 5 µm. See also Supplementary Fig. S5.', 'hash': '792cccdbefeb1443575c3042ee3e4373fb5ed287ace4a833a4295a64cc939598'}, {'image_id': 'dsv01306', 'image_file_name': 'dsv01306.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01306.jpg', 'caption': 'Heterochromatin clustering in the Zscan4+ cells. (A) Co-immunostaining of ES cells with an HP1α antibody (green) and a Zscan4 antibody (not shown). Red, DAPI. Scale bars, 5 µm. More examples are shown in Supplementary Fig. S2A. (B) Size distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average areas of each focus was 3.6 and 1.5 µm2 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 40. (C) Number distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average numbers of foci in each nucleus were 2.4 and 4.7 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 60. See also Supplementary Fig. S6.', 'hash': 'a48a336a491dcddb96d7aca6248e45510047b8f1546ddbecc427f0b2f93fe631'}, {'image_id': 'dsv01301', 'image_file_name': 'dsv01301.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01301.jpg', 'caption': 'Zscan4-associated heterochromatin transcription in mouse ES cells. (A) A schematic presentation for whole transcriptome analyses of ES cells in the Zscan4+ cells and Zscan4− cells. (B) FACS sorting of ES cells into Emerald-positive cells (Em+, Zscan4+ cells) and Emerald-negative cells (Em−, Zscan4− cells). (C) Expression levels of Zscan4-related genes (Zscan4, Tmem92, Tcstv3, Gm428) in FACS-sorted Em+ cells compared with Em− cells. The expression levels were normalized by GAPDH. Error bars, S.D. (D) Abundance of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ cells relative to that in Em− cells. Error bars, S.E. See also Supplementary Fig. S1. This figure is available in black and white in print and in colour at DNA Research online.', 'hash': '1691422f71d53aae0abb6dbcea8484c838de5a001114450f77ce5167cc77cfe1'}, {'image_id': 'dsv01307', 'image_file_name': 'dsv01307.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01307.jpg', 'caption': 'Active roles of Zscan4 in the heterochromatin regulation revealed by Zscan4 knockdown and Zscan4 overexpression experiments. (A, B) Inducible knockdown of Zscan4 expression (Zscan4 KD). (A) Left panel: Immunostaining analyses of the ES cells expressing a Dox-inducible shRNA against Zscan4, cultured for 8 days in the absence (−Dox) or the presence of 2 µg/ml Dox (+Dox), with the antibody against Zscan4 (red). DNA is counterstained with DAPI (red). Right panel: The percentage of cells stained with a Zscan4 antibody was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (B) The percentage of the cells with H3K27 acetylation in heterochromatin was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (C, D) Inducible overexpression of Zscan4 using tet-Zscan4 cells (Zscan4 OE). (C) Western blots using an anti-Flag antibody, anti-Zscan4 antibody, and anti-histone H3 antibody (loading control). (D) Left panel: The percentage of Zscan4+ cells in the Dox+ and Dox− conditions. *P < 0.01 versus +Dox, t-test. Right panel: Immunostaining of tet-Zscan4 ES cells with an anti-Zscan4, anti-CREST, and anti-H3K27ac. The nuclei of the Zscan4+ cells showed the hyperacetylation of H3K27 in clustered centromeric regions (arrows). (E) A schematic summary of heterochromatin dynamics during Z4 event. Tel., telomeres, Pericent., pericentromeres, Retro., retrotransposons, ZAGs, Z4 event-associated genes. See also Supplementary Fig. S7.', 'hash': '63d7c9c37ac4fce17a9db36a0271c4b7c2d6d74ad534184cf09f5152a428863e'}]
|
{'dsv01301': ['To characterize the transcriptome specific to the transient Z4 event, we carried out RNA-sequencing (RNA-seq) analyses with isolated Zscan4+ and Zscan4− cells. To separate the two kinds of cells, we FACS-sorted the MC1-ZE7 cell line,17 in which an Emerald fluorescence protein (Em) was controlled by the Zscan4 promoter, distinguishing strongly Em+ (i.e. Zscan4+) from Em− (i.e. Zscan4−) cells (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>A and B). Over 35 million reads per sample were mapped to non-repetitive regions of the mouse genome in two biological replications, detecting 24,538 genes (RefSeq). We found that among 476 differentially expressed genes (fold change > 2, FDR < 0.05), nearly all (469) were more highly expressed in Zscan4A and B). Over 35 million reads per sample were mapped to non-repetitive regions of the mouse genome in two biological replications, detecting 24,538 genes (RefSeq). We found that among 476 differentially expressed genes (fold change > 2, FDR < 0.05), nearly all (469) were more highly expressed in Zscan4+ cells than in Zscan4− cells (Supplementary Table S2). This result indicates that a specific molecular program is activated and superimposed on the regular mESC program. The findings were consistent with previous microarray analyses.18 For brevity, we refer to genes specifically up-regulated during Z4 event as ‘Z4 event-associated genes (ZAGs).’ The ZAGs included many preimplantation embryo genes such as Zscan4, Tmem92, Gm428, and Tcstv3, whose differential expression was also confirmed by quantitative PCR (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>C). During Z4 event, essentially all Zscan4 paralogues were abundantly transcribed from their canonical transcription start sites; six newly identified copies of Tmem92 were also abundantly transcribed (C). During Z4 event, essentially all Zscan4 paralogues were abundantly transcribed from their canonical transcription start sites; six newly identified copies of Tmem92 were also abundantly transcribed (Supplementary Fig. S1A), suggesting that expression of these genes is strictly controlled even though they are only expressed for a very short time.\nFigure\xa01.Zscan4-associated heterochromatin transcription in mouse ES cells. (A) A schematic presentation for whole transcriptome analyses of ES cells in the Zscan4+ cells and Zscan4− cells. (B) FACS sorting of ES cells into Emerald-positive cells (Em+, Zscan4+ cells) and Emerald-negative cells (Em−, Zscan4− cells). (C) Expression levels of Zscan4-related genes (Zscan4, Tmem92, Tcstv3, Gm428) in FACS-sorted Em+ cells compared with Em− cells. The expression levels were normalized by GAPDH. Error bars, S.D. (D) Abundance of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ cells relative to that in Em− cells. Error bars, S.E. See also Supplementary Fig. S1. This figure is available in black and white in print and in colour at DNA Research online.', 'We also mapped the RNA-seq reads to repetitive regions of the mouse genome (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>A). The analysis revealed significant increases of transcripts from repetitive sequences that are usually packed in silenced chromatin (constitutive heterochromatin), including major satellites, minor satellites, and telomeres during Z4 event (Fig.\xa0A). The analysis revealed significant increases of transcripts from repetitive sequences that are usually packed in silenced chromatin (constitutive heterochromatin), including major satellites, minor satellites, and telomeres during Z4 event (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>D). Increases of transcription from many retrotransposons were also detected (D). Increases of transcription from many retrotransposons were also detected (Supplementary Fig. S1B). Comparable activation of retrotransposons has already been reported in MuERV-L-marked mESCs (2C state).13'], 'dsv01302': ['The unusual transcriptional burst from constitutive heterochromatin during Z4 event prompted us to examine the histone modifications involved in the regulation of transcriptionally active chromatin: histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9, 14, and 27 acetylation (H3K9ac, H3K14ac, and H3K27ac). We found higher levels of these active histone modifications, especially H3K27ac, in the Zscan4+ cells compared with Zscan4– cells (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>A and A and Supplementary\nFig. S2A). This finding was further supported by the quantification of fluorescence intensity (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>B, B, P < 0.001) and immunoblot analyses (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>C).\nC).\nFigure\xa02.Activation of heterochromatin in the Zscan4+ cells. (A) ES cells were co-immunostained for Zscan4 (not shown) and euchromatin markers—H3K4me3, H3K9ac, H3K14ac, and H3K27ac (green). DNA was counterstained with DAPI (red). Arrows indicate DNA-dense heterochromatin foci. Scale bars, 5 µm. (B) Fluorescence intensities of euchromatin markers in the Zscan4+ cells compared with those in the Zscan4− cells. n = 15 for each group. Error bars, S.D. *P < 0.01, **P < 0.001. (C) Immunoblot analyses of Zscan4, H3K9ac, H3K14ac, H3K18ac, H3K27ac, H3K4me2, H3K4me3, H3K9me3, HP1α, and pan-H3 marker. The MC1-ZE7 cells were FACS-sorted into cells with Em+ (i.e. Zscan4+ cells) and cells with Em− (i.e. Zscan4− cells) and analysed by the immunoblotting. See also Supplementary Fig. S2.', 'As expected, we also found that specifically in the Zscan4+ cells, histone acetylations—particularly H3K27ac—localized not only in euchromatin but also in heterochromatin—DAPI-dense regions (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>A and A and Supplementary Fig. S2A). This was further confirmed by colocalization with major satellite (Supplementary Fig. S2B) and heterochromatin-specific protein—HP1α (heterochromatin protein 1α) (Supplementary Fig. S2C). The association of heterochromatin with active histone modifications is consistent with a burst of constitutive heterochromatin transcription during Z4 event.', 'The results thus far indicate that heterochromatin is transiently in an activated conformation in a Z4 event-specific manner. We suspected that Z4 event-specific activation of heterochromatin has some unique features. A hint came from the immunohistochemical analysis of histone modifications during Z4 event (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>A). We noticed the clustering of heterochromatin in a Z4 event-specific manner and further investigated the distribution of heterochromatin in nuclei by costaining for Zscan4 and HP1α. In the great majority (∼95%) of ESCs, which were Zscan4A). We noticed the clustering of heterochromatin in a Z4 event-specific manner and further investigated the distribution of heterochromatin in nuclei by costaining for Zscan4 and HP1α. In the great majority (∼95%) of ESCs, which were Zscan4−, heterochromatin recognized by HP1α appeared as discrete multiple foci scattered in the nucleoplasm and overlapping regions of high DNA density detected by DAPI (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>A and A and Supplementary Fig. S6A). This pattern of heterochromatin localization is commonly observed in mESCs and other cell types.40 In contrast, during Z4 event the heterochromatin appeared in larger and fewer clusters that also overlapped with the regions of high DNA density, mostly perinucleolar (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>A and A and Supplementary Fig. S6A). These observations were confirmed by quantitative morphometric analyses of a large number of cells: heterochromatin foci in Zscan4+ cells were significantly larger (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>B, B, P < 0.001) and fewer (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>C, C, P < 0.001) compared with those in Zscan4− cells. The clustering and relocalization of heterochromatin during Z4 event were also confirmed by immunostaining for H3K9me3 and H4K20me3—histone modifications associated with heterochromatin (Supplementary Fig. S6B and C).\nFigure\xa06.Heterochromatin clustering in the Zscan4+ cells. (A) Co-immunostaining of ES cells with an HP1α antibody (green) and a Zscan4 antibody (not shown). Red, DAPI. Scale bars, 5 µm. More examples are shown in Supplementary Fig. S2A. (B) Size distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average areas of each focus was 3.6 and 1.5 µm2 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 40. (C) Number distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average numbers of foci in each nucleus were 2.4 and 4.7 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 60. See also Supplementary Fig. S6.'], 'dsv01303': ['Among the histone modifications we examined thus far, H3K27ac showed the greatest up-regulation and the most specific nuclear localization in a Z4 event-specific manner. Therefore, we decided to identify the genomic localization of H3K27ac by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). To compare the genome-wide H3K27ac distributions reliably, we carried out ChIP-seq in duplicate using two independently FACS-sorted samples of Em+ and Em− cells (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>A). The independently replicated ChIP-seq results showed remarkable consistency, indicating the high specificity of H3K27ac ChIP-seq signals. We first analysed sequence reads that matched to repetitive sequences. Consistent with the immunostaining analyses, the number of sequence reads matched to major satellites and telomeres was 4- and 2-fold higher in the EmA). The independently replicated ChIP-seq results showed remarkable consistency, indicating the high specificity of H3K27ac ChIP-seq signals. We first analysed sequence reads that matched to repetitive sequences. Consistent with the immunostaining analyses, the number of sequence reads matched to major satellites and telomeres was 4- and 2-fold higher in the Em+ cells than in the Em− cells, respectively (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>B). Similarly, retrotransposons were more abundantly marked with H3K27ac in EmB). Similarly, retrotransposons were more abundantly marked with H3K27ac in Em+ cells than in Em− cells (Supplementary Fig. S3A). These results further support the involvement of H3K27ac in the transcriptional burst from repetitive sequences during Z4 event.\nFigure\xa03.Correlation between histone hyperacetylations and gene expression in the Zscan4+ cells. (A) MC1-ZE7 cells were FACS-sorted into Em+ and Em− cells and analysed by the ChIP-seq using an anti-H3K27ac antibody. (B) Fractions (%) of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ or Em− cells. Error bars, S.E. (C) A scatterplot showing the comparison of H3K27ac peaks between the Em+ and Em− cells. Red dots (1,429), named ‘zH3K27ac peaks’, indicate H3K27ac peaks with significantly more (>3-fold) sequence reads in the Em+ cells compared with the Em− cells. (D) A plot showing the correlation between the gene expression differences in Em+ versus Em− cells (x-axis) and the proportion of genes with at least one zH3K27ac peak within 100 Kb from TSS, identified with a sliding window of 500 genes (y-axis). (E) Localizations of ZAGs (purple) and zH3K27ac peaks (blue) on mouse genomes. See also Supplementary Fig. S3.', 'We next analysed sequence reads matching non-repetitive sequences. H3K27ac peaks with significantly more (>3-fold) sequence reads in the Em+ cells compared with that in the Em− cells were identified, resulting in 1,429 peaks—tentatively named ‘Z4 event-associated H3K27ac peaks (zH3K27ac peaks)’ (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>C). The majority of these zH3K27ac peaks were located in intronic and intergenic regions (C). The majority of these zH3K27ac peaks were located in intronic and intergenic regions (Supplementary Fig. S3B) and away from transcription start sites (TSSs) (Supplementary Fig. S3C). These results are consistent with H3K27ac function as a transcriptional active mark at enhancers.34 Indeed, the locations of zH3K27ac peaks were positively correlated with genes whose expression was higher in Em+ cells than in Em− cells, that is ZAGs (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>D and E), suggesting that the expression of ZAGs is also regulated by H3K27ac at their enhancers. Overlap between ZAGs and zH3K27ac was even more clearly observed when more stringent thresholds were used: 150 (59%) of 253 ZAGs (>5-fold EmD and E), suggesting that the expression of ZAGs is also regulated by H3K27ac at their enhancers. Overlap between ZAGs and zH3K27ac was even more clearly observed when more stringent thresholds were used: 150 (59%) of 253 ZAGs (>5-fold Em+/Em− expression difference) were located within 0.5 Mb from zH3K27ac peaks (P = 6 × 10−6).', 'In addition to the constitutive heterochromatin, we also observed ‘zHC’ that comprise a few hundred ‘ZAGs’, including Zscan4, which show a burst of transcription during Z4 event. Although zHC is not located in the constitutive heterochromatin region and forms scattered clusters throughout the genome (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>E), we show that in regular mESCs zHC is transcriptionally silenced and shares features of heterochromatin such as high levels of DNA methylation and the lack of activating histone marks. However, as summarized in Fig.\xa0E), we show that in regular mESCs zHC is transcriptionally silenced and shares features of heterochromatin such as high levels of DNA methylation and the lack of activating histone marks. However, as summarized in Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E, during the Z4 event, zHC also shows a burst of transcription, with increased levels of activating histone marks, H3K27ac, and reduced levels of DNA methylation (Fig.\xa0E, during the Z4 event, zHC also shows a burst of transcription, with increased levels of activating histone marks, H3K27ac, and reduced levels of DNA methylation (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E). Because H3K27ac marks distal enhancers associated with active genesE). Because H3K27ac marks distal enhancers associated with active genes34 and is important for recruiting Pol II to regulatory regions to initiate transcription,38 it is likely that the burst of transcription of ZAGs from the zHC is controlled by H3K27ac.'], 'dsv01304': ['To address this question, we examined the association of zH3K27ac peaks with previously published chromatin features in mouse ES cells—essentially equivalent to Zscan4− cells (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>A and A and Supplementary Table S3, Fig. S4). Remarkably, zH3K27ac peaks were found to be enriched for heterochromatin features, that is high levels of DNA methylation;35 Lamin B1 binding;36 and H3K9me2 marks.37 These results suggest that not only the repetitive sequences such as major satellites (pericentromeres) and telomeres, but also zH3K27ac peaks such as ZAGs are enveloped in heterochromatin in the Zscan4− cells. In other words, pericentromeres, telomeres, retrotransposons, and unique sets of genes such as ZAGs are usually in heterochromatin in mESCs, but are transiently activated during Z4 event.\nFigure\xa04.DNA demethylation coupled with histone hyperacetylation in heterochromatin in Zscan4+ cells. (A) Proportion of H3K27ac peaks with the high levels of DNA methylation (>50% methylated CpGs per total CpGs by the bisulfite sequencing),35 the average log-enrichment in binding of Lamin B1,36 the average log-enrichment of H3K9me2, and the log-enrichment of H3K27ac, in a sliding window of 300 H3K27ac peaks, which were sorted by the difference in the H3K27ac between Em+ and Em− cells. (B) Comparison of DNA methylation levels with the changes in H3K27ac levels between Em+ and Em− cells. H3K27ac peaks were sorted by the decreasing log ratio of H3K27ac in the Em+ versus Em− cells (x-axis), and then the proportion of peaks with DNA methylation (y-axis) was estimated in a sliding window of 300 peaks. (C) Representative examples of genes with H3K27 hyperacetylation (H3K27ac ChIP-seq, this study) and DNA demethylation (by the HELP assay, this study) in the Em+ cells compared with the Em− cells. (D) Bisulfite sequencing analyses of Tmem92 and Tdpoz4 regions (blue bars in Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>C) were performed on EmC) were performed on Em+ and Em− cells. Open and filled circles indicate unmethylated or methylated CpG sites, respectively. The percentage of methylated CpGs per total CpGs is presented below each data set. See also Supplementary Fig. S4.', 'To further examine the epigenetic changes specific to the Z4 event, we carried out DNA methylation analysis by the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay on Em+ and Em− cells. As expected, zH3K27 peaks were enriched with high levels of DNA methylation (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>B). However, the levels of DNA methylation in the zH3K27ac peaks were slightly reduced in EmB). However, the levels of DNA methylation in the zH3K27ac peaks were slightly reduced in Em+ cells compared with Em− cells (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>B). For example, the regulatory regions of representative ZAGs such as Tmem92 and Tdpoz4 showed H3K27ac and DNA hypomethylation (Fig.\xa0B). For example, the regulatory regions of representative ZAGs such as Tmem92 and Tdpoz4 showed H3K27ac and DNA hypomethylation (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>C)—a finding confirmed independently by bisulfite sequencing of the regions (Fig.\xa0C)—a finding confirmed independently by bisulfite sequencing of the regions (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>D). Thus, both repetitive sequences and non-repetitive ZAGs are transcriptionally silenced and heterochromatinized in Zscan4D). Thus, both repetitive sequences and non-repetitive ZAGs are transcriptionally silenced and heterochromatinized in Zscan4− cells (usual mESCs), but become transcriptionally active, acquiring H3K27ac—active histone modifications and DNA demethylation. For brevity, we call the subset of facultative heterochromatin where zH3K27ac and ZAGs are located ‘zHC’.'], 'dsv01305': ['Next, protein mixtures from each column fraction were immunoprecipitated (IP) with anti-Flag antibody and subjected to mass spectrometry analyses. After removing common contaminants often detected by Flag-tag-based IP mass spectrometry, the number of identified peptides for each protein by mass spectrometry was tabulated for each protein fraction (Supplementary Fig. S5B). The analyses revealed that Zscan4 complexes contained primarily repressing chromatin remodelling complexes (HDAC1, HDAC2, LSD1/KDM1A, NuRD, Sin3A), but also showed evidence of activating chromatin remodelling complexes (SWI/SNF). Zscan4 complexes also contained KAP1—one of the key proteins in the regulation of heterochromatin. Consistent with the mass spectrometry data, protein mixtures pulled down by Flag-IP contained Zscan4, LSD1/KDM1A, MTA1 (NuRD), BRG1 (SWI/SNF), and KAP1 (Fig.\xa0<xref ref-type="fig" rid="dsv01305">5</xref>A); and protein mixtures pulled down by LSD1-IP, MTA1-IP, and BRG1-IP, respectively, all contained Zscan4, MTA1, BRG1, HDAC1, and KAP1. These results suggest that both activating and repressing chromatin remodelling complexes are associated with Zscan4 protein.\nA); and protein mixtures pulled down by LSD1-IP, MTA1-IP, and BRG1-IP, respectively, all contained Zscan4, MTA1, BRG1, HDAC1, and KAP1. These results suggest that both activating and repressing chromatin remodelling complexes are associated with Zscan4 protein.\nFigure\xa05.Both activating and repressing chromatin remodelling complexes localize in heterochromatin in the Zscan4+ cells. (A) Immunoblot analyses of Flag-Zscan4, Lsd1/Kdm1a, Mta1, Brg1, Hdac1, and Kap1/Trim28 proteins after immunoprecipitating the nuclear extracts of tet-Zscan4 ES cells by antibodies against Flag-tag, Lsd1/Kdm1a, Mta1, and Brg1. The tet-Zscan4 ES cells were cultured in the Dox+ (without Zscan4 overexpression) and Dox− (with Zscan4 overexpression) for 3 days. *, possible cross-reactive polypeptides. **, non-specific bands. (B) Triple immunostaining analyses of ES cells with the Zscan4 antibody (red), the CREST antibody (white, an anti-centromere protein), and various antibodies indicated (green). Blue, DAPI. Arrows indicate the clustered centromeres. Scale bars, 5 µm. See also Supplementary Fig. S5.', 'To further verify Zscan4-dependent colocalization of these chromatin remodelling complexes on heterochromatin, we carried out the immunostaining analyses of these proteins in mouse ES cells (Fig.\xa0<xref ref-type="fig" rid="dsv01305">5</xref>B). For the activating chromatin remodelling complexes, the staining of BRG1 was similar for Zscan4B). For the activating chromatin remodelling complexes, the staining of BRG1 was similar for Zscan4+ and Zscan4− cells, but the histone acetyltransferases (HATs), p300 and CBP, which are known to specifically mediate the acetylation of H3K27,38 accumulated in the heterochromatin labeled with anti-centromere antibody in a Z4 event-specific manner. For the repressing chromatin remodelling complexes, HDAC1, HDAC2, MTA1, MTA2, Rbbp4, and Rbbp7, all of which are components of the NuRD complex,39 were localized in the heterochromatin in a Z4 event-specific manner. The staining of LSD1 was observed in both euchromatin and heterochromatin, but was stronger in the Zscan4+ cells than in Zscan4− cells. As a control, KAP1, heterochromatin marker, was detected in heterochromatin both in the Zscan4+ and Zscan4− cells. Interestingly, Zscan4 staining showed its localization not only in pericentromeric heterochromatin, but also in euchromatic regions, suggesting the detection of zHC, which are located in euchromatin. Alternatively, these results may indicate that Zscan4 functions not only in heterochromatin, but also in euchromatin. Overall, the heterochromatin is associated with both activating and repressing chromatin remodelling complexes during Z4 event (though a single site probably moves through a cycle of derepression and rerepression; see below).'], 'dsv01307': ['For the loss-of-function assays, we generated a mESC line carrying a Dox-inducible shRNA (short hairpin RNA) directed against Zscan4. We confirmed that Dox-induced shRNA expression for 8 days decreased the fraction of Zscan4+ mESCs (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>A). In these Zscan4 knockdown cell colonies, the number of cells displaying H3K27 acetylation in heterochromatin was significantly reduced (Fig.\xa0A). In these Zscan4 knockdown cell colonies, the number of cells displaying H3K27 acetylation in heterochromatin was significantly reduced (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>B). Furthermore, the expression of major satellites was decreased by ∼50% in Zscan4 knockdown cells (B). Furthermore, the expression of major satellites was decreased by ∼50% in Zscan4 knockdown cells (Supplementary Fig. S7A, P < 0.01).\nFigure\xa07.Active roles of Zscan4 in the heterochromatin regulation revealed by Zscan4 knockdown and Zscan4 overexpression experiments. (A, B) Inducible knockdown of Zscan4 expression (Zscan4 KD). (A) Left panel: Immunostaining analyses of the ES cells expressing a Dox-inducible shRNA against Zscan4, cultured for 8 days in the absence (−Dox) or the presence of 2 µg/ml Dox (+Dox), with the antibody against Zscan4 (red). DNA is counterstained with DAPI (red). Right panel: The percentage of cells stained with a Zscan4 antibody was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (B) The percentage of the cells with H3K27 acetylation in heterochromatin was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (C, D) Inducible overexpression of Zscan4 using tet-Zscan4 cells (Zscan4 OE). (C) Western blots using an anti-Flag antibody, anti-Zscan4 antibody, and anti-histone H3 antibody (loading control). (D) Left panel: The percentage of Zscan4+ cells in the Dox+ and Dox− conditions. *P < 0.01 versus +Dox, t-test. Right panel: Immunostaining of tet-Zscan4 ES cells with an anti-Zscan4, anti-CREST, and anti-H3K27ac. The nuclei of the Zscan4+ cells showed the hyperacetylation of H3K27 in clustered centromeric regions (arrows). (E) A schematic summary of heterochromatin dynamics during Z4 event. Tel., telomeres, Pericent., pericentromeres, Retro., retrotransposons, ZAGs, Z4 event-associated genes. See also Supplementary Fig. S7.', 'For the gain-of-function assays, we examined whether ectopic overexpression of Zscan4 can induce these epigenetic changes in mESCs, using the tet-Zscan4 ES cell line.30 As expected, the overexpression of Zscan4, which was confirmed by the western blot (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>C), increased the number of cells stained with Zscan4 antibodies (Fig.\xa0C), increased the number of cells stained with Zscan4 antibodies (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>D). Zscan4D). Zscan4+ cells were also strongly stained with the H3K27ac in the heterochromatin (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>D) and the H3K9ac (D) and the H3K9ac (Supplementary Fig. S7B). Furthermore, ChIP-qPCR analyses showed that the overexpression of Zscan4 up-regulated H3K27ac on Tmem92, Tdpoz3/4, Zscan4c/d regions (Supplementary Fig. S7C, P < 0.05). Next, we carried out MeDIP-seq (methylated DNA immunoprecipitation followed by sequencing) analyses and found that the overexpression of Zscan4 induced the demethylation of DNAs in telomeric and major satellite regions—and to some extent in minor satellite regions (Supplementary Fig. S7D). Subsequent analyses of non-repeated genome regions revealed that the overexpression of Zscan4 also induced the demethylation of DNAs in the zH3K27ac peaks (Supplementary Fig. S7E).', 'The Z4 event—a short burst of Zscan4 transcription—occurs very infrequently in usual mESC culture conditions, so that only 1–5% of mESCs are undergoing Z4 event at a given time.16–18 However, essentially all mESCs undergo Z4 event at least once within nine passages.17 In this paper, we have carried out detailed molecular analyses of the Z4 event, finding that it coincides with rapid and unusual molecular changes in chromatin, particularly in heterochromatin (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E).E).', 'Constitutive heterochromatin is usually considered silenced for transcription. However, as summarized in Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E, during the Z4 event, constitutive heterochromatin shows a burst of transcription. Generally, gene activation is accompanied by ‘open’ chromatin with enrichment of active marks and loss of repressive marks on their regulatory regions.E, during the Z4 event, constitutive heterochromatin shows a burst of transcription. Generally, gene activation is accompanied by ‘open’ chromatin with enrichment of active marks and loss of repressive marks on their regulatory regions.42 In fact, we saw increased levels of activating histone marks, H3K27ac, reduced levels of DNA methylation, along with clustering around nucleoli (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E). At the same time, repressive marks, H3K9me2, and DNA methylation were still abundant in the acetylated active heterochromatin. These results suggest that the transcriptional burst is induced from ‘open’ heterochromatin, but the heterochromatin immediately returns to its silent conformation.E). At the same time, repressive marks, H3K9me2, and DNA methylation were still abundant in the acetylated active heterochromatin. These results suggest that the transcriptional burst is induced from ‘open’ heterochromatin, but the heterochromatin immediately returns to its silent conformation.', 'The rapid and profound changes in heterochromatin during Z4 event are rather unusual, even considering the already unusual chromatin features of regular mESCs,43 such as a bivalent structure,44,45 dynamic plasticity,46 and non-CpG methylation.47 These chromatin changes specific to Z4 event suggest that mESCs undergo rapid derepression followed by the immediate rerepression of both constitutive heterochromatin and zHC during the Z4 event (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E). This notion is further supported by our finding that both activating chromatin remodelling complexes (HATs, SWI/SNF) and repressing chromatin remodelling complexes (HDAC1, HDAC2, LSD1/KDM1A, NuRD) gather on heterochromatin during Z4 event. Simultaneous modifications of both constitutive heterochromatin and zHC are also intriguing, as it has been shown that different enzymes are involved in the histone methylation of constitutive heterochromatin and facultative heterochromatin (including zHC): Suv39h1/h2 for constitutive heterochromatin and G9a for facultative heterochromatin.E). This notion is further supported by our finding that both activating chromatin remodelling complexes (HATs, SWI/SNF) and repressing chromatin remodelling complexes (HDAC1, HDAC2, LSD1/KDM1A, NuRD) gather on heterochromatin during Z4 event. Simultaneous modifications of both constitutive heterochromatin and zHC are also intriguing, as it has been shown that different enzymes are involved in the histone methylation of constitutive heterochromatin and facultative heterochromatin (including zHC): Suv39h1/h2 for constitutive heterochromatin and G9a for facultative heterochromatin.48 It remains to be clarified whether these enzymes are involved in Z4 events.']}
|
Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells
|
[
"heterochromatin",
"pericentromere",
"embryonic stem cells"
] |
DNA Res
|
1446274800
|
Prediction of complex traits using molecular genetic information is an active area in quantitative genetics research. In the postgenomic era, many types of -omic (e.g., transcriptomic, epigenomic, methylomic, and proteomic) data are becoming increasingly available. Therefore, evaluating the utility of this massive amount of information in prediction of complex traits is of interest. DNA methylation, the covalent change of a DNA molecule without affecting its underlying sequence, is one quantifiable form of epigenetic modification. We used methylation information for predicting plant height (PH) in Arabidopsis thaliana nonparametrically, using reproducing kernel Hilbert spaces (RKHS) regression. Also, we used different criteria for selecting smaller sets of probes, to assess how representative probes could be used in prediction instead of using all probes, which may lessen computational burden and lower experimental costs. Methylation information was used for describing epigenetic similarities between individuals through a kernel matrix, and the performance of predicting PH using this similarity matrix was reasonably good. The predictive correlation reached 0.53 and the same value was attained when only preselected probes were used for prediction. We created a kernel that mimics the genomic relationship matrix in genomic best linear unbiased prediction (G-BLUP) and estimated that, in this particular data set, epigenetic variation accounted for 65% of the phenotypic variance. Our results suggest that methylation information can be useful in whole-genome prediction of complex traits and that it may help to enhance understanding of complex traits when epigenetics is under examination.
|
[
"Arabidopsis",
"DNA Methylation",
"Epigenesis, Genetic",
"Phenotype",
"Proteomics",
"Quantitative Trait Loci"
] |
other
|
PMC4596397
| null |
86
|
[
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DNA Res. 2015 Oct 31; 22(5):307-318
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Active roles of Zscan4 in the heterochromatin regulation revealed by Zscan4 knockdown and Zscan4 overexpression experiments. (A, B) Inducible knockdown of Zscan4 expression (Zscan4 KD). (A) Left panel: Immunostaining analyses of the ES cells expressing a Dox-inducible shRNA against Zscan4, cultured for 8 days in the absence (−Dox) or the presence of 2 µg/ml Dox (+Dox), with the antibody against Zscan4 (red). DNA is counterstained with DAPI (red). Right panel: The percentage of cells stained with a Zscan4 antibody was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (B) The percentage of the cells with H3K27 acetylation in heterochromatin was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (C, D) Inducible overexpression of Zscan4 using tet-Zscan4 cells (Zscan4 OE). (C) Western blots using an anti-Flag antibody, anti-Zscan4 antibody, and anti-histone H3 antibody (loading control). (D) Left panel: The percentage of Zscan4+ cells in the Dox+ and Dox− conditions. *P < 0.01 versus +Dox, t-test. Right panel: Immunostaining of tet-Zscan4 ES cells with an anti-Zscan4, anti-CREST, and anti-H3K27ac. The nuclei of the Zscan4+ cells showed the hyperacetylation of H3K27 in clustered centromeric regions (arrows). (E) A schematic summary of heterochromatin dynamics during Z4 event. Tel., telomeres, Pericent., pericentromeres, Retro., retrotransposons, ZAGs, Z4 event-associated genes. See also Supplementary Fig. S7.
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multiple panels: images & plots
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[
"Microscopy",
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"Immuno Assays"
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[{'image_id': 'dsv01304', 'image_file_name': 'dsv01304.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01304.jpg', 'caption': 'DNA demethylation coupled with histone hyperacetylation in heterochromatin in Zscan4+ cells. (A) Proportion of H3K27ac peaks with the high levels of DNA methylation (>50% methylated CpGs per total CpGs by the bisulfite sequencing),35 the average log-enrichment in binding of Lamin B1,36 the average log-enrichment of H3K9me2, and the log-enrichment of H3K27ac, in a sliding window of 300 H3K27ac peaks, which were sorted by the difference in the H3K27ac between Em+ and Em− cells. (B) Comparison of DNA methylation levels with the changes in H3K27ac levels between Em+ and Em− cells. H3K27ac peaks were sorted by the decreasing log ratio of H3K27ac in the Em+ versus Em− cells (x-axis), and then the proportion of peaks with DNA methylation (y-axis) was estimated in a sliding window of 300 peaks. (C) Representative examples of genes with H3K27 hyperacetylation (H3K27ac ChIP-seq, this study) and DNA demethylation (by the HELP assay, this study) in the Em+ cells compared with the Em− cells. (D) Bisulfite sequencing analyses of Tmem92 and Tdpoz4 regions (blue bars in Fig.\xa04C) were performed on Em+ and Em− cells. Open and filled circles indicate unmethylated or methylated CpG sites, respectively. The percentage of methylated CpGs per total CpGs is presented below each data set. See also Supplementary Fig. S4.', 'hash': '4a788707167b477c161850677dc17714a409a8b1705ba09e9463c68a5d699b00'}, {'image_id': 'dsv01303', 'image_file_name': 'dsv01303.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01303.jpg', 'caption': 'Correlation between histone hyperacetylations and gene expression in the Zscan4+ cells. (A) MC1-ZE7 cells were FACS-sorted into Em+ and Em− cells and analysed by the ChIP-seq using an anti-H3K27ac antibody. (B) Fractions (%) of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ or Em− cells. Error bars, S.E. (C) A scatterplot showing the comparison of H3K27ac peaks between the Em+ and Em− cells. Red dots (1,429), named ‘zH3K27ac peaks’, indicate H3K27ac peaks with significantly more (>3-fold) sequence reads in the Em+ cells compared with the Em− cells. (D) A plot showing the correlation between the gene expression differences in Em+ versus Em− cells (x-axis) and the proportion of genes with at least one zH3K27ac peak within 100 Kb from TSS, identified with a sliding window of 500 genes (y-axis). (E) Localizations of ZAGs (purple) and zH3K27ac peaks (blue) on mouse genomes. See also Supplementary Fig. S3.', 'hash': '13d7302e0b8c559f941d5b73aa98c8d78c6b15477214c0f9a59a71cf24c877ba'}, {'image_id': 'dsv01302', 'image_file_name': 'dsv01302.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01302.jpg', 'caption': 'Activation of heterochromatin in the Zscan4+ cells. (A) ES cells were co-immunostained for Zscan4 (not shown) and euchromatin markers—H3K4me3, H3K9ac, H3K14ac, and H3K27ac (green). DNA was counterstained with DAPI (red). Arrows indicate DNA-dense heterochromatin foci. Scale bars, 5 µm. (B) Fluorescence intensities of euchromatin markers in the Zscan4+ cells compared with those in the Zscan4− cells. n = 15 for each group. Error bars, S.D. *P < 0.01, **P < 0.001. (C) Immunoblot analyses of Zscan4, H3K9ac, H3K14ac, H3K18ac, H3K27ac, H3K4me2, H3K4me3, H3K9me3, HP1α, and pan-H3 marker. The MC1-ZE7 cells were FACS-sorted into cells with Em+ (i.e. Zscan4+ cells) and cells with Em− (i.e. Zscan4− cells) and analysed by the immunoblotting. See also Supplementary Fig. S2.', 'hash': '86e17cee66d139f859cf65816c53798c635430809df1002892ff7ad4b791afb2'}, {'image_id': 'dsv01305', 'image_file_name': 'dsv01305.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01305.jpg', 'caption': 'Both activating and repressing chromatin remodelling complexes localize in heterochromatin in the Zscan4+ cells. (A) Immunoblot analyses of Flag-Zscan4, Lsd1/Kdm1a, Mta1, Brg1, Hdac1, and Kap1/Trim28 proteins after immunoprecipitating the nuclear extracts of tet-Zscan4 ES cells by antibodies against Flag-tag, Lsd1/Kdm1a, Mta1, and Brg1. The tet-Zscan4 ES cells were cultured in the Dox+ (without Zscan4 overexpression) and Dox− (with Zscan4 overexpression) for 3 days. *, possible cross-reactive polypeptides. **, non-specific bands. (B) Triple immunostaining analyses of ES cells with the Zscan4 antibody (red), the CREST antibody (white, an anti-centromere protein), and various antibodies indicated (green). Blue, DAPI. Arrows indicate the clustered centromeres. Scale bars, 5 µm. See also Supplementary Fig. S5.', 'hash': '792cccdbefeb1443575c3042ee3e4373fb5ed287ace4a833a4295a64cc939598'}, {'image_id': 'dsv01306', 'image_file_name': 'dsv01306.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01306.jpg', 'caption': 'Heterochromatin clustering in the Zscan4+ cells. (A) Co-immunostaining of ES cells with an HP1α antibody (green) and a Zscan4 antibody (not shown). Red, DAPI. Scale bars, 5 µm. More examples are shown in Supplementary Fig. S2A. (B) Size distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average areas of each focus was 3.6 and 1.5 µm2 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 40. (C) Number distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average numbers of foci in each nucleus were 2.4 and 4.7 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 60. See also Supplementary Fig. S6.', 'hash': 'a48a336a491dcddb96d7aca6248e45510047b8f1546ddbecc427f0b2f93fe631'}, {'image_id': 'dsv01301', 'image_file_name': 'dsv01301.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01301.jpg', 'caption': 'Zscan4-associated heterochromatin transcription in mouse ES cells. (A) A schematic presentation for whole transcriptome analyses of ES cells in the Zscan4+ cells and Zscan4− cells. (B) FACS sorting of ES cells into Emerald-positive cells (Em+, Zscan4+ cells) and Emerald-negative cells (Em−, Zscan4− cells). (C) Expression levels of Zscan4-related genes (Zscan4, Tmem92, Tcstv3, Gm428) in FACS-sorted Em+ cells compared with Em− cells. The expression levels were normalized by GAPDH. Error bars, S.D. (D) Abundance of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ cells relative to that in Em− cells. Error bars, S.E. See also Supplementary Fig. S1. This figure is available in black and white in print and in colour at DNA Research online.', 'hash': '1691422f71d53aae0abb6dbcea8484c838de5a001114450f77ce5167cc77cfe1'}, {'image_id': 'dsv01307', 'image_file_name': 'dsv01307.jpg', 'image_path': '../data/media_files/PMC4596397/dsv01307.jpg', 'caption': 'Active roles of Zscan4 in the heterochromatin regulation revealed by Zscan4 knockdown and Zscan4 overexpression experiments. (A, B) Inducible knockdown of Zscan4 expression (Zscan4 KD). (A) Left panel: Immunostaining analyses of the ES cells expressing a Dox-inducible shRNA against Zscan4, cultured for 8 days in the absence (−Dox) or the presence of 2 µg/ml Dox (+Dox), with the antibody against Zscan4 (red). DNA is counterstained with DAPI (red). Right panel: The percentage of cells stained with a Zscan4 antibody was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (B) The percentage of the cells with H3K27 acetylation in heterochromatin was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (C, D) Inducible overexpression of Zscan4 using tet-Zscan4 cells (Zscan4 OE). (C) Western blots using an anti-Flag antibody, anti-Zscan4 antibody, and anti-histone H3 antibody (loading control). (D) Left panel: The percentage of Zscan4+ cells in the Dox+ and Dox− conditions. *P < 0.01 versus +Dox, t-test. Right panel: Immunostaining of tet-Zscan4 ES cells with an anti-Zscan4, anti-CREST, and anti-H3K27ac. The nuclei of the Zscan4+ cells showed the hyperacetylation of H3K27 in clustered centromeric regions (arrows). (E) A schematic summary of heterochromatin dynamics during Z4 event. Tel., telomeres, Pericent., pericentromeres, Retro., retrotransposons, ZAGs, Z4 event-associated genes. See also Supplementary Fig. S7.', 'hash': '63d7c9c37ac4fce17a9db36a0271c4b7c2d6d74ad534184cf09f5152a428863e'}]
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{'dsv01301': ['To characterize the transcriptome specific to the transient Z4 event, we carried out RNA-sequencing (RNA-seq) analyses with isolated Zscan4+ and Zscan4− cells. To separate the two kinds of cells, we FACS-sorted the MC1-ZE7 cell line,17 in which an Emerald fluorescence protein (Em) was controlled by the Zscan4 promoter, distinguishing strongly Em+ (i.e. Zscan4+) from Em− (i.e. Zscan4−) cells (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>A and B). Over 35 million reads per sample were mapped to non-repetitive regions of the mouse genome in two biological replications, detecting 24,538 genes (RefSeq). We found that among 476 differentially expressed genes (fold change > 2, FDR < 0.05), nearly all (469) were more highly expressed in Zscan4A and B). Over 35 million reads per sample were mapped to non-repetitive regions of the mouse genome in two biological replications, detecting 24,538 genes (RefSeq). We found that among 476 differentially expressed genes (fold change > 2, FDR < 0.05), nearly all (469) were more highly expressed in Zscan4+ cells than in Zscan4− cells (Supplementary Table S2). This result indicates that a specific molecular program is activated and superimposed on the regular mESC program. The findings were consistent with previous microarray analyses.18 For brevity, we refer to genes specifically up-regulated during Z4 event as ‘Z4 event-associated genes (ZAGs).’ The ZAGs included many preimplantation embryo genes such as Zscan4, Tmem92, Gm428, and Tcstv3, whose differential expression was also confirmed by quantitative PCR (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>C). During Z4 event, essentially all Zscan4 paralogues were abundantly transcribed from their canonical transcription start sites; six newly identified copies of Tmem92 were also abundantly transcribed (C). During Z4 event, essentially all Zscan4 paralogues were abundantly transcribed from their canonical transcription start sites; six newly identified copies of Tmem92 were also abundantly transcribed (Supplementary Fig. S1A), suggesting that expression of these genes is strictly controlled even though they are only expressed for a very short time.\nFigure\xa01.Zscan4-associated heterochromatin transcription in mouse ES cells. (A) A schematic presentation for whole transcriptome analyses of ES cells in the Zscan4+ cells and Zscan4− cells. (B) FACS sorting of ES cells into Emerald-positive cells (Em+, Zscan4+ cells) and Emerald-negative cells (Em−, Zscan4− cells). (C) Expression levels of Zscan4-related genes (Zscan4, Tmem92, Tcstv3, Gm428) in FACS-sorted Em+ cells compared with Em− cells. The expression levels were normalized by GAPDH. Error bars, S.D. (D) Abundance of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ cells relative to that in Em− cells. Error bars, S.E. See also Supplementary Fig. S1. This figure is available in black and white in print and in colour at DNA Research online.', 'We also mapped the RNA-seq reads to repetitive regions of the mouse genome (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>A). The analysis revealed significant increases of transcripts from repetitive sequences that are usually packed in silenced chromatin (constitutive heterochromatin), including major satellites, minor satellites, and telomeres during Z4 event (Fig.\xa0A). The analysis revealed significant increases of transcripts from repetitive sequences that are usually packed in silenced chromatin (constitutive heterochromatin), including major satellites, minor satellites, and telomeres during Z4 event (Fig.\xa0<xref ref-type="fig" rid="dsv01301">1</xref>D). Increases of transcription from many retrotransposons were also detected (D). Increases of transcription from many retrotransposons were also detected (Supplementary Fig. S1B). Comparable activation of retrotransposons has already been reported in MuERV-L-marked mESCs (2C state).13'], 'dsv01302': ['The unusual transcriptional burst from constitutive heterochromatin during Z4 event prompted us to examine the histone modifications involved in the regulation of transcriptionally active chromatin: histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9, 14, and 27 acetylation (H3K9ac, H3K14ac, and H3K27ac). We found higher levels of these active histone modifications, especially H3K27ac, in the Zscan4+ cells compared with Zscan4– cells (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>A and A and Supplementary\nFig. S2A). This finding was further supported by the quantification of fluorescence intensity (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>B, B, P < 0.001) and immunoblot analyses (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>C).\nC).\nFigure\xa02.Activation of heterochromatin in the Zscan4+ cells. (A) ES cells were co-immunostained for Zscan4 (not shown) and euchromatin markers—H3K4me3, H3K9ac, H3K14ac, and H3K27ac (green). DNA was counterstained with DAPI (red). Arrows indicate DNA-dense heterochromatin foci. Scale bars, 5 µm. (B) Fluorescence intensities of euchromatin markers in the Zscan4+ cells compared with those in the Zscan4− cells. n = 15 for each group. Error bars, S.D. *P < 0.01, **P < 0.001. (C) Immunoblot analyses of Zscan4, H3K9ac, H3K14ac, H3K18ac, H3K27ac, H3K4me2, H3K4me3, H3K9me3, HP1α, and pan-H3 marker. The MC1-ZE7 cells were FACS-sorted into cells with Em+ (i.e. Zscan4+ cells) and cells with Em− (i.e. Zscan4− cells) and analysed by the immunoblotting. See also Supplementary Fig. S2.', 'As expected, we also found that specifically in the Zscan4+ cells, histone acetylations—particularly H3K27ac—localized not only in euchromatin but also in heterochromatin—DAPI-dense regions (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>A and A and Supplementary Fig. S2A). This was further confirmed by colocalization with major satellite (Supplementary Fig. S2B) and heterochromatin-specific protein—HP1α (heterochromatin protein 1α) (Supplementary Fig. S2C). The association of heterochromatin with active histone modifications is consistent with a burst of constitutive heterochromatin transcription during Z4 event.', 'The results thus far indicate that heterochromatin is transiently in an activated conformation in a Z4 event-specific manner. We suspected that Z4 event-specific activation of heterochromatin has some unique features. A hint came from the immunohistochemical analysis of histone modifications during Z4 event (Fig.\xa0<xref ref-type="fig" rid="dsv01302">2</xref>A). We noticed the clustering of heterochromatin in a Z4 event-specific manner and further investigated the distribution of heterochromatin in nuclei by costaining for Zscan4 and HP1α. In the great majority (∼95%) of ESCs, which were Zscan4A). We noticed the clustering of heterochromatin in a Z4 event-specific manner and further investigated the distribution of heterochromatin in nuclei by costaining for Zscan4 and HP1α. In the great majority (∼95%) of ESCs, which were Zscan4−, heterochromatin recognized by HP1α appeared as discrete multiple foci scattered in the nucleoplasm and overlapping regions of high DNA density detected by DAPI (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>A and A and Supplementary Fig. S6A). This pattern of heterochromatin localization is commonly observed in mESCs and other cell types.40 In contrast, during Z4 event the heterochromatin appeared in larger and fewer clusters that also overlapped with the regions of high DNA density, mostly perinucleolar (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>A and A and Supplementary Fig. S6A). These observations were confirmed by quantitative morphometric analyses of a large number of cells: heterochromatin foci in Zscan4+ cells were significantly larger (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>B, B, P < 0.001) and fewer (Fig.\xa0<xref ref-type="fig" rid="dsv01306">6</xref>C, C, P < 0.001) compared with those in Zscan4− cells. The clustering and relocalization of heterochromatin during Z4 event were also confirmed by immunostaining for H3K9me3 and H4K20me3—histone modifications associated with heterochromatin (Supplementary Fig. S6B and C).\nFigure\xa06.Heterochromatin clustering in the Zscan4+ cells. (A) Co-immunostaining of ES cells with an HP1α antibody (green) and a Zscan4 antibody (not shown). Red, DAPI. Scale bars, 5 µm. More examples are shown in Supplementary Fig. S2A. (B) Size distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average areas of each focus was 3.6 and 1.5 µm2 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 40. (C) Number distribution of nuclear foci stained with an HP1α antibody in the Zscan4+ cells (red bars) and in the Zscan4− cells (blue bars). Average numbers of foci in each nucleus were 2.4 and 4.7 in the Zscan4+ cells and in the Zscan4− cells, respectively. n = 60. See also Supplementary Fig. S6.'], 'dsv01303': ['Among the histone modifications we examined thus far, H3K27ac showed the greatest up-regulation and the most specific nuclear localization in a Z4 event-specific manner. Therefore, we decided to identify the genomic localization of H3K27ac by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). To compare the genome-wide H3K27ac distributions reliably, we carried out ChIP-seq in duplicate using two independently FACS-sorted samples of Em+ and Em− cells (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>A). The independently replicated ChIP-seq results showed remarkable consistency, indicating the high specificity of H3K27ac ChIP-seq signals. We first analysed sequence reads that matched to repetitive sequences. Consistent with the immunostaining analyses, the number of sequence reads matched to major satellites and telomeres was 4- and 2-fold higher in the EmA). The independently replicated ChIP-seq results showed remarkable consistency, indicating the high specificity of H3K27ac ChIP-seq signals. We first analysed sequence reads that matched to repetitive sequences. Consistent with the immunostaining analyses, the number of sequence reads matched to major satellites and telomeres was 4- and 2-fold higher in the Em+ cells than in the Em− cells, respectively (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>B). Similarly, retrotransposons were more abundantly marked with H3K27ac in EmB). Similarly, retrotransposons were more abundantly marked with H3K27ac in Em+ cells than in Em− cells (Supplementary Fig. S3A). These results further support the involvement of H3K27ac in the transcriptional burst from repetitive sequences during Z4 event.\nFigure\xa03.Correlation between histone hyperacetylations and gene expression in the Zscan4+ cells. (A) MC1-ZE7 cells were FACS-sorted into Em+ and Em− cells and analysed by the ChIP-seq using an anti-H3K27ac antibody. (B) Fractions (%) of sequence reads matched to major satellites, minor satellites, and telomeres in the Em+ or Em− cells. Error bars, S.E. (C) A scatterplot showing the comparison of H3K27ac peaks between the Em+ and Em− cells. Red dots (1,429), named ‘zH3K27ac peaks’, indicate H3K27ac peaks with significantly more (>3-fold) sequence reads in the Em+ cells compared with the Em− cells. (D) A plot showing the correlation between the gene expression differences in Em+ versus Em− cells (x-axis) and the proportion of genes with at least one zH3K27ac peak within 100 Kb from TSS, identified with a sliding window of 500 genes (y-axis). (E) Localizations of ZAGs (purple) and zH3K27ac peaks (blue) on mouse genomes. See also Supplementary Fig. S3.', 'We next analysed sequence reads matching non-repetitive sequences. H3K27ac peaks with significantly more (>3-fold) sequence reads in the Em+ cells compared with that in the Em− cells were identified, resulting in 1,429 peaks—tentatively named ‘Z4 event-associated H3K27ac peaks (zH3K27ac peaks)’ (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>C). The majority of these zH3K27ac peaks were located in intronic and intergenic regions (C). The majority of these zH3K27ac peaks were located in intronic and intergenic regions (Supplementary Fig. S3B) and away from transcription start sites (TSSs) (Supplementary Fig. S3C). These results are consistent with H3K27ac function as a transcriptional active mark at enhancers.34 Indeed, the locations of zH3K27ac peaks were positively correlated with genes whose expression was higher in Em+ cells than in Em− cells, that is ZAGs (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>D and E), suggesting that the expression of ZAGs is also regulated by H3K27ac at their enhancers. Overlap between ZAGs and zH3K27ac was even more clearly observed when more stringent thresholds were used: 150 (59%) of 253 ZAGs (>5-fold EmD and E), suggesting that the expression of ZAGs is also regulated by H3K27ac at their enhancers. Overlap between ZAGs and zH3K27ac was even more clearly observed when more stringent thresholds were used: 150 (59%) of 253 ZAGs (>5-fold Em+/Em− expression difference) were located within 0.5 Mb from zH3K27ac peaks (P = 6 × 10−6).', 'In addition to the constitutive heterochromatin, we also observed ‘zHC’ that comprise a few hundred ‘ZAGs’, including Zscan4, which show a burst of transcription during Z4 event. Although zHC is not located in the constitutive heterochromatin region and forms scattered clusters throughout the genome (Fig.\xa0<xref ref-type="fig" rid="dsv01303">3</xref>E), we show that in regular mESCs zHC is transcriptionally silenced and shares features of heterochromatin such as high levels of DNA methylation and the lack of activating histone marks. However, as summarized in Fig.\xa0E), we show that in regular mESCs zHC is transcriptionally silenced and shares features of heterochromatin such as high levels of DNA methylation and the lack of activating histone marks. However, as summarized in Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E, during the Z4 event, zHC also shows a burst of transcription, with increased levels of activating histone marks, H3K27ac, and reduced levels of DNA methylation (Fig.\xa0E, during the Z4 event, zHC also shows a burst of transcription, with increased levels of activating histone marks, H3K27ac, and reduced levels of DNA methylation (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E). Because H3K27ac marks distal enhancers associated with active genesE). Because H3K27ac marks distal enhancers associated with active genes34 and is important for recruiting Pol II to regulatory regions to initiate transcription,38 it is likely that the burst of transcription of ZAGs from the zHC is controlled by H3K27ac.'], 'dsv01304': ['To address this question, we examined the association of zH3K27ac peaks with previously published chromatin features in mouse ES cells—essentially equivalent to Zscan4− cells (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>A and A and Supplementary Table S3, Fig. S4). Remarkably, zH3K27ac peaks were found to be enriched for heterochromatin features, that is high levels of DNA methylation;35 Lamin B1 binding;36 and H3K9me2 marks.37 These results suggest that not only the repetitive sequences such as major satellites (pericentromeres) and telomeres, but also zH3K27ac peaks such as ZAGs are enveloped in heterochromatin in the Zscan4− cells. In other words, pericentromeres, telomeres, retrotransposons, and unique sets of genes such as ZAGs are usually in heterochromatin in mESCs, but are transiently activated during Z4 event.\nFigure\xa04.DNA demethylation coupled with histone hyperacetylation in heterochromatin in Zscan4+ cells. (A) Proportion of H3K27ac peaks with the high levels of DNA methylation (>50% methylated CpGs per total CpGs by the bisulfite sequencing),35 the average log-enrichment in binding of Lamin B1,36 the average log-enrichment of H3K9me2, and the log-enrichment of H3K27ac, in a sliding window of 300 H3K27ac peaks, which were sorted by the difference in the H3K27ac between Em+ and Em− cells. (B) Comparison of DNA methylation levels with the changes in H3K27ac levels between Em+ and Em− cells. H3K27ac peaks were sorted by the decreasing log ratio of H3K27ac in the Em+ versus Em− cells (x-axis), and then the proportion of peaks with DNA methylation (y-axis) was estimated in a sliding window of 300 peaks. (C) Representative examples of genes with H3K27 hyperacetylation (H3K27ac ChIP-seq, this study) and DNA demethylation (by the HELP assay, this study) in the Em+ cells compared with the Em− cells. (D) Bisulfite sequencing analyses of Tmem92 and Tdpoz4 regions (blue bars in Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>C) were performed on EmC) were performed on Em+ and Em− cells. Open and filled circles indicate unmethylated or methylated CpG sites, respectively. The percentage of methylated CpGs per total CpGs is presented below each data set. See also Supplementary Fig. S4.', 'To further examine the epigenetic changes specific to the Z4 event, we carried out DNA methylation analysis by the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay on Em+ and Em− cells. As expected, zH3K27 peaks were enriched with high levels of DNA methylation (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>B). However, the levels of DNA methylation in the zH3K27ac peaks were slightly reduced in EmB). However, the levels of DNA methylation in the zH3K27ac peaks were slightly reduced in Em+ cells compared with Em− cells (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>B). For example, the regulatory regions of representative ZAGs such as Tmem92 and Tdpoz4 showed H3K27ac and DNA hypomethylation (Fig.\xa0B). For example, the regulatory regions of representative ZAGs such as Tmem92 and Tdpoz4 showed H3K27ac and DNA hypomethylation (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>C)—a finding confirmed independently by bisulfite sequencing of the regions (Fig.\xa0C)—a finding confirmed independently by bisulfite sequencing of the regions (Fig.\xa0<xref ref-type="fig" rid="dsv01304">4</xref>D). Thus, both repetitive sequences and non-repetitive ZAGs are transcriptionally silenced and heterochromatinized in Zscan4D). Thus, both repetitive sequences and non-repetitive ZAGs are transcriptionally silenced and heterochromatinized in Zscan4− cells (usual mESCs), but become transcriptionally active, acquiring H3K27ac—active histone modifications and DNA demethylation. For brevity, we call the subset of facultative heterochromatin where zH3K27ac and ZAGs are located ‘zHC’.'], 'dsv01305': ['Next, protein mixtures from each column fraction were immunoprecipitated (IP) with anti-Flag antibody and subjected to mass spectrometry analyses. After removing common contaminants often detected by Flag-tag-based IP mass spectrometry, the number of identified peptides for each protein by mass spectrometry was tabulated for each protein fraction (Supplementary Fig. S5B). The analyses revealed that Zscan4 complexes contained primarily repressing chromatin remodelling complexes (HDAC1, HDAC2, LSD1/KDM1A, NuRD, Sin3A), but also showed evidence of activating chromatin remodelling complexes (SWI/SNF). Zscan4 complexes also contained KAP1—one of the key proteins in the regulation of heterochromatin. Consistent with the mass spectrometry data, protein mixtures pulled down by Flag-IP contained Zscan4, LSD1/KDM1A, MTA1 (NuRD), BRG1 (SWI/SNF), and KAP1 (Fig.\xa0<xref ref-type="fig" rid="dsv01305">5</xref>A); and protein mixtures pulled down by LSD1-IP, MTA1-IP, and BRG1-IP, respectively, all contained Zscan4, MTA1, BRG1, HDAC1, and KAP1. These results suggest that both activating and repressing chromatin remodelling complexes are associated with Zscan4 protein.\nA); and protein mixtures pulled down by LSD1-IP, MTA1-IP, and BRG1-IP, respectively, all contained Zscan4, MTA1, BRG1, HDAC1, and KAP1. These results suggest that both activating and repressing chromatin remodelling complexes are associated with Zscan4 protein.\nFigure\xa05.Both activating and repressing chromatin remodelling complexes localize in heterochromatin in the Zscan4+ cells. (A) Immunoblot analyses of Flag-Zscan4, Lsd1/Kdm1a, Mta1, Brg1, Hdac1, and Kap1/Trim28 proteins after immunoprecipitating the nuclear extracts of tet-Zscan4 ES cells by antibodies against Flag-tag, Lsd1/Kdm1a, Mta1, and Brg1. The tet-Zscan4 ES cells were cultured in the Dox+ (without Zscan4 overexpression) and Dox− (with Zscan4 overexpression) for 3 days. *, possible cross-reactive polypeptides. **, non-specific bands. (B) Triple immunostaining analyses of ES cells with the Zscan4 antibody (red), the CREST antibody (white, an anti-centromere protein), and various antibodies indicated (green). Blue, DAPI. Arrows indicate the clustered centromeres. Scale bars, 5 µm. See also Supplementary Fig. S5.', 'To further verify Zscan4-dependent colocalization of these chromatin remodelling complexes on heterochromatin, we carried out the immunostaining analyses of these proteins in mouse ES cells (Fig.\xa0<xref ref-type="fig" rid="dsv01305">5</xref>B). For the activating chromatin remodelling complexes, the staining of BRG1 was similar for Zscan4B). For the activating chromatin remodelling complexes, the staining of BRG1 was similar for Zscan4+ and Zscan4− cells, but the histone acetyltransferases (HATs), p300 and CBP, which are known to specifically mediate the acetylation of H3K27,38 accumulated in the heterochromatin labeled with anti-centromere antibody in a Z4 event-specific manner. For the repressing chromatin remodelling complexes, HDAC1, HDAC2, MTA1, MTA2, Rbbp4, and Rbbp7, all of which are components of the NuRD complex,39 were localized in the heterochromatin in a Z4 event-specific manner. The staining of LSD1 was observed in both euchromatin and heterochromatin, but was stronger in the Zscan4+ cells than in Zscan4− cells. As a control, KAP1, heterochromatin marker, was detected in heterochromatin both in the Zscan4+ and Zscan4− cells. Interestingly, Zscan4 staining showed its localization not only in pericentromeric heterochromatin, but also in euchromatic regions, suggesting the detection of zHC, which are located in euchromatin. Alternatively, these results may indicate that Zscan4 functions not only in heterochromatin, but also in euchromatin. Overall, the heterochromatin is associated with both activating and repressing chromatin remodelling complexes during Z4 event (though a single site probably moves through a cycle of derepression and rerepression; see below).'], 'dsv01307': ['For the loss-of-function assays, we generated a mESC line carrying a Dox-inducible shRNA (short hairpin RNA) directed against Zscan4. We confirmed that Dox-induced shRNA expression for 8 days decreased the fraction of Zscan4+ mESCs (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>A). In these Zscan4 knockdown cell colonies, the number of cells displaying H3K27 acetylation in heterochromatin was significantly reduced (Fig.\xa0A). In these Zscan4 knockdown cell colonies, the number of cells displaying H3K27 acetylation in heterochromatin was significantly reduced (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>B). Furthermore, the expression of major satellites was decreased by ∼50% in Zscan4 knockdown cells (B). Furthermore, the expression of major satellites was decreased by ∼50% in Zscan4 knockdown cells (Supplementary Fig. S7A, P < 0.01).\nFigure\xa07.Active roles of Zscan4 in the heterochromatin regulation revealed by Zscan4 knockdown and Zscan4 overexpression experiments. (A, B) Inducible knockdown of Zscan4 expression (Zscan4 KD). (A) Left panel: Immunostaining analyses of the ES cells expressing a Dox-inducible shRNA against Zscan4, cultured for 8 days in the absence (−Dox) or the presence of 2 µg/ml Dox (+Dox), with the antibody against Zscan4 (red). DNA is counterstained with DAPI (red). Right panel: The percentage of cells stained with a Zscan4 antibody was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (B) The percentage of the cells with H3K27 acetylation in heterochromatin was significantly reduced by the Dox treatment (*P < 0.01, t-test). Error bars indicate S.D. (C, D) Inducible overexpression of Zscan4 using tet-Zscan4 cells (Zscan4 OE). (C) Western blots using an anti-Flag antibody, anti-Zscan4 antibody, and anti-histone H3 antibody (loading control). (D) Left panel: The percentage of Zscan4+ cells in the Dox+ and Dox− conditions. *P < 0.01 versus +Dox, t-test. Right panel: Immunostaining of tet-Zscan4 ES cells with an anti-Zscan4, anti-CREST, and anti-H3K27ac. The nuclei of the Zscan4+ cells showed the hyperacetylation of H3K27 in clustered centromeric regions (arrows). (E) A schematic summary of heterochromatin dynamics during Z4 event. Tel., telomeres, Pericent., pericentromeres, Retro., retrotransposons, ZAGs, Z4 event-associated genes. See also Supplementary Fig. S7.', 'For the gain-of-function assays, we examined whether ectopic overexpression of Zscan4 can induce these epigenetic changes in mESCs, using the tet-Zscan4 ES cell line.30 As expected, the overexpression of Zscan4, which was confirmed by the western blot (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>C), increased the number of cells stained with Zscan4 antibodies (Fig.\xa0C), increased the number of cells stained with Zscan4 antibodies (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>D). Zscan4D). Zscan4+ cells were also strongly stained with the H3K27ac in the heterochromatin (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>D) and the H3K9ac (D) and the H3K9ac (Supplementary Fig. S7B). Furthermore, ChIP-qPCR analyses showed that the overexpression of Zscan4 up-regulated H3K27ac on Tmem92, Tdpoz3/4, Zscan4c/d regions (Supplementary Fig. S7C, P < 0.05). Next, we carried out MeDIP-seq (methylated DNA immunoprecipitation followed by sequencing) analyses and found that the overexpression of Zscan4 induced the demethylation of DNAs in telomeric and major satellite regions—and to some extent in minor satellite regions (Supplementary Fig. S7D). Subsequent analyses of non-repeated genome regions revealed that the overexpression of Zscan4 also induced the demethylation of DNAs in the zH3K27ac peaks (Supplementary Fig. S7E).', 'The Z4 event—a short burst of Zscan4 transcription—occurs very infrequently in usual mESC culture conditions, so that only 1–5% of mESCs are undergoing Z4 event at a given time.16–18 However, essentially all mESCs undergo Z4 event at least once within nine passages.17 In this paper, we have carried out detailed molecular analyses of the Z4 event, finding that it coincides with rapid and unusual molecular changes in chromatin, particularly in heterochromatin (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E).E).', 'Constitutive heterochromatin is usually considered silenced for transcription. However, as summarized in Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E, during the Z4 event, constitutive heterochromatin shows a burst of transcription. Generally, gene activation is accompanied by ‘open’ chromatin with enrichment of active marks and loss of repressive marks on their regulatory regions.E, during the Z4 event, constitutive heterochromatin shows a burst of transcription. Generally, gene activation is accompanied by ‘open’ chromatin with enrichment of active marks and loss of repressive marks on their regulatory regions.42 In fact, we saw increased levels of activating histone marks, H3K27ac, reduced levels of DNA methylation, along with clustering around nucleoli (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E). At the same time, repressive marks, H3K9me2, and DNA methylation were still abundant in the acetylated active heterochromatin. These results suggest that the transcriptional burst is induced from ‘open’ heterochromatin, but the heterochromatin immediately returns to its silent conformation.E). At the same time, repressive marks, H3K9me2, and DNA methylation were still abundant in the acetylated active heterochromatin. These results suggest that the transcriptional burst is induced from ‘open’ heterochromatin, but the heterochromatin immediately returns to its silent conformation.', 'The rapid and profound changes in heterochromatin during Z4 event are rather unusual, even considering the already unusual chromatin features of regular mESCs,43 such as a bivalent structure,44,45 dynamic plasticity,46 and non-CpG methylation.47 These chromatin changes specific to Z4 event suggest that mESCs undergo rapid derepression followed by the immediate rerepression of both constitutive heterochromatin and zHC during the Z4 event (Fig.\xa0<xref ref-type="fig" rid="dsv01307">7</xref>E). This notion is further supported by our finding that both activating chromatin remodelling complexes (HATs, SWI/SNF) and repressing chromatin remodelling complexes (HDAC1, HDAC2, LSD1/KDM1A, NuRD) gather on heterochromatin during Z4 event. Simultaneous modifications of both constitutive heterochromatin and zHC are also intriguing, as it has been shown that different enzymes are involved in the histone methylation of constitutive heterochromatin and facultative heterochromatin (including zHC): Suv39h1/h2 for constitutive heterochromatin and G9a for facultative heterochromatin.E). This notion is further supported by our finding that both activating chromatin remodelling complexes (HATs, SWI/SNF) and repressing chromatin remodelling complexes (HDAC1, HDAC2, LSD1/KDM1A, NuRD) gather on heterochromatin during Z4 event. Simultaneous modifications of both constitutive heterochromatin and zHC are also intriguing, as it has been shown that different enzymes are involved in the histone methylation of constitutive heterochromatin and facultative heterochromatin (including zHC): Suv39h1/h2 for constitutive heterochromatin and G9a for facultative heterochromatin.48 It remains to be clarified whether these enzymes are involved in Z4 events.']}
|
Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells
|
[
"heterochromatin",
"pericentromere",
"embryonic stem cells"
] |
DNA Res
|
1446274800
|
Prediction of complex traits using molecular genetic information is an active area in quantitative genetics research. In the postgenomic era, many types of -omic (e.g., transcriptomic, epigenomic, methylomic, and proteomic) data are becoming increasingly available. Therefore, evaluating the utility of this massive amount of information in prediction of complex traits is of interest. DNA methylation, the covalent change of a DNA molecule without affecting its underlying sequence, is one quantifiable form of epigenetic modification. We used methylation information for predicting plant height (PH) in Arabidopsis thaliana nonparametrically, using reproducing kernel Hilbert spaces (RKHS) regression. Also, we used different criteria for selecting smaller sets of probes, to assess how representative probes could be used in prediction instead of using all probes, which may lessen computational burden and lower experimental costs. Methylation information was used for describing epigenetic similarities between individuals through a kernel matrix, and the performance of predicting PH using this similarity matrix was reasonably good. The predictive correlation reached 0.53 and the same value was attained when only preselected probes were used for prediction. We created a kernel that mimics the genomic relationship matrix in genomic best linear unbiased prediction (G-BLUP) and estimated that, in this particular data set, epigenetic variation accounted for 65% of the phenotypic variance. Our results suggest that methylation information can be useful in whole-genome prediction of complex traits and that it may help to enhance understanding of complex traits when epigenetics is under examination.
|
[
"Arabidopsis",
"DNA Methylation",
"Epigenesis, Genetic",
"Phenotype",
"Proteomics",
"Quantitative Trait Loci"
] |
other
|
PMC4596397
| null |
86
|
[
"{'Citation': 'Arnold P., Schöler A., Pachkov M., Balwierz P. J., Jørgensen H., et al. , 2013. \\u2003Modeling of epigenome dynamics identifies transcription factors that mediate Polycomb targeting. Genome Res. 23(1): 60–73.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3530684'}, {'@IdType': 'pubmed', '#text': '22964890'}]}}",
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] |
DNA Res. 2015 Oct 31; 22(5):307-318
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NO-CC CODE
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CSPP proteins are required for stabilization of NPHP8 but not NPHP4 at the basal body but NPHP8 is not required for ciliogenesis. Immunofluorescence images of serum-starved hTERT-RPE1 cells transfected with siRNAs targeting GFP (control; A, top left), NPHP8 (SMARTpool; A, bottom left), CSPP/CSPP-L (SMARTpool; A, top right) or CSPP-L alone (A, bottom right) stained for the centriole and ciliary marker acetylated tubulin (red), NPHP8 (green), and DNA (blue). Magnifications of indicated areas are presented to the right (A) or below (B) of each merged overlay image. Centrioles are not stained by the acetylated tubulin-specific antibody due to formaldehyde fixation. The concentration of NPHP8 proteins at the basal body is diminished in cilia-defective CSPP or CSPP-L siRNA-targeting transfectants (right). In contrast, ciliogenesis itself can occur independently of NPHP8 protein expression (A, bottom left; and B) and leads to statistically significant longer average cilia length (B). Examples of the NPHP8 concentration-dependent cilia length increase are shown to the left, and a quantification of this defect is shown in the bar diagram (error bars depict SE of the mean cilia length measured in three independent experiments). (C) NPHP8 (red) is not required for the ciliary localization of CSPP-L (green) to the cilia axoneme (acetylated tubulin, blue) because NPHP8 (red)-depleted cells showed similar axonemal localization of CSPP-L (green) as in control transfectants. (D) CSPP-L is required for the recruitment or maintenance of NPHP8 (red) but not NPHP4 (green) at the basal body (acetylated tubulin, blue). Also see Supplemental Figure 6.
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multiple
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multiple panels: images & plots
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[
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"Immuno Assays"
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[
427,
548
] |
[{'image_id': 'zmk0151095190007', 'image_file_name': 'zmk0151095190007.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190007.jpg', 'caption': 'CSPP and CSPP-L interact with NPHP proteins of the basal body/transition zone. (A) Western blots of immunoprecipitates from total cell lysates of hTERT-RPE1 cells. NPHP8 is identified as a CSPP-L interacting protein by immunoblotting for NPHP8-specific antibody (right; NPHP8 antibody as in Arts et al., 2007). Left, control immunoblotting for CSPP-L. (B) The interaction of CSPP-L and NPHP8 proteins also is detected by reciprocal immunoprecipitations from bovine retinal extracts (NPHP8 antibody as in Khanna et al., 2009). (C) Western blot of immunoprecipitates from total cell lysates of HEK293T transfected with indicated expression plasmids. Immunoprecipitation (IP) of Myc-tagged CSPP isoforms with a Myc-tag–specific antibody coimmunoprecipitated FLAG-tagged NPHP8 as detected by immunoblotting (IB) with a FLAG-tag–specific antibody (top). Inversely, immunoprecipitation of FLAG-NPHP8 with a FLAG-tag–specific antibody coimmunoprecipitated Myc-tagged CSPP isoforms as detected by immunoblotting with a Myc-tag–specific antibody (bottom). Neither the Myc nor the FLAG antibody showed cross-reactivity to the other tag. (D) Western blot of immunoprecipitates with a FLAG-tag–specific antibody from total cell lysates of HEK293T transfected with indicated expression plasmids. Immunoblotting with either FLAG- or Myc-tag–specific antibodies reveals that coimmunoprecipitation of HA-CSPP by FLAG-NPHP8 is lost when the common C-terminal domain of CSPP and CSPP-L is deleted (HA-CSPPdel585-876). The FLAG-tag–specific antibody shows no cross-reactivity for the HA-tag. (E) Western blot of immunoprecipitates with a Myc-tag–specific antibody from total cell lysates of HEK293T transfected with indicated expression plasmids. Efficient coprecipitation of FLAG-NPHP4 by Myc-tagged CSPP isoforms is dependent on the coexpression of FLAG-NPHP8, suggesting ternary complex formation.', 'hash': 'f4c488fb9222e0032db73798e6d27b86f8997ef43a2cbec90116d8a89a9f072a'}, {'image_id': 'zmk0151095190008', 'image_file_name': 'zmk0151095190008.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190008.jpg', 'caption': 'CSPP proteins are required for stabilization of NPHP8 but not NPHP4 at the basal body but NPHP8 is not required for ciliogenesis. Immunofluorescence images of serum-starved hTERT-RPE1 cells transfected with siRNAs targeting GFP (control; A, top left), NPHP8 (SMARTpool; A, bottom left), CSPP/CSPP-L (SMARTpool; A, top right) or CSPP-L alone (A, bottom right) stained for the centriole and ciliary marker acetylated tubulin (red), NPHP8 (green), and DNA (blue). Magnifications of indicated areas are presented to the right (A) or below (B) of each merged overlay image. Centrioles are not stained by the acetylated tubulin-specific antibody due to formaldehyde fixation. The concentration of NPHP8 proteins at the basal body is diminished in cilia-defective CSPP or CSPP-L siRNA-targeting transfectants (right). In contrast, ciliogenesis itself can occur independently of NPHP8 protein expression (A, bottom left; and B) and leads to statistically significant longer average cilia length (B). Examples of the NPHP8 concentration-dependent cilia length increase are shown to the left, and a quantification of this defect is shown in the bar diagram (error bars depict SE of the mean cilia length measured in three independent experiments). (C) NPHP8 (red) is not required for the ciliary localization of CSPP-L (green) to the cilia axoneme (acetylated tubulin, blue) because NPHP8 (red)-depleted cells showed similar axonemal localization of CSPP-L (green) as in control transfectants. (D) CSPP-L is required for the recruitment or maintenance of NPHP8 (red) but not NPHP4 (green) at the basal body (acetylated tubulin, blue). Also see Supplemental Figure 6.', 'hash': '0cc58fadff76d322e92b7b66df3ebeeb24b26a6eb801497e0c56664e03167473'}, {'image_id': 'zmk0151095190006', 'image_file_name': 'zmk0151095190006.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190006.jpg', 'caption': 'CSPP and CSPP-L colocalize with NPHP proteins at the transition zone and at the basal body. Immunofluorescence images of hTERT-RPE1 cells 72 h after serum starvation. (A) Cells were stained for NPHP8 (green) and CSPP/CSPP-L (red). Boxes indicate magnified areas displayed in bottom panels. (B) The centrosome/basal body of a ciliated hTERT-RPE1 cell stained for the marker of the transition zone, NPHP1 (green), and CSPP/CSPP-L. CSPP/CSPP-L is detected at centrioles and the lower cilia axoneme showing colocalization with NPHP1 in individual confocal planes (bottom). (C) Immunofluorescence images of serum-starved hTERT-RPE1 cells stained for DNA (blue), CSPP-L (green), and NPHP4 (red) showing colocalization of both proteins at the basal body.', 'hash': '320429126b90d1240c73c756742a58476373d39c06716f557d2db6cf90bef571'}, {'image_id': 'zmk0151095190001', 'image_file_name': 'zmk0151095190001.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190001.jpg', 'caption': 'Generation of a CSPP/CSPP-L–specific antibody. (A) Schematic representation of CSPP and CSPP-L proteins and bacterially expressed constructs used for antibody generation. Bold bars indicate coiled-coil regions. (B) Western blot using the generated monoclonal CSPP/CSPP-L–specific antibody for detection of CSPP isoforms in total cell lysates of HEK293T cells transfected with indicated plasmids. (C) Immunofluorescence detection of CSPP-Legfp (green) in transiently transfected HeLa cells using the generated monoclonal CSPP/CSPP-L–specific antibody (red). (D) Immunofluorescence staining against CSPP/CSPP-L (red) and γ-tubulin (green) of HeLa cells 72 h posttransfection with either GFP or CSPP/CSPP-L mRNA targeting siRNA.', 'hash': 'fccc79d0b92325a9daef4ad83094f6c9a4839d32dc37ca399b19ee833f33849c'}, {'image_id': 'zmk0151095190002', 'image_file_name': 'zmk0151095190002.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190002.jpg', 'caption': 'CSPP proteins localize to centrioles, cilia, and the midbody. (A) Immunofluorescence images of hTERT-RPE1 cells stably expressing the GFP tagged centriolar protein centrin 2 (green) stained for CSPP/CSPP-L (red). Panels below show magnified montages of centrosomes indicated in the merged image. CSPP proteins colocalize with CENT2gfp at centrioles but show additional staining at the tip of one centriole. (B) Immunofluorescence images of hTERT-RPE1 cells 72 h after serum starvation stained for the ciliary marker IFT88 (green) and glutamylated tubulin (red; left) or CSPP/CSPP-L (red; right). Bottom panels show magnified images of indicated cilia in the merged image. CSPP proteins are detected at centrioles and along the lower cilia axoneme. (C) Immunofluorescence images of hTERT-RPE1 cells in G2 phase (left cell) and telophase/cytokinesis (right cell) stained for CSPP/CSPP-L (red) and the interflagellar transport protein IFT88 (green). Phase-contrast imaging reveals localization of CSPP/CSPP-L to the phase-dense midbody. Panels to the right show magnified images of indicated centrosomes.', 'hash': 'ea4d5748340e38d6029f857401b2ca3d1611031c325e28ae48e9189ac0310886'}, {'image_id': 'zmk0151095190005', 'image_file_name': 'zmk0151095190005.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190005.jpg', 'caption': 'Knockdown of CSPP proteins impairs serum starvation induced ciliogenesis in hTERT-RPE1 cells. Immunofluorescence images of siRNA-transfected hTERT-RPE1 cells stained for the centrosomal marker γ-tubulin (green) and the cilia marker acetylated tubulin (red). CSPP/CSPP-L mRNA targeting siRNA transfectants but not control transfectants show defects in ciliogenesis. Images of larger field (A) and close-up view on representative primary cilia (B) show severely shortened cilia axonemes in CSPP/CSPP-L siRNA transfectants. (C) Quantification of the efficacy of ciliogenesis in response to serum starvation after siRNA transfection with indicated siRNAs. Depletion of pericentrin and CSPP/CSPP-L efficiently inhibit cilia formation compared with control cells or GFP siRNA transfected cells. Individual siRNA molecules targeting both CSPP isoforms or CSPP-L alone result in similar ciliogenesis defects. At least 300 cells were scored in three independent transfections of each siRNA. Error bars indicate SD. Also see Supplemental Figure 4.', 'hash': '77690a0fa9ffa60e7355cfe824c53e2451a07755fb054304b08ce1c3e023845d'}, {'image_id': 'zmk0151095190004', 'image_file_name': 'zmk0151095190004.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190004.jpg', 'caption': 'CSPP proteins are expressed in ciliated renal, retinal, and respiratory epithelia cells. Sections of formalin fixed tissues were stained by immunohistochemistry with either the mouse monoclonal CSPP/CSPP-L–specific antibody (A) or the rabbit polyclonal CSPP-L–specific antibody (B–D) (brown). Nuclei were detected by counterstaining with hematoxylin (blue). (A) Staining of human renal tissue section with the CSPP/CSPP-L–specific mAb by immunohistochemistry shows expression of CSPP isoforms (brown) in tubular epithelia cells, whereas cells of glomeruli and the mesenchymal cells showed no staining. (B) Detection of CSPP-L in renal tissue sections from male BALB/C nu/nu mice shows identical staining pattern to that observed in human kidney sections. (C) CSPP-L shows a distinct expression pattern in murine retina sections. Photoreceptor cells of the outer nuclear layer show subcellular accumulation of CSPP-L mainly at the connecting cilium comprising transition zone between inner and outer segment and partially at regions of synapses to the bipolar cells of the inner nuclear layer. The outer segment is negative for CSPP-L. Right, magnification of indicated area and schematic view of cellular organization. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment; RPE, retina pigment epithelium; CB, choroidal border. (D) High CSPP-L expression is detected in bronchiole lining ciliated respiratory epithelia cells. Also see Supplemental Figure 4.', 'hash': '818a7bc742dbd37d7d66e043dcb3fc1878a21810c4cbd2a87861c9548de812e9'}, {'image_id': 'zmk0151095190003', 'image_file_name': 'zmk0151095190003.jpg', 'image_path': '../data/media_files/PMC2912343/zmk0151095190003.jpg', 'caption': 'Identification of CSPP-L as a ciliary protein. (A) Schematic presentation of CSPP and CSPP-L isoforms with labeling of regions used for generation of the monoclonal CSPP/CSPP-L–specific and the polyclonal CSPP-L–specific antibodies. The affinity-purified CSPP-L–specific antibody detects Myc-tagged CSPP-L and an endogenous protein of equivalent molecular weight but not Myc-tagged CSPP in Western blots of total cell lysates of transiently transfected HEK293T cells. Immunoblotting of the same lysates with the monoclonal CSPP/CSPP-L–specific antibody is shown as control. (B) The polyclonal CSPP-L–specific antibody detects and immunoprecipitates an endogenous protein of 150 kDa in total cell lysates of hTERT-RPE1 cells that is also detected by the monoclonal CSPP/CSPP-L–specific antibody. (C) Immunofluorescence images of serum starved hTERT-RPE1 cells stained for CSPP-L (green) and acetylated tubulin (red) showing localization of CSPP-L to the basal body and the cilia axoneme. Panels to the right show magnified images of indicated cilia. (D) Immunofluorescence images of serum-starved hTERT-RPE1 cells stained with the polyclonal CSPP-L–specific (green) and the CSPP/CSPP-L–specific mAb (red). Colocalization is observed at the basal body and in patches at the lower cilia axoneme (arrow). (E) Costaining for the ciliary marker acetylated tubulin in serum starved hTERT-RPE1 cells transiently expressing either EGFP, CSPP-egfp, or CSPP-L-egfp (green) show both EGFP-fusion proteins specifically enriched at basal bodies and along cilia axonemes, including the ciliary tip structure (see Supplemental Figure 3). CSPP-L-egfp transfectants show statistically significant longer cilia axonemes than CSPP-egfp and EGFP transfectants (error bars depict SE of the mean cilia length measured in three independent experiments).', 'hash': '2da58bf2b81235f99790e5d3d52b3baf24003447621fa8e148ca9dea064e00bb'}]
|
{'zmk0151095190001': ['A mAb directed against the common C terminus of CSPP and CSPP-L was generated by immunization of mice with a bacterially expressed GST-fusion protein that made up the C-terminal 379 aa of CSPP and CSPP-L. Individual clones were screened for reactivity against a bacterially expressed His-tagged fusion protein making up the common C-terminal 291 aa and further tested for reactivity against both CSPP isoforms ectopically expressed in HEK293T cells (<xref ref-type="fig" rid="zmk0151095190001">Figure 1</xref>A). A clone, k22-11, was identified whose antibodies specifically detected both ectopically expressed CSPP isoforms by means of Western blot analysis and immunofluorescence (A). A clone, k22-11, was identified whose antibodies specifically detected both ectopically expressed CSPP isoforms by means of Western blot analysis and immunofluorescence (<xref ref-type="fig" rid="zmk0151095190001">Figure 1</xref>, B and C). The sensitivity of this antibody was too low to detect endogenous CSPP isoforms by Western blot of total cell lysates (, B and C). The sensitivity of this antibody was too low to detect endogenous CSPP isoforms by Western blot of total cell lysates (<xref ref-type="fig" rid="zmk0151095190001">Figure 1</xref>B) but sufficient to detect a centrosomal antigen by immunofluorescence analysis of HeLa cells (B) but sufficient to detect a centrosomal antigen by immunofluorescence analysis of HeLa cells (<xref ref-type="fig" rid="zmk0151095190001">Figure 1</xref>C). The immunofluorescence staining consistently showed a two barrel like staining pattern embedded within γ-tubulin containing foci. Importantly, the mAb staining was lost in cells depleted for CSPP isoforms by transfection with an individual siRNA against a sequence common to CSPP and CSPP-L mRNAs as well as siRNA pool targeting both isoforms (C). The immunofluorescence staining consistently showed a two barrel like staining pattern embedded within γ-tubulin containing foci. Importantly, the mAb staining was lost in cells depleted for CSPP isoforms by transfection with an individual siRNA against a sequence common to CSPP and CSPP-L mRNAs as well as siRNA pool targeting both isoforms (<xref ref-type="fig" rid="zmk0151095190001">Figure 1</xref>D and Supplemental Figure 1).D and Supplemental Figure 1).'], 'zmk0151095190002': ['The observed centrosomal staining pattern suggested that CSPP isoforms localized to the centrioles. We therefore analyzed the localization of CSPP isoforms in hTERT-RPE1 cells that stably express GFP-centrin 2 (CETN2) fusion protein, a structural component of the centrioles. CSPP isoforms colocalized with the centriolar marker. However, frequently extracentriolar staining, extending from one of the centrioles reminiscent of cilia was observed (<xref ref-type="fig" rid="zmk0151095190002">Figure 2</xref>A). We therefore investigated the localization of CSPP isoforms in hTERT-RPE1 cells that had been serum starved for 48 h to induce primary cilia formation. CSPP isoforms not only localized to centrioles but also extended into the lower cilia axoneme as shown by costaining for the intraflagellar transport protein 88 (IFT88), which decorates the cilia axoneme and the transition zone. In contrast to IFT88, CSPP proteins were only detected at the lower part of the cilia axoneme (A). We therefore investigated the localization of CSPP isoforms in hTERT-RPE1 cells that had been serum starved for 48 h to induce primary cilia formation. CSPP isoforms not only localized to centrioles but also extended into the lower cilia axoneme as shown by costaining for the intraflagellar transport protein 88 (IFT88), which decorates the cilia axoneme and the transition zone. In contrast to IFT88, CSPP proteins were only detected at the lower part of the cilia axoneme (<xref ref-type="fig" rid="zmk0151095190002">Figure 2</xref>B).B).', 'In cycling cells, IFT88 localizes to the distal appendages of the mother centriole (<xref ref-type="fig" rid="zmk0151095190002">Figure 2</xref>C). In C). In <xref ref-type="fig" rid="zmk0151095190002">Figure 2</xref>C, one interphase and one mitotic (telophase/cytokinesis) cell are depicted. The interphase cell displays duplicated and separated centriole pairs, which both are stained by the CSPP antibody although only one centriole stains at the distal end for IFT88. During telophase, CSPP isoforms are still localized to centrioles but also stained midbodies that were identified by phase-contrast imaging. The expression and localization of CSPP-L to centrosomes and midbodies also was observed in unciliated cells such as human B lymphocytes (Supplemental Figure 2).C, one interphase and one mitotic (telophase/cytokinesis) cell are depicted. The interphase cell displays duplicated and separated centriole pairs, which both are stained by the CSPP antibody although only one centriole stains at the distal end for IFT88. During telophase, CSPP isoforms are still localized to centrioles but also stained midbodies that were identified by phase-contrast imaging. The expression and localization of CSPP-L to centrosomes and midbodies also was observed in unciliated cells such as human B lymphocytes (Supplemental Figure 2).'], 'zmk0151095190003': ['During the course of our study, a polyclonal antibody against the CSPP-L specific N terminus became available. This polyclonal antibody specifically detects Myc-tagged CSPP-L but not CSPP in immuno blots of total cell lysates of HEK293T transfectants, and it detects an endogenous protein of the size of CSPP-L in total cell lysates of untransfected HEK293T cells (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>A). Furthermore, this polyclonal antibody immunoprecipitated an endogenous protein of approximately 150 kDa from total cell lysates of hTERT-RPE1 cells. The immunoprecipitated protein was stained by the mAb that is directed against the common C-terminal end of CSPP and CSPP-L (A). Furthermore, this polyclonal antibody immunoprecipitated an endogenous protein of approximately 150 kDa from total cell lysates of hTERT-RPE1 cells. The immunoprecipitated protein was stained by the mAb that is directed against the common C-terminal end of CSPP and CSPP-L (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>B). We conclude that the polyclonal antibody is specific to CSPP-L. Immunofluorescence staining of serum-starved hTERT-RPE1 cells for CSPP-L and acetylated tubulin, a tubulin modification found on centrioles and the cilia axoneme, showed that CSPP-L localized to the centrosome and the cilia axoneme (B). We conclude that the polyclonal antibody is specific to CSPP-L. Immunofluorescence staining of serum-starved hTERT-RPE1 cells for CSPP-L and acetylated tubulin, a tubulin modification found on centrioles and the cilia axoneme, showed that CSPP-L localized to the centrosome and the cilia axoneme (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>C). Notably, the cilia staining seemed not to be limited to the lower axoneme as observed with the mAb. CSPP-L decorated patches along the whole axoneme. Furthermore, costaining with the mAb against the common C-terminal domain of both isoforms and the CSPP-L specific antibody not only showed colocalization at centrioles and the lower axoneme but also additional staining of CSPP-L around the centrosome and along the upper cilia axoneme (C). Notably, the cilia staining seemed not to be limited to the lower axoneme as observed with the mAb. CSPP-L decorated patches along the whole axoneme. Furthermore, costaining with the mAb against the common C-terminal domain of both isoforms and the CSPP-L specific antibody not only showed colocalization at centrioles and the lower axoneme but also additional staining of CSPP-L around the centrosome and along the upper cilia axoneme (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>D).D).', 'We next examined the localization of ectopically expressed enhanced green fluorescent protein (egfp)-tagged CSPP isoforms in transiently transfected, serum-starved hTERT-RPE1 cells. Both isoforms specifically concentrated around the basal body and within the cilia axoneme (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>E and Supplemental Figure 3). Notably, the axonemes of in particular CSPP-Legfp transfectants seemed elongated in live transfectants. This increase in cilia length was confirmed in fixed cells by costaining for the ciliary marker acetylated tubulin. The average length of the cilia axoneme in CSPP-Legfp transfectants (4.9 μm; SEM = 0.2 μm) was found to be significantly longer than in CSPP-egfp (3.4 μm; SEM = 0.2 μm) and EGFP (3.1 μm; SEM = 0.1 μm) control transfectants (E and Supplemental Figure 3). Notably, the axonemes of in particular CSPP-Legfp transfectants seemed elongated in live transfectants. This increase in cilia length was confirmed in fixed cells by costaining for the ciliary marker acetylated tubulin. The average length of the cilia axoneme in CSPP-Legfp transfectants (4.9 μm; SEM = 0.2 μm) was found to be significantly longer than in CSPP-egfp (3.4 μm; SEM = 0.2 μm) and EGFP (3.1 μm; SEM = 0.1 μm) control transfectants (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>E). Expression of truncated CSPP/CSPP-L constructs identified the common central coiled-coil (amino acids 295–708 in CSPP-L, 1–503 in CSPP) as being required for ciliary localization, whereas the common C-terminal domain of both isoforms only enriched around centrosomes. The CSPP-L specific N-terminal domain (amino acids 1-295 in CSPP-L) neither showed ciliary localization but decorated stress fibers in live cells and colocalized with actin fibers in fixed cells (Supplemental Figure 3). Collectively, these data identify CSPP proteins as novel ciliary proteins and identify a CSPP-L–specific actin or stress fiber targeting protein domain.E). Expression of truncated CSPP/CSPP-L constructs identified the common central coiled-coil (amino acids 295–708 in CSPP-L, 1–503 in CSPP) as being required for ciliary localization, whereas the common C-terminal domain of both isoforms only enriched around centrosomes. The CSPP-L specific N-terminal domain (amino acids 1-295 in CSPP-L) neither showed ciliary localization but decorated stress fibers in live cells and colocalized with actin fibers in fixed cells (Supplemental Figure 3). Collectively, these data identify CSPP proteins as novel ciliary proteins and identify a CSPP-L–specific actin or stress fiber targeting protein domain.', 'The immunofluorescence staining suggests that CSPP proteins can occur in different protein pools localized to 1) centrosomes, 2) midspindel/midbodies, and 3) the cilia axoneme. Both antibodies used in this study detected endogenous CSPP isoforms at the centrosome, the midbody and along the cilia axoneme. However, not all CSPP proteins stained by the affinity-purified polyclonal CSPP-L–specific antibody also were stained by the mAb that is directed against the common C-terminal domain of both CSPP isoforms (<xref ref-type="fig" rid="zmk0151095190003">Figure 3</xref>). This discrepancy may be explained by either the lower affinity of the mAb compared with the CSPP-L–specific polyclonal antibody or epitope masking of the C-terminal domain by the formation of protein complexes such as with NPHP8. Alternatively, because we have shown previously that CSPP proteins can be phosphorylated at serine residues (). This discrepancy may be explained by either the lower affinity of the mAb compared with the CSPP-L–specific polyclonal antibody or epitope masking of the C-terminal domain by the formation of protein complexes such as with NPHP8. Alternatively, because we have shown previously that CSPP proteins can be phosphorylated at serine residues (Patzke et al., 2005), posttranslational modification could mask the epitope detected by the mAb.'], 'zmk0151095190004': ['Supportive for a cilary function, we observed that CSPP1 mRNA is predominantly expressed in ciliated tissues during mouse embryogenesis (Supplemental Figure 4). We therefore examined the expression of CSPP proteins in three independent biopsies of normal human kidney using the monoclonal CSPP antibody. CSPP protein expression was specifically detected in tubular epithelia cells of the nephron, whereas mesenchymal cells and cells of the blood filtrating glomeruli did not express CSPP proteins (<xref ref-type="fig" rid="zmk0151095190004">Figure 4</xref>A). An identical staining pattern was obtained using the polyclonal CSPP-L–specific antibody (data not shown). This antibody also showed cross-reactivity to murine CSPP-L and resulted in a similar staining pattern in kidney sections of BALBc nu/nu mice (A). An identical staining pattern was obtained using the polyclonal CSPP-L–specific antibody (data not shown). This antibody also showed cross-reactivity to murine CSPP-L and resulted in a similar staining pattern in kidney sections of BALBc nu/nu mice (<xref ref-type="fig" rid="zmk0151095190004">Figure 4</xref>B).B).', 'Next, we analyzed CSPP-L expression in the murine retina (<xref ref-type="fig" rid="zmk0151095190004">Figure 4</xref>B). CSPP-L expression could be detected in cells of the pigmented epithelium, the sensory photoreceptor cells of the outer nuclear layer, and the signal transducing cells of the inner nuclear layer, whereas the mesenchyme of the choroid (underlying the pigmented epithelium) showed no CSPP-L expression. Interestingly, the strongest CSPP-L expression was detected in cells of the outer nuclear layer and showed a characteristic subcellular localization to the connecting cilium that composes the transition zone between the inner segment and the outer segment (B). CSPP-L expression could be detected in cells of the pigmented epithelium, the sensory photoreceptor cells of the outer nuclear layer, and the signal transducing cells of the inner nuclear layer, whereas the mesenchyme of the choroid (underlying the pigmented epithelium) showed no CSPP-L expression. Interestingly, the strongest CSPP-L expression was detected in cells of the outer nuclear layer and showed a characteristic subcellular localization to the connecting cilium that composes the transition zone between the inner segment and the outer segment (<xref ref-type="fig" rid="zmk0151095190004">Figure 4</xref>C). A second zone of concentrated CSPP-L expression is detected in the apical part of the outer plexiform layer adjacent to the outer nuclear layer where synapses between bipolar cells of the inner nuclear layer and the photoreceptor cells of the outer nuclear layer are formed. Notably, both neuronal cell types, the bipolar cells and the ganglia cells, also showed CSPP-L expression. This localization is very similar to what is reported for NPHP8 and NPHP4 (C). A second zone of concentrated CSPP-L expression is detected in the apical part of the outer plexiform layer adjacent to the outer nuclear layer where synapses between bipolar cells of the inner nuclear layer and the photoreceptor cells of the outer nuclear layer are formed. Notably, both neuronal cell types, the bipolar cells and the ganglia cells, also showed CSPP-L expression. This localization is very similar to what is reported for NPHP8 and NPHP4 (Arts et al., 2007). NPHP8 and NPHP4 belong to a group of eleven genes (NPHP1–11) identified by positional cloning to harbor causative mutations in patients suffering from NPHP. Individual NPHP proteins have been functionally associated with centrosomes, cilia, and cell–cell junctions. It is thus thought that NPHP is a result of the deregulation of ciliary/centrosome–associated signaling pathways in tubular epithelia cells, leading to cyst formation. The loss of cilia associated functions of NPHP4 and NPHP8 proteins is frequently associated with an extra-renal phenotype, primarily retinal degeneration and neurological disorders.', 'Finally, gene expression in the mouse embryo (Supplemental Figure 4) and transcriptional profiling of mucociliary differentiation in human airway epithelial cells indicated CSPP expression in multiciliary epithelium (Ross et al., 2007). Immunohistochemistry staining of tissue sections of murine respiratory bronchioles identified the highly polarized, multiciliated bronchial epithelia cells to express CSPP-L (<xref ref-type="fig" rid="zmk0151095190004">Figure 4</xref>D). This result was further confirmed by immunofluorescence staining of isolated murine trachea epithelia cells (Supplemental Figure 4).D). This result was further confirmed by immunofluorescence staining of isolated murine trachea epithelia cells (Supplemental Figure 4).'], 'zmk0151095190005': ['To explore the putative ciliary function of CSPP proteins, we tested their requirement for cilia formation in hTERT-RPE1 cells in response to serum starvation. hTERT-RPE1 cells were consecutively transfected with siRNA targeting both CSPP isoforms or targeting the CSPP-L isoform alone (<xref ref-type="fig" rid="zmk0151095190005">Figure 5</xref> and Supplemental Figure 5). To achieve an efficient knockdown, cells were allowed to grow in serum containing medium for 48h after a first siRNA transfection before being subjected to a second transfection that was followed by serum starvation for 48 h to induce ciliogenesis. As controls, cells were transfected with siRNA targeting GFP or the centrosomal protein PCNT. PCNT has been shown previously to be required for ciliogenesis in hTERT-RPE1 cells ( and Supplemental Figure 5). To achieve an efficient knockdown, cells were allowed to grow in serum containing medium for 48h after a first siRNA transfection before being subjected to a second transfection that was followed by serum starvation for 48 h to induce ciliogenesis. As controls, cells were transfected with siRNA targeting GFP or the centrosomal protein PCNT. PCNT has been shown previously to be required for ciliogenesis in hTERT-RPE1 cells (Mikule et al., 2007). The knockdown of CSPP isoforms inhibited ciliogenesis to a similar degree as the knockdown of pericentrin, whereas ciliogenesis was unimpaired in GFP siRNA transfectants (<xref ref-type="fig" rid="zmk0151095190005">Figure 5</xref>, A and B). Fifty percent (SD = 5%) of the cells transfected with SMARTpool siRNAs targeting , A and B). Fifty percent (SD = 5%) of the cells transfected with SMARTpool siRNAs targeting CSPP1 transcripts displayed either no cilia or cilia with severely shortened axonemes (<xref ref-type="fig" rid="zmk0151095190005">Figure 5</xref>C). A similar loss of ciliogenesis was observed in hTERT-RPE1 cells transfected with the CSPP and CSPP-L targeting single siRNA used in previous studies (C). A similar loss of ciliogenesis was observed in hTERT-RPE1 cells transfected with the CSPP and CSPP-L targeting single siRNA used in previous studies (Patzke et al., 2005), thus excluding potential unspecificity of the siRNA mix. Interestingly, a single siRNA specific to the mRNA of the larger CSPP-L isoform also impaired ciliogenesis (<xref ref-type="fig" rid="zmk0151095190005">Figure 5</xref>C and Supplemental Figure 5A), indicating that this isoform alone is a major contributor to ciliogenesis in hTERT-RPE1 cells.C and Supplemental Figure 5A), indicating that this isoform alone is a major contributor to ciliogenesis in hTERT-RPE1 cells.', 'Our results show that CSPP proteins not only are expressed in NPHP protein-expressing cell types in renal, retinal, and respiratory tissue sections but also that they interact directly with NPHP8 and indirectly with NPHP4. Furthermore, the interaction of endogenous CSPP proteins with NPHP8 is not only detected in cell lines but also confirmed in bovine retina extracts. Our results suggest that CSPP proteins are required for correct localization of NPHP8 to the tip of the basal body that is forming the transition zone. In hTERT-RPE1 cells the targeted knockdown of CSPP-L alone is sufficient to impair ciliogenesis and to decrease the recruitment of NPHP8 to centrosomes, indicating its sole significance in this process in this cell line (<xref ref-type="fig" rid="zmk0151095190005">Figures 5</xref>C and C and <xref ref-type="fig" rid="zmk0151095190008">8</xref>A). Our finding that ciliogenesis is not impaired by NPHP8 knockdown indicates that loss of NPHP8 from the transition zone alone is not sufficient to explain the CSPP-L siRNA-mediated ciliogenesis defect. This finding is consistent with the observation of primary cilia in primary mouse embryonic fibroblasts derived from the NPHP8 knockout mouse (A). Our finding that ciliogenesis is not impaired by NPHP8 knockdown indicates that loss of NPHP8 from the transition zone alone is not sufficient to explain the CSPP-L siRNA-mediated ciliogenesis defect. This finding is consistent with the observation of primary cilia in primary mouse embryonic fibroblasts derived from the NPHP8 knockout mouse (Vierkotten et al., 2007). The observed increase in cilia length might reflect a homeostatic response in which hTERT-RPE1 cells try to compensate for lack of signaling through the cilium as NPHP8 has been shown to be required for Shh signaling. Alternatively, it could be the consequence of imbalanced sorting of cilia length determining proteins (e.g., Rab8; Nachury et al., 2007) over the transition zone, because the NPHP8 interactor NPHP4 and its interactor NPHP1 also are suggested to regulate transport through the transition zone (Winkelbauer et al., 2005; Jauregui et al., 2008). However, NPHP8 seems to be expendable for the localization of NPHP4 and CSPP-L to the primary cilium. Thus, NPHP8 might act antagonistically on CSPP-L, which we found not only to be required for ciliogenesis but also to positively regulate cilia length upon ectopic expression. Interestingly, NPHP8 has been found to interact with RPGR (Khanna et al., 2009), which in turn is associated with NPHP6 (CEP290; Chang et al., 2006) that has been shown to regulate ciliogenesis via recruitment of Rab8 (Kim et al., 2008; Tsang et al., 2008). Although our study did not unravel the mechanism of CSPP/CSPP-L dependence in ciliogenesis, we identified a second arm of the NPHP network feeding into the control of ciliogenesis or eventually cilia homeostasis.'], 'zmk0151095190006': ['The cell type-specific expression, the requirement for ciliogenesis and the intriguing localization to the lower cilia axoneme throughout the transition zone suggested a possible involvement of CSPP isoforms in human ciliopathies. We therefore tested known ciliopathy related proteins that localize to the basal body and the transition zone for colocalization and interaction with CSPP or CSPP-L. These include NPHP1 (Fliegauf et al., 2006), NPHP4 (interacts with NPHP1; Mollet et al., 2002. 2005; Otto et al., 2002), and the retinitis pigmentosa GTPase regulator-interacting protein 1-like protein NPHP8 (interacts with NPHP4; Delous et al., 2007; Arts et al., 2007). NPHP8 and NPHP1 localized specifically to the transition zone that is formed at the tip of the mother centriole/basal body, colocalizing with CSPP proteins (<xref ref-type="fig" rid="zmk0151095190006">Figure 6</xref>, A and B). NPHP4 showed a less focused localization being detected around both centrioles and to minor degree at the transition zone as shown by costaining with the CSPP-L–specific antibody (, A and B). NPHP4 showed a less focused localization being detected around both centrioles and to minor degree at the transition zone as shown by costaining with the CSPP-L–specific antibody (<xref ref-type="fig" rid="zmk0151095190006">Figure 6</xref>C).C).', 'The interaction of NPHP8 with CSPP proteins still allowed ternary complex formation with NPHP4, which in turn is a known NPHP1 interacting protein (Mollet et al., 2005). The partial colocalization of CSPP-L and NPHP4 as well as NPHP1 at the basal body/transition zone (<xref ref-type="fig" rid="zmk0151095190006">Figure 6</xref>E) may be supportive for the existence of CSPP-L–NPHP8–NPHP4 (–NPHP1) complex formation in cells. Furthermore, CSPP proteins are enriched at the connecting cilium and at synapses of rod and cone cells in the outer nuclear layer (E) may be supportive for the existence of CSPP-L–NPHP8–NPHP4 (–NPHP1) complex formation in cells. Furthermore, CSPP proteins are enriched at the connecting cilium and at synapses of rod and cone cells in the outer nuclear layer (<xref ref-type="fig" rid="zmk0151095190004">Figure 4</xref>), thus closely resembling reported staining pattern of NPHP8 and NPHP4 (see supplemental figure 5 in ), thus closely resembling reported staining pattern of NPHP8 and NPHP4 (see supplemental figure 5 in Arts et al., 2007). Also, NPHP1 is a known constituent of the retinal connecting cilium as well as of the transition zone of the renal and respiratory cilia (Fliegauf et al., 2006; Jiang et al., 2009). Notably, in addition to their ciliary localization, these proteins also have been shown to localize to cell–cell and cell–matrix contacts where they complex with proteins involved in cytoskeleton organization to facilitate epithelial morphogenesis (Donaldson et al., 2000; Mollet et al., 2005; Delous et al., 2009); and importantly, NPHP associated mutations in NPHP8 weaken or abrogate their interaction with NPHP4 (Arts et al., 2007; Delous et al., 2007). Although little is known about the dynamic behavior of these proteins, in a unifying concept NPHP8 might modulate cilia growth and polarization on two levels: indirectly through modulation of transport across the transition zone thereby affecting planar-cell-polarity signaling pathways and directly through interactions with cytoskeleton affecting proteins such as NPHP4, NPHP6, and Rab8 via RPGR, and CSPP proteins identified here (Supplemental Figure 7). Our results may suggest that CSPP-L and NPHP8 can form a functional unit of which (in hTERT-RPE1 cells) CSPP-L is required for cilia formation and NPHP8 recruitment or maintenance. Once the cilium is formed, NPHP8 might antagonistically act on CSPP-L to regulate cilia length. This might involve further interactions with other proteins such as NPHP4. However, additional work is required to investigate whether, when, and where these proteins can occur as multiprotein complexes together with CSPP in vivo. Furthermore the identification of putative binding partners of CSPP-L within the ciliary axoneme and its tip region will be a prerequisite to understand its effect on axoneme length control.'], 'zmk0151095190007': ['We screened for possible protein–protein interactions in hTERT-RPE1 cells and identified NPHP8 as a CSPP-L–interacting protein by means of coimmunoprecipitation with the CSPP-L specific antibody (<xref ref-type="fig" rid="zmk0151095190007">Figure 7</xref>A). This interaction was further confirmed in bovine retina extracts by reciprocal coimmunoprecipitation experiments by using NPHP8-specific and CSPP-L–specific antibodies, respectively (A). This interaction was further confirmed in bovine retina extracts by reciprocal coimmunoprecipitation experiments by using NPHP8-specific and CSPP-L–specific antibodies, respectively (<xref ref-type="fig" rid="zmk0151095190007">Figure 7</xref>B). Notably, bovine CSPP-L migrated at a slightly higher molecular weight, which may indicate posttranslational modification or the existence of further splice variants. Next, differentially tagged proteins were transiently overexpressed in HEK293T cells and tested for interaction by coimmunoprecipitation from total cell lysates to further characterize the nature of the interaction of NPHP8 and CSPP proteins. FLAG-NPHP8 was confirmed as a CSPP-myc– and CSPP-L-myc–interacting protein. Immunoprecipitation of either myc-tagged CSPP isoform coimmunoprecipitated a FLAG-tagged construct of the C-terminal part of NPHP8 (amino acid residues 411-1055; B). Notably, bovine CSPP-L migrated at a slightly higher molecular weight, which may indicate posttranslational modification or the existence of further splice variants. Next, differentially tagged proteins were transiently overexpressed in HEK293T cells and tested for interaction by coimmunoprecipitation from total cell lysates to further characterize the nature of the interaction of NPHP8 and CSPP proteins. FLAG-NPHP8 was confirmed as a CSPP-myc– and CSPP-L-myc–interacting protein. Immunoprecipitation of either myc-tagged CSPP isoform coimmunoprecipitated a FLAG-tagged construct of the C-terminal part of NPHP8 (amino acid residues 411-1055; Arts et al., 2007) and vice versa (<xref ref-type="fig" rid="zmk0151095190007">Figure 7</xref>C). Furthermore, this result indicated that CSPP and CSPP-L share a NPHP8 binding domain. To identify this domain, the FLAG-NPHP8 construct was transiently coexpressed with either full-length or truncated HA-tagged variants of CSPP lacking either the N- or the C-terminal domain (C). Furthermore, this result indicated that CSPP and CSPP-L share a NPHP8 binding domain. To identify this domain, the FLAG-NPHP8 construct was transiently coexpressed with either full-length or truncated HA-tagged variants of CSPP lacking either the N- or the C-terminal domain (Patzke et al., 2005) and subjected to immunoprecipitation using a FLAG-tag–specific antibody. Immunoprecipitated FLAG-NPHP8 copurified both full-length HA-CSPP and the construct lacking the N-terminal domain, whereas the interaction was lost upon deletion of the C-terminal domain. This result supports the specificity of the interaction, because both HA-tagged and Myc-tagged CSPP can be coprecipitated, and identifies the common C-terminal 292 aa of CSPP and CSPP-L to be required for the interaction with NPHP8 (<xref ref-type="fig" rid="zmk0151095190007">Figure 7</xref>D).D).', 'NPHP8 is a NPHP4-interacting protein (Delous et al., 2007; Arts et al., 2007). It is therefore possible that CSPP could complex with NPHP4 directly or could form a ternary complex with NPHP8. To test this hypothesis, individual Myc-tagged CSPP isoforms were transiently expressed in HEK293T cells together with either FLAG-tagged NPHP4 (FLAG-NPHP4) alone or with a combination of FLAG-NPHP4 and FLAG-NPHP8 (<xref ref-type="fig" rid="zmk0151095190007">Figure 7</xref>E). CSPP isoforms were immunoprecipitated from total lysates with a Myc-tag–specific antibody and tested for coprecipitation of FLAG-tagged NPHP4 or NPHP8. CSPP and CSPP-L poorly copurified FLAG-NPHP4 in the absence of FLAG-NPHP8, whereas coexpression of FLAG-NPHP8 increased significantly the efficacy of FLAG-NPHP4 copurification. This result indicated that CSPP and CSPP-L can form a ternary complex with NPHP4 via its binding to NPHP8.E). CSPP isoforms were immunoprecipitated from total lysates with a Myc-tag–specific antibody and tested for coprecipitation of FLAG-tagged NPHP4 or NPHP8. CSPP and CSPP-L poorly copurified FLAG-NPHP4 in the absence of FLAG-NPHP8, whereas coexpression of FLAG-NPHP8 increased significantly the efficacy of FLAG-NPHP4 copurification. This result indicated that CSPP and CSPP-L can form a ternary complex with NPHP4 via its binding to NPHP8.', 'Finally, recombinant CSPP proteins were able to form a ternary complex with NPHP4 in the presence of NPHP8 (<xref ref-type="fig" rid="zmk0151095190007">Figure 7</xref>). We therefore also investigated whether the localization of NPHP4 to basal bodies would be affected in CSPP/CSPP-L–depleted cells (). We therefore also investigated whether the localization of NPHP4 to basal bodies would be affected in CSPP/CSPP-L–depleted cells (<xref ref-type="fig" rid="zmk0151095190008">Figure 8</xref>D and Supplemental Figure 6). The localization of NPHP4 seemed unchanged in cilia-defective basal bodies of CSPP/CSPP-L siRNA transfectants displaying decreased NPHP8 levels (D and Supplemental Figure 6). The localization of NPHP4 seemed unchanged in cilia-defective basal bodies of CSPP/CSPP-L siRNA transfectants displaying decreased NPHP8 levels (<xref ref-type="fig" rid="zmk0151095190008">Figure 8</xref>D). Similarly, the localization of NPHP4 was found unchanged in NPHP8-depleted cells (Supplemental Figure 6). These results suggest that CSPP-L specifically recruits or maintains NPHP8 at the basal body.D). Similarly, the localization of NPHP4 was found unchanged in NPHP8-depleted cells (Supplemental Figure 6). These results suggest that CSPP-L specifically recruits or maintains NPHP8 at the basal body.'], 'zmk0151095190008': ['NPHP8 is a known regulator of cilia-mediated signaling, and the NPHP8 knockout mouse displays decreased cilia numbers in tissues undergoing morphogenic transitions, although primary mouse embryonic fibroblasts derived from this mouse are competent in ciliogenesis (Vierkotten et al., 2007). We therefore investigated whether loss of CSPP/CSPP-L would perturb the localization of NPHP8. Whereas control siRNA transfectants displayed normal cilia and normal localization of NPHP8 to the base of the primary cilium (the area between centrioles and the cilia axoneme that is devoid of staining for acetylated tubulin), NPHP8 staining is severely diminished in the cilia defective CSPP/CSPP-L SMARTpool siRNA or CSPP-L siRNA transfectants (<xref ref-type="fig" rid="zmk0151095190008">Figure 8</xref>A). Thus, CSPP-L either recruits or maintains NPHP8 at the transition zone. Yet, interestingly, knockdown of NPHP8 did not impair ciliogenesis in serum-starved hTERT-RPE1, and cilia seemed longer than in control transfectants. We therefore measured cilia length in control and NPHP8 siRNA transfectants (A). Thus, CSPP-L either recruits or maintains NPHP8 at the transition zone. Yet, interestingly, knockdown of NPHP8 did not impair ciliogenesis in serum-starved hTERT-RPE1, and cilia seemed longer than in control transfectants. We therefore measured cilia length in control and NPHP8 siRNA transfectants (<xref ref-type="fig" rid="zmk0151095190008">Figure 8</xref>B). The average cilia length of presumably untransfected cells of the NPHP8 siRNA transfection, which displayed high mean NPHP8 signal intensity, was measured to 3.7 μm (SEM = 0.5 μm). Although longer than cilia length of control cells, 2.4 μm (SEM = 0.1 μm), this difference was not statistically significant. In contrast, the average cilia length of cells that lost NPHP8 signal in the NPHP8 siRNA transfection was 6.3 μm (SEM = 0.3 μm) and showed a statistically significant difference to control transfectants and to cells displaying near normal NPHP8 signal intensities in the NPHP8 siRNA transfection. Notably, CSPP-L still decorated the prolonged cilia axonemes of NPHP8-depleted cells (B). The average cilia length of presumably untransfected cells of the NPHP8 siRNA transfection, which displayed high mean NPHP8 signal intensity, was measured to 3.7 μm (SEM = 0.5 μm). Although longer than cilia length of control cells, 2.4 μm (SEM = 0.1 μm), this difference was not statistically significant. In contrast, the average cilia length of cells that lost NPHP8 signal in the NPHP8 siRNA transfection was 6.3 μm (SEM = 0.3 μm) and showed a statistically significant difference to control transfectants and to cells displaying near normal NPHP8 signal intensities in the NPHP8 siRNA transfection. Notably, CSPP-L still decorated the prolonged cilia axonemes of NPHP8-depleted cells (<xref ref-type="fig" rid="zmk0151095190008">Figure 8</xref>C).C).']}
|
CSPP Is a Ciliary Protein Interacting with Nephrocystin 8 and Required for Cilia Formation
| null |
Mol Biol Cell
|
1280646000
|
Lafora progressive myoclonus epilepsy is a fatal neurodegenerative disorder caused by defects in the function of at least two proteins: laforin, a dual-specificity protein phosphatase, and malin, an E3-ubiquitin ligase. In this study, we report that a functional laforin-malin complex promotes the ubiquitination of AMP-activated protein kinase (AMPK), a serine/threonine protein kinase that acts as a sensor of cellular energy status. This reaction occurs when any of the three AMPK subunits (alpha, beta, and gamma) are expressed individually in the cell, and it also occurs on AMPK beta when it is part of a heterotrimeric complex. We also report that the laforin-malin complex promotes the formation of K63-linked ubiquitin chains, which are not involved in proteasome degradation. On the contrary, this modification increases the steady-state levels of at least AMPK beta subunit, possibly because it leads to the accumulation of this protein into inclusion bodies. These results suggest that the modification introduced by the laforin-malin complex could affect the subcellular distribution of AMPK beta subunits.
|
[
"AMP-Activated Protein Kinases",
"Animals",
"Carrier Proteins",
"Cell Line",
"Humans",
"Lafora Disease",
"Leupeptins",
"Lysine",
"Mice",
"Multiprotein Complexes",
"Proteasome Endopeptidase Complex",
"Proteasome Inhibitors",
"Protein Multimerization",
"Protein Processing, Post-Translational",
"Protein Stability",
"Protein Transport",
"Protein Tyrosine Phosphatases, Non-Receptor",
"Substrate Specificity",
"Ubiquitin",
"Ubiquitin-Protein Ligases",
"Ubiquitination"
] |
other
|
PMC2912343
| null |
46
|
[
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] |
Mol Biol Cell. 2010 Aug 1; 21(15):2555-2567
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Carm1 leads to decreased Par3 mRNA expression and protein apicalization. (A) Separated eight-cell blastomeres injected with Carm1 or Carm1(E267Q) and DsRed mRNAs at the two-cell stage were pooled into noninjected (NonInj) and injected (+) samples used for qRT-PCR. Normalized averages of biological and technical triplicate are shown. Blastocyst cDNA was used as a positive control. As a disaggregation control, cDNA from 25 intact eight-cell embryos and 25 disaggregated eight-cell embryos was used in the same way. Normalized averages of biological duplicate and technical triplicate are shown for these groups. Error bars, SEM. Par3 transcripts were lower in Carm1-injected clones than in Carm1(E267Q)-injected and both noninjected samples (two-way ANOVA, *p < 0.001). (B–G) Embryos treated as in A were fixed at the eight-cell stage and immunostained for Par3. Weaker Par3 domains (arrowheads) were associated with Carm1 injected clones (54/60 blastomeres, n = 15 embryos), than noninjected clones of the same embryos (arrows; B–D). This trend was not observed in the 28 blastomeres of seven control embryos (E–G). Scale bars, 20 μm. Z value indicates plane of view (/n). (D and G) Graphs indicating the mean fluorescence intensity of Par3 domains, measured for each cell in alternate Z-sections using Image J and calculated for the Carm1/Carm1(E267Q)-injected and noninjected cell populations. Error bars, SEM. Student's t test, **p < 0.01.
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e49163d6810d37bc383b6858af2f0c930116b9b3b3df9264272db425eaeb53ab
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
472,
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[{'image_id': 'zmk0151095390003', 'image_file_name': 'zmk0151095390003.jpg', 'image_path': '../data/media_files/PMC2912351/zmk0151095390003.jpg', 'caption': "Carm1 leads to decreased Par3 mRNA expression and protein apicalization. (A) Separated eight-cell blastomeres injected with Carm1 or Carm1(E267Q) and DsRed mRNAs at the two-cell stage were pooled into noninjected (NonInj) and injected (+) samples used for qRT-PCR. Normalized averages of biological and technical triplicate are shown. Blastocyst cDNA was used as a positive control. As a disaggregation control, cDNA from 25 intact eight-cell embryos and 25 disaggregated eight-cell embryos was used in the same way. Normalized averages of biological duplicate and technical triplicate are shown for these groups. Error bars, SEM. Par3 transcripts were lower in Carm1-injected clones than in Carm1(E267Q)-injected and both noninjected samples (two-way ANOVA, *p < 0.001). (B–G) Embryos treated as in A were fixed at the eight-cell stage and immunostained for Par3. Weaker Par3 domains (arrowheads) were associated with Carm1 injected clones (54/60 blastomeres, n = 15 embryos), than noninjected clones of the same embryos (arrows; B–D). This trend was not observed in the 28 blastomeres of seven control embryos (E–G). Scale bars, 20 μm. Z value indicates plane of view (/n). (D and G) Graphs indicating the mean fluorescence intensity of Par3 domains, measured for each cell in alternate Z-sections using Image J and calculated for the Carm1/Carm1(E267Q)-injected and noninjected cell populations. Error bars, SEM. Student's t test, **p < 0.01.", 'hash': 'e49163d6810d37bc383b6858af2f0c930116b9b3b3df9264272db425eaeb53ab'}, {'image_id': 'zmk0151095390004', 'image_file_name': 'zmk0151095390004.jpg', 'image_path': '../data/media_files/PMC2912351/zmk0151095390004.jpg', 'caption': "Carm1 leads to increased EMK1 mRNA levels and changes to EMK1 protein distribution. (A) Separated eight-cell blastomeres injected with Carm1 or Carm1(E267Q) and DsRed mRNAs at the two-cell stage were pooled into noninjected (NonInj) and injected (+) samples used for qRT-PCR. Normalized averages of biological and technical triplicate are shown. Blastocyst cDNA was used as a positive control. As a disaggregation control, cDNA from 25 intact eight-cell embryos and 25 disaggregated eight-cell embryos was used in the same way. Normalized averages of biological duplicate and technical triplicate are shown for these groups. Error bars, SEM. EMK1 transcripts were higher in Carm1-injected clones than Carm1(E267Q)-injected and both noninjected clones (two-way ANOVA, *p = 0.013). (B–G) Embryos treated as in A were fixed at the eight-cell stage and immunostained for EMK1. Stronger EMK1 basolateral domains (arrows) were associated with Carm1-positive clones (36/46 blastomeres, n = 12 embryos) than the control clones in the same embryos (B–D). This trend was not observed in 104 blastomeres analyzed from 13 control embryos (E–G). Scale bars, 20 μm. Z value indicates plane of view (/n). (D and G) Graphs indicating the mean fluorescence intensity of EMK1 domains, measured for each cell in alternate Z-sections using Image J and calculated for the Carm1/Carm1(E267Q)-injected and noninjected cell populations. Error bars, SEM. Student's t test, **p = 0.024.", 'hash': 'a080924eaab14e4768bf03c7b348b50e10e91b58c199bdada5ec747a5616b022'}, {'image_id': 'zmk0151095390005', 'image_file_name': 'zmk0151095390005.jpg', 'image_path': '../data/media_files/PMC2912351/zmk0151095390005.jpg', 'caption': "Carm1 leads to increased expression of the aPKCζ antagonist, PKCII. (A–H) aPKCζ protein in two-cell (n = 5), 4-cell (n = 7), mid-eight-cell (n = 28), and early 32-cell (n = 8) embryos. (I) aPKCζ and PKCII expression measured at several time points, plotted as % of peak expression. (J) Normalized levels of aPKCζ and PKCII transcripts measured by qRT-PCR using lung cDNA. Averages of biological duplicate and technical triplicate are shown. Error bars, SEM. (K–P) Embryos treated as in A were examined for aPKCζ at the eight-cell stage. Examples illustrate stronger apical aPKCζ domains associated with Carm1-injected clones (outlined in red; 85/96 blastomeres, n = 24 embryos) than the noninjected clones in the same embryos (K–L). This trend was not observed in the 84 blastomeres from 11 control embryos (N and O). Scale bars, 20 μm. (M, P) Graphs indicating the mean fluorescence intensity of aPKCζ domains, measured for each cell in alternate Z-sections using Image J and calculated for the Carm1/Carm1(E267Q)-injected and noninjected cell populations. Error bars, SEM. Student's t test, **p < 0.01. (Q) Separated eight-cell blastomeres injected with Carm1 or Carm1(E267Q) and DsRed mRNAs at the two-cell stage were pooled into noninjected (NonInj) and injected (+) samples used for qRT-PCR. Normalized averages of biological and technical triplicate are shown. Error bars, SEM. aPKCζ and PKCII transcripts were higher in Carm1-injected clones than Carm1(E267Q)-injected and both noninjected clones. Two-way ANOVA, *p = 0.0005, **p = 0.003.", 'hash': '81f347fdac5496c98cd4dfb4fb707220e1c47d549e658ee6c6f38142bf169b2d'}, {'image_id': 'zmk0151095390002', 'image_file_name': 'zmk0151095390002.jpg', 'image_path': '../data/media_files/PMC2912351/zmk0151095390002.jpg', 'caption': 'Carm1 leads to changes in the frequency and direction of cell movement. two-cell blastomeres of H2B-EGFP embryos were injected with Carm1 (Carm1+) or Carm1(E267Q) (E267Q+) and DsRed mRNAs, and their development was tracked as in Figure 1. (A) Example illustrating one 16-cell blastomere (red and purple asterisks) dividing symmetrically (time-point 100) producing one daughter (red arrow) moving from an outer to inner position, before dividing again (time-point 142). Z-value indicates plane of view (n = 15), illustrating that the moving cell begins at the top of the embryo, coming to occupy a deeper position within the embryo in this plane as well as in the X-Y plane. (B) Number and direction of movements of noninjected (NonInj) and injected (+) 16- and 32-cell-stage clones under experimental (Carm1+DsRed) and control (Carm1(E267Q)+DsRed) conditions. (C) Total number of cell movements in groups of embryos injected with Carm1+dsRed or Carm1(E267Q)+DsRed mRNAs at the two-cell stage. (D) Table summarizing data in B and C. Clones are indicated as positive (+) or negative (NonInj) for Carm1 or Carm1(E267Q) for overexpression. Parentheses indicate cumulative numbers of 16- and 32-cell blastomeres (n) and % of these that alter their position. Legend indicates outcomes of χ2 tests performed on data outlined in corresponding colors.', 'hash': '4b3f2ec43b313fd6034545497e8d62c4cb57c1193dfdb6ff5f3fa0fe5fe07734'}, {'image_id': 'zmk0151095390001', 'image_file_name': 'zmk0151095390001.jpg', 'image_path': '../data/media_files/PMC2912351/zmk0151095390001.jpg', 'caption': "Carm1 increases blastomere contribution to the ICM and frequency of asymmetric division. two-cell blastomeres of H2B-EGFP embryos were injected with Carm1 (A) or Carm1(E267Q) (B) and DsRed mRNAs. After time-lapse microscopy, cells were scored as inner/outer and DsRed positive and negative. Student's t tests: *p = 0.00022, ^ p = 0.00091. (C) H2B-EGFP–expressing embryos were injected as in A, time-lapse imaged to the blastocyst stage and tracked using Simi Biocell software. DIC and GFP Z-stack images were used to determine positions of blastomeres every 15 min. Examples where Carm1+DsRed mRNAs were injected are shown. (D) Average proportions of asymmetric and symmetric divisions during 4th and 5th cleavage for noninjected (top) and injected (bottom) clones in experimental (Carm1, n = 14) and control (E267Q, n = 12) embryos. Error bars, SEM. Student's t test: **p < 0.001. Scale bar, 10 μm.", 'hash': '1bff5aea5e22d2428da5425e366eb0bba713f5a7d6b4768135e7f35dd989ed9e'}, {'image_id': 'zmk0151095390006', 'image_file_name': 'zmk0151095390006.jpg', 'image_path': '../data/media_files/PMC2912351/zmk0151095390006.jpg', 'caption': 'Carm1 leads to a down-regulation of Cdx2. (A) Separated eight-cell blastomeres injected with Carm1 or Carm1(E267Q) and DsRed mRNAs at the two-cell stage were pooled into noninjected (NonInj) and injected (+) samples used for qRT-PCR. Normalized averages of biological and technical triplicate are shown. Error bars, SEM. Cdx2 transcripts were lower in Carm1-injected clones than Carm1(E267Q)-injected and both noninjected clones. Two-way ANOVA; *p < 0.001. (B and C) Eight-cell embryos treated as in A were fixed and immunostained for Cdx2. Six blastomeres in the example are Cdx2-positive, one weakly so (arrowhead). Of these, four derive from the noninjected clone. Scale bar, 20 μm. (D) Frequencies of Cdx2-positive nuclei in Carm1 overexpressing (Carm1+) and noninjected blastomeres (NonInj) from 19 embryos.', 'hash': 'b6ea8013e515c0395ef34a2574057f5433c2ce38c525e2694f7643255b3657fb'}]
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{'zmk0151095390002': ['Fluorescence and DIC Z-stacks of embryos from the zygote (24 h after human chorionic gonadotropin [hCG]) or two-cell (44 h after hCG) to blastocyst stage were collected on 15 focal planes every 15 min for ∼96 and 72 h of continuous embryo culture, respectively. The images were processed as described previously (Bischoff et al., 2008). All cells were followed in 4D using SIMI Biocell software (http://www.simi.com/en/products/biocell/index.html/; Schnabel et al., 1997): The 3D coordinates of every nucleus were taken every 2–3 frames, including one frame before and one after cell cleavages. Between each of these “fixed” points, cell movement was intercalated by SIMI Biocell software. Cell behavior was defined using the position of daughter cells shortly after mitosis and at the end of their cell cycle to determine whether they had moved from or toward the outside, as in our previous study (Bischoff et al., 2008). If the DIC images indicated a cell with a clear outside surface (e.g., highlighted in <xref ref-type="fig" rid="zmk0151095390002">Figure 2</xref>) and if there were no surrounding nuclei in the fluorescence images, including consideration for nuclei above or below in the z-plane, then a cell was defined as outer and vice versa for cells that did not meet these criteria. By following every cell in the recording and using the DsRed fluorescence signal at the two-cell stage to determine which blastomere had been injected with ) and if there were no surrounding nuclei in the fluorescence images, including consideration for nuclei above or below in the z-plane, then a cell was defined as outer and vice versa for cells that did not meet these criteria. By following every cell in the recording and using the DsRed fluorescence signal at the two-cell stage to determine which blastomere had been injected with Carm1 or Carm1(E267Q) mRNA, complete lineages of the behavior of all cells up to the blastocyst stage were generated. This dataset allowed the position, fate allocation and division orientation of all cells to be determined individually.', 'To identify whether such cell repositioning happens after Carm1 up-regulation, we scored the position (inner or outer) of daughter cells both immediately after their mitotic division and at the end of their cell cycle, to check whether this had changed (<xref ref-type="fig" rid="zmk0151095390002">Figure 2</xref>A; see A; see Materials and Methods). This revealed that in control, Carm1(E267Q) embryos, cell movement was observed in 2.3% of all cells (<xref ref-type="fig" rid="zmk0151095390002">Figure 2</xref>, B and D; n = 576 when all 16- and 32-cell blastomeres are considered together). Twenty-five percent (3/12) of these movements occurred during the 16-cell stage, two of which were from an inside to an outside position: one from a noninjected and the other a , B and D; n = 576 when all 16- and 32-cell blastomeres are considered together). Twenty-five percent (3/12) of these movements occurred during the 16-cell stage, two of which were from an inside to an outside position: one from a noninjected and the other a Carm1(E267Q)-injected clone. Of the movements occurring at the 32-cell stage, six of nine were from an inner to an outer position, the noninjected and injected clones making up similar proportions of these movements. Hence, cell movement defined in these terms occurs as often as in unmanipulated embryos and in similar “directions” (Bischoff et al., 2008). In contrast, the frequency of cell movement significantly increased in embryos in which Carm1 was up-regulated, when compared with both Carm1(E267Q)-injected and unmanipulated embryos: 8.7% of all cells were observed to move in Carm1-injected embryos (n = 672; 16- and 32-cell blastomeres; <xref ref-type="fig" rid="zmk0151095390002">Figure 2</xref>, C and D) and most of these cell movements (77%, n = 59) occurred during the 32-cell stage. Strikingly, we found that cells with elevated levels of Carm1 tended to move in the opposite direction to their noninjected counterparts in the same embryos (i.e., from an outer to inner position; , C and D) and most of these cell movements (77%, n = 59) occurred during the 32-cell stage. Strikingly, we found that cells with elevated levels of Carm1 tended to move in the opposite direction to their noninjected counterparts in the same embryos (i.e., from an outer to inner position; <xref ref-type="fig" rid="zmk0151095390002">Figure 2</xref>, B and D). Moreover, the frequency of cell movement in the noninjected clone indicated that cells of the noninjected clones moving in the opposite direction might compensate for the behavior of cells of the , B and D). Moreover, the frequency of cell movement in the noninjected clone indicated that cells of the noninjected clones moving in the opposite direction might compensate for the behavior of cells of the Carm1-injected clone. In agreement with this, no extra cells were found in the ICM of Carm1 embryos compared with Carm1(E267Q) embryos (p = 0.69; <xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>, A and B). Together with the analysis of division orientation, these results provide evidence that elevation of Carm1 leads cells to adopt ICM positions through both increased frequency of asymmetric divisions and cell movement., A and B). Together with the analysis of division orientation, these results provide evidence that elevation of Carm1 leads cells to adopt ICM positions through both increased frequency of asymmetric divisions and cell movement.'], 'zmk0151095390001': ['After imaging, the total number of cells per embryo in the Carm1 group was, on average, 31.5 (±1.0), of which 19.2 (±4.9) were outer (TE) and 12.2 (±4.9) were inner (ICM) cells. The number of cells derived from the Carm1-injected clone was similar to that derived from the noninjected clone (<xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>A). However, when we analyzed the contribution of individual cells to the ICM, we found that the significant majority (74.9%) were derived from the blastomere in which the Carm1 level was increased (A). However, when we analyzed the contribution of individual cells to the ICM, we found that the significant majority (74.9%) were derived from the blastomere in which the Carm1 level was increased (<xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>A). The opposite was true when we analyzed the contribution of clones to the TE. The proportion of outer cells derived from blastomeres with artificially higher levels of Carm1 was on average 35.4%; thus, the significant majority (64.6%) of the TE was derived from the clone in which Carm1 levels remained unchanged. This tendency was not observed when A). The opposite was true when we analyzed the contribution of clones to the TE. The proportion of outer cells derived from blastomeres with artificially higher levels of Carm1 was on average 35.4%; thus, the significant majority (64.6%) of the TE was derived from the clone in which Carm1 levels remained unchanged. This tendency was not observed when Carm1(E267Q) mRNA was overexpressed in place of Carm1: approximately half (47.8%) the progeny of the Carm1(E267Q)-injected blastomere contributed to the ICM and half (52.2%) to the TE (<xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>B). Thus, elevated expression of Carm1 in a two-cell blastomere leads its progeny to contribute predominantly to the ICM (Student\'s B). Thus, elevated expression of Carm1 in a two-cell blastomere leads its progeny to contribute predominantly to the ICM (Student\'s t test, p = 0.00022), in agreement with previous studies (Torres-Padilla et al., 2007).', 'To understand whether differences in cell dynamics between the Carm1-injected and noninjected cells within the same embryo accounted for the former\'s more extensive ICM allocation, we analyzed the behavior of all cells in 4D, until their allocation to the ICM and TE at the early blastocyst stage. Inner cells are largely generated through asymmetric division of eight- and 16-cell blastomeres and the division of inside cells from the 16-cell stage. To assess whether Carm1 up-regulation was associated with a change in the proportion of asymmetric/symmetric divisions, we used Simi Biocell reconstructions to determine the division orientation of the progeny of both two-cell blastomeres. These were classified by scoring the positions of daughter cells relative to each other and to the embryo surface one frame before and one after their mitotic division in both DIC and fluorescence images. This allowed the direct comparison of the proportions of asymmetric/symmetric divisions between Carm1/Carm1(E267Q)-injected and noninjected clones (<xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>C, Supplemental Movie).C, Supplemental Movie).', 'We found that during the 4th cleavage, the clone of cells in which Carm1 levels were up-regulated took more asymmetric divisions in comparison to the noninjected clone (on average, 60.0 and 52.8%, respectively (p = 0.0007; <xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>D). Similarly, the proportion of asymmetric divisions taken by the D). Similarly, the proportion of asymmetric divisions taken by the Carm1-injected clone was higher than that of the Carm1(E267Q)-injected clone, which was 51.9% (p = 0.0024). In contrast, when we compared the proportion of asymmetric divisions between noninjected clones of Carm1 and Carm1(E267Q) groups, we found them to be statistically equivalent (52.8 and 55.5%, respectively, p = 0.43). Similarly, the proportion of asymmetric divisions was statistically equal in Carm1(E267Q)-injected and noninjected blastomeres of the same embryos (52.3 and 55.1%, respectively; p = 0.097).', 'Carm1 leads to changes in the frequency and direction of cell movement. two-cell blastomeres of H2B-EGFP embryos were injected with Carm1 (Carm1+) or Carm1(E267Q) (E267Q+) and DsRed mRNAs, and their development was tracked as in <xref ref-type="fig" rid="zmk0151095390001">Figure 1</xref>. (A) Example illustrating one 16-cell blastomere (red and purple asterisks) dividing symmetrically (time-point 100) producing one daughter (red arrow) moving from an outer to inner position, before dividing again (time-point 142). Z-value indicates plane of view (n = 15), illustrating that the moving cell begins at the top of the embryo, coming to occupy a deeper position within the embryo in this plane as well as in the X-Y plane. (B) Number and direction of movements of noninjected (NonInj) and injected (+) 16- and 32-cell-stage clones under experimental (. (A) Example illustrating one 16-cell blastomere (red and purple asterisks) dividing symmetrically (time-point 100) producing one daughter (red arrow) moving from an outer to inner position, before dividing again (time-point 142). Z-value indicates plane of view (n = 15), illustrating that the moving cell begins at the top of the embryo, coming to occupy a deeper position within the embryo in this plane as well as in the X-Y plane. (B) Number and direction of movements of noninjected (NonInj) and injected (+) 16- and 32-cell-stage clones under experimental (Carm1+DsRed) and control (Carm1(E267Q)+DsRed) conditions. (C) Total number of cell movements in groups of embryos injected with Carm1+dsRed or Carm1(E267Q)+DsRed mRNAs at the two-cell stage. (D) Table summarizing data in B and C. Clones are indicated as positive (+) or negative (NonInj) for Carm1 or Carm1(E267Q) for overexpression. Parentheses indicate cumulative numbers of 16- and 32-cell blastomeres (n) and % of these that alter their position. Legend indicates outcomes of χ2 tests performed on data outlined in corresponding colors.'], 'zmk0151095390003': ['To gain further insight into the underlying reasons for our observations on cell dynamics, we examined whether the expression levels and/or distribution of polarity molecules known to govern cell position might be affected by Carm1 up-regulation. We first focused on the cell polarity marker Par3. To compare the expression levels of Par3 between cells in which Carm1 was up-regulated and those in which it was not in the same embryos, we performed qRT-PCR in three biological samples of cDNA derived from 100 Carm1-injected and 100 noninjected blastomeres of 25 disaggregated eight-cell embryos. In control experiments, Carm1(E267Q) mRNA replaced that of Carm1, as above. To determine whether the disaggregation of blastomeres might have any effect on Par3 transcript levels, we measured those of whole and disaggregated (but otherwise unmanipulated) eight-cell embryos at the same developmental time point. Low levels of Par3 transcript were found in all eight-cell cDNA samples (<xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>A). There was no significant difference between A). There was no significant difference between Par3 levels detected in cDNA derived from whole and disaggregated eight-cell embryos (<xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>A, Student\'s A, Student\'s t test, p = 0.35). Similarly, there was no significant difference in these transcript levels between Carm1(E267Q)-injected cells and their noninjected counterparts (Student\'s t test, p = 0.62). In contrast, the levels of Par3 were 85% lower in the Carm1-injected eight-cell clone than in its noninjected counterpart, and, indeed, both control samples (two-way ANOVA; p < 0.001, <xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>A). To determine the extent to which this could also be seen at the protein level, we examined the distribution of Par3 by immunofluorescence. An apical distribution of Par3 was detected in mid-eight-cell blastomeres (A). To determine the extent to which this could also be seen at the protein level, we examined the distribution of Par3 by immunofluorescence. An apical distribution of Par3 was detected in mid-eight-cell blastomeres (<xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>B), in agreement with one previous report (B), in agreement with one previous report (Plusa et al., 2005) but in contrast to another (Vinot et al., 2005). Furthermore, in agreement with the results of the qRT-PCR experiments, these apical domains of Par3 appeared attenuated in most (90%, 54/60) Carm1-injected blastomeres (<xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>, B and C). In agreement with this, when we quantified the intensity of Par3 domains using Image J, they were on average significantly less in the , B and C). In agreement with this, when we quantified the intensity of Par3 domains using Image J, they were on average significantly less in the Carm1-injected blastomeres compared with their noninjected counterparts in the same embryos (p < 0.01; <xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>D). This was not observed in D). This was not observed in Carm1(E267Q)-injected control embryos (<xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>, E–G)., E–G).'], 'zmk0151095390004': ['To determine whether the changes in cell behavior described above could also reflect the organization of the basolateral pole of eight-cell blastomeres, we examined the distribution of the mammalian homologue of Par1, EMK1, which localizes basolaterally at this stage (Vinot et al., 2005) and is implicated in polarity and cell division regulation in epithelial cells (Bohm et al., 1997). To compare the expression levels of EMK1 between Carm1-injected and noninjected clones in the same embryos, we carried out experiments similar to the ones described for Par3. We found that EMK1 was detectable in all eight-cell and blastocyst cDNA samples (<xref ref-type="fig" rid="zmk0151095390004">Figure 4</xref>A). We could not find any significant differences in A). We could not find any significant differences in EMK1 expression between Carm1(E267Q)-injected and noninjected clones of the same embryos (Student\'s t test, p = 0.8). In contrast we found that EMK1 transcript levels were 55% higher in Carm1-injected than in noninjected cells of the same embryos (<xref ref-type="fig" rid="zmk0151095390004">Figure 4</xref>A). Thus, A). Thus, EMK1 expression was significantly higher when Carm1 levels were up-regulated than in all other samples analyzed (two-way ANOVA, p = 0.013). To determine the extent to which this could also be seen at the protein level, we examined the distribution of EMK1 protein. Although EMK1 was present throughout the cell, it showed a basolateral accumulation in eight-cell blastomeres as described previously (Vinot et al., 2005). In none of the 13 embryos expressing Carm1(E267Q) were we able to detect any obvious difference in EMK1 distribution between injected (n = 52) and (n = 52) noninjected clones (<xref ref-type="fig" rid="zmk0151095390004">Figure 4</xref>, E–G). However, the fluorescent signal of EMK1 appeared stronger, particularly at the basolateral region, in the majority (75%, 36/48) of , E–G). However, the fluorescent signal of EMK1 appeared stronger, particularly at the basolateral region, in the majority (75%, 36/48) of Carm1-injected blastomeres (n = 48) compared with their noninjected neighbors (n = 48; <xref ref-type="fig" rid="zmk0151095390004">Figure 4</xref>, B and C). When we quantified this, we found that the intensity of the EMK1 signal was indeed significantly greater in , B and C). When we quantified this, we found that the intensity of the EMK1 signal was indeed significantly greater in Carm1-injected blastomeres than in their noninjected counterparts in the same embryos (p = 0.024; <xref ref-type="fig" rid="zmk0151095390004">Figure 4</xref>D). Together these results suggest that elevated expression of Carm1 affects the expression and distribution of Par3 and EMK1 in a reciprocal manner, a decrease in Par3 expression and apical distribution being associated with an increase in EMK1 expression and basolateral distribution. Thus, elevation of Carm1 leads to an alteration of the expression and distribution of molecules known to regulate mammalian cell polarity and associate with changes in division asymmetry (D). Together these results suggest that elevated expression of Carm1 affects the expression and distribution of Par3 and EMK1 in a reciprocal manner, a decrease in Par3 expression and apical distribution being associated with an increase in EMK1 expression and basolateral distribution. Thus, elevation of Carm1 leads to an alteration of the expression and distribution of molecules known to regulate mammalian cell polarity and associate with changes in division asymmetry (Bohm et al., 1997; Plusa et al., 2005).'], 'zmk0151095390005': ['Because of the role demonstrated for the λ isoform of aPKC in affecting division orientation during mouse development (Plusa et al., 2005) and pole size in Xenopus embryos (Chalmers et al., 2005), we wondered whether the increased frequency of asymmetric division and cell internalization associated with Carm1 up-regulation is also associated with changes in the expression or distribution of aPKCζ. To this end, we first characterized the distribution of aPKCζ protein from the two-cell stage to the late morula stage in normal development (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>). Initially, aPKCζ appears to be distributed uniformly at the cell cortex until the eight-cell stage (). Initially, aPKCζ appears to be distributed uniformly at the cell cortex until the eight-cell stage (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>, A and B). However, this changes after compaction, when aPKCζ distribution becomes distinctively apical at the mid-eight-cell stage (, A and B). However, this changes after compaction, when aPKCζ distribution becomes distinctively apical at the mid-eight-cell stage (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>C) and is more evenly distributed over the apical cell membrane from the late eight-cell stage onward; it also shows a detectable concentration at the outer surface of the developing morula (C) and is more evenly distributed over the apical cell membrane from the late eight-cell stage onward; it also shows a detectable concentration at the outer surface of the developing morula (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>D). We also quantified D). We also quantified aPKCζ expression in total RNA extracts of intact, unmanipulated embryos through developmental time. This revealed a peak in aPKCζ levels concomitant with its change in distribution at the eight-cell stage (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>I).I).', 'To compare aPKCζ distribution between cells in which Carm1 was elevated and their noninjected neighbors, mid-eight-cell embryos expressing Carm1 and DsRed mRNAs in the progeny of one two-cell blastomere were fixed and stained with antibody specific to the kinase-containing C-terminus of aPKCζ. This revealed that in 89% (85/96, n = 24 embryos) of blastomeres with higher levels of Carm1, the apical domains of aPKCζ appeared more concentrated than in the noninjected neighboring blastomeres (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>, K–N). Such differences were not apparent between the injected and noninjected clones of , K–N). Such differences were not apparent between the injected and noninjected clones of Carm1(E267Q) embryos (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>, O and P; n = 11 embryos). Indeed, when we quantified the intensities of aPKCζ domains, they were significantly higher in the blastomeres in which Carm1 was elevated than their counterparts with nonelevated Carm1 levels in the same embryos (p < 0.01; , O and P; n = 11 embryos). Indeed, when we quantified the intensities of aPKCζ domains, they were significantly higher in the blastomeres in which Carm1 was elevated than their counterparts with nonelevated Carm1 levels in the same embryos (p < 0.01; <xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>M), a trend we did not observe in M), a trend we did not observe in Carm1(E267Q)-injected control embryos (<xref ref-type="fig" rid="zmk0151095390003">Figure 3</xref>P). To test this further, we analyzed P). To test this further, we analyzed aPKCζ transcript levels using eight-cell cDNA samples as above: Levels in cells with elevated Carm1 were 56% higher than in the noninjected cells of the same embryos, a difference not seen between injected and noninjected cells in Carm1(E267Q) embryos (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>Q; two-way ANOVA, p = 0.0005).Q; two-way ANOVA, p = 0.0005).', 'In some respects, this was an unexpected result, as the effect on Par3/EMK1 expression and distribution suggested a “depolarizing” phenotype for Carm1 up-regulation. Moreover, down-regulation of aPKCλ promotes asymmetric division and cell internalization (Plusa et al., 2005), similar to the effects described here for up-regulation of Carm1. However, although overexpression of aPKCλ is sufficient to induce cell protrusion in Xenopus embryos, overexpression of a truncated version of the protein, lacking the kinase domain, does not produce this phenotype (Chalmers et al., 2005). This drew our attention to a previously reported isoform, PKCII, showing 98% amino acid identity with aPKCζ but lacking the kinase domain (Parkinson et al., 2004). Because the interaction domains of these proteins are functionally identical, PKCII is proposed to regulate the activity of aPKCζ by competing for sites of activity in mammalian epithelial cell culture (Parkinson et al., 2004). To date, there have been no reports of whether a similar mechanism might occur in the mouse embryo, so we wanted to examine whether PKCII expression could be positively affected by Carm1 overexpression. Using primers specific for this particular isoform and lung cDNA as a positive control, we found that expression of PKCII is indeed detectable in the mouse embryo, although at lower levels than aPKCζ (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>, I and J). This finding led us to analyze changes in , I and J). This finding led us to analyze changes in PKCII expression levels throughout preimplantation development, which revealed a peak in its expression at the late eight-cell stage (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>I). To determine whether a change in I). To determine whether a change in PKCII expression could be associated with Carm1 up-regulation, we next compared the level of PKCII transcript in eight-cell stage cells with higher levels of Carm1 with that of noninjected clones in the same embryos. This revealed that PKCII levels were 77-fold higher in cells in which Carm1 was elevated (<xref ref-type="fig" rid="zmk0151095390005">Figure 5</xref>Q). Such a difference was not seen between injected and noninjected blastomeres in Q). Such a difference was not seen between injected and noninjected blastomeres in Carm1(E267Q) embryos. Thus PKCII levels in Carm1-injected cells were significantly higher than in all other samples (p = 0.0032; <xref ref-type="fig" rid="zmk0151095390006">Figure 6</xref>K). This striking increase in K). This striking increase in PKCII expression observed upon Carm1 up-regulation could affect normal aPKCζ function at the apical pole.'], 'zmk0151095390006': ['The transcription factor Cdx2 is key for TE formation (Niwa et al., 2005; Strumpf et al., 2005; Jedrusik et al., 2008; Nishioka et al., 2009) and its down-regulation increases the probability with which cells take asymmetric rather than symmetric division (Jedrusik et al., 2008). We therefore wondered whether Carm1 up-regulation affects the level of Cdx2 expression. To address this, we measured Cdx2 transcript levels in cells in which Carm1 was up-regulated and compared them with those of noninjected cells of the same embryos, as above. This revealed that the expression of Cdx2 was 70% lower in cells with higher levels of Carm1 (<xref ref-type="fig" rid="zmk0151095390006">Figure 6</xref>A). This was in contrast to the levels of A). This was in contrast to the levels of Cdx2 observed between injected and noninjected blastomeres in Carm1(E267Q) embryos, which were statistically comparable. Thus, Carm1 overexpression results in a significant reduction in Cdx2 expression (two-way ANOVA, p < 0.001).', 'Because Cdx2 expression shows heterogeneity at the eight-cell stage, we extended the above analysis and examined the proportions of Cdx2-positive nuclei in blastomeres with different levels of Carm1 at the eight-cell stage (<xref ref-type="fig" rid="zmk0151095390006">Figure 6</xref>, B and C). From 19 embryos in which Carm1 was overexpressed, 60 eight-cell blastomeres were Cdx2-positive (, B and C). From 19 embryos in which Carm1 was overexpressed, 60 eight-cell blastomeres were Cdx2-positive (<xref ref-type="fig" rid="zmk0151095390006">Figure 6</xref>D). Of these, only 22 were derived from these cells in which D). Of these, only 22 were derived from these cells in which Carm1 was injected (χ2 = 4.28, p = 0.039). Taken together, these results provide evidence that Cdx2 is expressed to a lesser extent upon Carm1 up-regulation, but also indicate that this association can be affected by the endogenous variability in Cdx2, previously observed among eight-cell blastomeres (Ralston and Rossant, 2008; Jedrusik et al., 2008). This may parallel the natural heterogeneity in Carm1-modified histone substrates observed at this time (Torres-Padilla et al., 2007).']}
|
Epigenetic Modification Affecting Expression of Cell Polarity and Cell Fate Genes to Regulate Lineage Specification in the Early Mouse Embryo
| null |
Mol Biol Cell
|
1280646000
|
The noncentrosomal cortical microtubules (CMTs) of plant cells self-organize into a parallel three-dimensional (3D) array that is oriented transverse to the cell elongation axis in wild-type plants and is oblique in some of the mutants that show twisted growth. To study the mechanisms of CMT array organization, we developed a 3D computer simulation model based on experimentally observed properties of CMTs. Our computer model accurately mimics transverse array organization and other fundamental properties of CMTs observed in rapidly elongating wild-type cells as well as the defective CMT phenotypes observed in the Arabidopsis mor1-1 and fra2 mutants. We found that CMT interactions, boundary conditions, and the bundling cutoff angle impact the rate and extent of CMT organization, whereas branch-form CMT nucleation did not significantly impact the rate of CMT organization but was necessary to generate polarity during CMT organization. We also found that the dynamic instability parameters from twisted growth mutants were not sufficient to generate oblique CMT arrays. Instead, we found that parameters regulating branch-form CMT nucleation and boundary conditions at the end walls are important for forming oblique CMT arrays. Together, our computer model provides new mechanistic insights into how plant CMTs self-organize into specific 3D arrangements.
|
[
"Arabidopsis",
"Cell Polarity",
"Computer Simulation",
"Microtubules",
"Models, Molecular",
"Mutation",
"Phenotype"
] |
other
|
PMC2912351
| null |
49
|
[
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Mol Biol Cell. 2010 Aug 1; 21(15):2649-2660
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NO-CC CODE
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At least one formin and Myo2p are required for correct Kar9p polarity. (A and B) Effect of inactivation of both yeast formins on Kar9p polarized localization in synchronized budded cells. bnr1Δ or bnr1Δ bni1ts cells were released from a G1 block at 23°C, and samples were taken to monitor budding and progression of the spindle pathway (data not shown). (A) At 45 min after release from G1, budded cells were shifted to 34°C, and still images were acquired for scoring Kar9p localization onto short spindles after a 15-min incubation (nbnr1Δ = 249; nbnr1Δbni1ts = 75), 30 min (nbnr1Δ = 281; nbnr1Δbni1ts = 325), and 45 min (nbnr1Δ = 83; nbnr1Δbni1ts = 305). For reference, Kar9p localization in asynchronous cultures is shown (n > 226 cells). The bni1ts allele bni1-FH2#1 (Sagot et al., 2002) was introduced into 15D background as described in Supplemental Data. Single bnr1Δ or bni1Δ mutants exhibited correct Kar9p polarization, whereas combined inactivation of both formins increased Kar9p symmetry. (B) Representative images of cells from the experiment shown in A after a 30-min incubation at 34°C. Overlays show CFP-Tub1p (in magenta) and Kar9p-GFP3 (in green). Bar, 2 μm. Arrowheads point to cells with disrupted Kar9p polarity. (C) Distribution of Kar9p modes of localization in logarithmic cultures of MYO2 or myo2-16 cells grown at 23°C (nMYO2 = 152; nmyo2-16 = 99) or after 1-h shift to 34°C (nMYO2 = 184; nmyo2-16 = 275). Only budded cells were scored. After a temperature shift, the myo2-16 mutant exhibited a marked disruption of Kar9p polarized distribution.
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multiple
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multiple panels: images & plots
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[
"Microscopy",
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"Immuno Assays"
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"bar plot",
"immunoblot"
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[{'image_id': 'zmk0151095280006', 'image_file_name': 'zmk0151095280006.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280006.jpg', 'caption': 'Efficient Kar9p repolarization to the SPBbud requires polarity determinants and actin cables. (A) Time course analysis of Kar9p repolarization after treatment with nocodazole in the indicated strains was carried out as described in Figure 5. For simplicity, the “one pole” category is depicted in the plot. For full details on the time course see Supplemental Figure S3A. Cell polarity mutants were delayed in Kar9p repolarization. (B) LatB prevented Kar9p repolarization after nocodazole washout. Time course analysis in wild-type cells was carried out as described in Figure 5 except that after removal of nocodazole, part of the culture was treated with either dimethyl sulfoxide (DMSO) or 100 μM LatB for the indicated times, and aliquots were drawn for scoring (n > 128). In the presence of LatB, symmetry persisted but unequal loading to the SPBs increased over time. (C and D) Time lapse analysis of Kar9p behavior during recovery from nocodazole treatment in a wild-type cell. (C) Unseparated SPBs became repositioned near the bud neck (0–10 min, arrows). At onset of SPB separation (5–10 min) Kar9p preferentially marked one SPB (arrow vs. arrowhead). Bias persisted during alignment (10–20 min). Numbers indicate time elapsed in minutes. Bars, 2 μm. (D) Label intensities for Spc42p-CFP at SPBs (SPB1 is indicated by the arrow in C) and Kar9p-GFP3 associated with each pole during the time lapse depicted in C. When SPB separation began at 5 min, Kar9p labeled SPBs unequally. At 10 min, SPBs are clearly separated and Kar9p label increased at SPB1. Further increase occurred by 20 min. (E) Summary of time lapse analysis for symmetry breaking upon recovery from nocodazole treatment in wild-type versus bud6Δ cells. In wild-type cells, 45.8% cells recorded (n = 24 cells) exhibited Kar9p symmetric label at onset of SPB separation that became polarized within 10 min. In bud6Δ cells, 69.6% (n = 24 cells recorded) exhibited symmetric label that took longer to repolarize. For further representative time lapse sequences see Supplemental Figure S3C.', 'hash': '79855d8002981f2f828d46122d6e0aea1e0e3005236bce90c8c9360d0fc5ed37'}, {'image_id': 'zmk0151095280001', 'image_file_name': 'zmk0151095280001.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280001.jpg', 'caption': 'Kar9p polarization to the SPBbud is disrupted in a bud6Δ bni1Δ mutant. (A) Representative images of wild-type (a–d) and bud6Δ bni1Δ cells (e–m) expressing Kar9p-GFP3 (in green) and CFP-Tub1p (in magenta), showing the extent of polarization of Kar9p-GFP3 along the spindle pathway. Wild-type cells exhibited Kar9p-GFP3 label on both poles at onset of spindle assembly (a and b, arrows and arrowheads). Label became polarized to the SPBbud in preanaphase spindles (c, arrows) and continued to favor the SPBbud in anaphase (d, arrow). bud6Δ bni1Δ cells did not coordinate polarization of Kar9p-GFP3 with spindle alignment. (e) Two cells with misaligned spindles marked at both poles by Kar9-GFP3. (f) A cell with a short spindle carrying symmetric Kar9-GFP3 label (arrow and arrowhead) despite spindle alignment. (g) A cell with a correctly aligned spindle and Kar9p-GFP3 label favoring the SPBbud (arrow). (h) Kar9p-GFP3 preferentially marks the SPBmother (arrowhead) despite spindle alignment. (i) Misaligned spindle symmetrically labeled by Kar9-GFP3. (j and k) Kar9p-GFP3 label is associated mainly with the SPBbud (arrows). (l) Two cells with elongated spindle exhibit Kar9p-GFP3 label associated with both poles despite alignment. (m) A cell containing a misaligned anaphase spindle with Kar9p-GFP3 present at both poles. In conclusion, loss of polarity persisted throughout anaphase and seemed unrelated to alignment or SPB identity. (B) Categories of Kar9p localization used in this study: one pole (a), asymmetric (b), partially symmetric (c), and symmetric (d). (C) Distribution of asynchronous cells according to their labeling by Kar9p-GFP3 as depicted in B. Cells with <1-μm-long spindles (n > 282 cells); 1- to 2.5-μm-long spindles (n > 290 cells) or elongated spindles (n > 270 cells) in asynchronous cultures were scored. (D) Distribution of preanaphase cells with correctly aligned spindles according to the relative bias of Kar9p-GFP3 toward the SPBbud or SPBmother, n > 390. The frequency of preanaphase spindle misalignment was 10.7% in wild-type cells, 25% in bud6Δ cells, 40% in bni1Δ cells, and 51.3% in bud6Δ bni1Δ cells. Only in bud6Δ bni1Δ cells Kar9p-GFP3 no longer favored the SPBbud. For a detailed breakdown of all categories scored, see Supplemental Figure S1. Bars, 2 μm.', 'hash': 'b5a15bb236f650914daaf8c43ed93d0c1fd9255504b6ed07677ee2dba17dea13'}, {'image_id': 'zmk0151095280008', 'image_file_name': 'zmk0151095280008.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280008.jpg', 'caption': 'A model for Kar9p polarization driven by actin cables, Myo2p, and aMTs. (A) Nocodazole treatment erases differential SPB history and allows Kar9p symmetric recruitment to a basal level. After removal of the drug, Kar9p recruitment increases after regrowth of aMTs. aMTs from one SPB may stochastically engage in Kar9p-mediated deliveries. These interactions can rapidly evoke a feedback loop that helps symmetry breaking, before (solid arrow) or after (dashed arrows) SPB separation. In a bud6Δ mutant, the reduction in actin cables may lead to a delay in SPB repositioning and symmetry breaking. (B) In an unperturbed wild-type cell, spindle polarity is primed by aMTs from the old SPB tethering the side-by-side SPBs to the incipient bud. The old SPB also engages in Kar9p-mediated transport through its existing aMTs. The new SPB can also recruit Kar9p to a basal level but lacks aMTs. A bias for Kar9p recruitment to the old SPB can become established before aMTs form at the new SPB. This bias prevails after SPB separation leading to the loss of Kar9p from the new SPB.', 'hash': 'e84f722581755afb43811f474477269f4aae9fbec828942d6727585b8cf51391'}, {'image_id': 'zmk0151095280007', 'image_file_name': 'zmk0151095280007.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280007.jpg', 'caption': 'Kar9p accumulation bias may be enforced by sustained cycles of aMT delivery along actin cables. (A) Kar9p behavior after recovery from treatment with LatB. Wild-type cells were synchronized and released from G1 to allow formation of a bud before treatment with LatB. Overlays of fluorescence images for Kar9p (in green) and Tub1p (in magenta) are shown. Linescans for fluorescence intensity depict Kar9p-GFP3 distribution along an aMT (with the spindle pole close to the origin and the plus end always to the right; distance in micrometers) for the indicated frames. Seesaw-like behavior of the spindle accompanied Kar9p-mediated transport from alternating poles because aMTs decorated by Kar9p displayed the characteristic angular movements. Initially, Kar9p was asymmetric, but a cycle of transport engaging the right pole (yellow arrows; 0–5 min) led to intensified label at that pole as the aMT depolymerized (5 min). Label decreased as the left pole (white arrows) engaged in deliveries (5–7 min). A second cycle from the right pole (14–18 min, yellow arrows) triggered an increase in Kar9p label at this pole. Both poles continued to move in response to alternating transits, although the left pole was favored, restoring the bias. Progressively, label disappeared from the right pole (28–31 min) and persisted on the left pole and further transports began to align the spindle (29–31 min, arrowheads). Numbers indicate time elapsed in minutes. Bars, 2 μm. (B) Inactivation of a myo2ts allele disrupting Kar9p binding to the cargo domain of Myo2p increased Kar9p symmetry. Plot for distribution of cells with short spindles labeled at one pole by Kar9p in asynchronous MYO2, myo2-17 (encoding a cargo domain mutant with defects in polarized secretion, yet able to bind Kar9p) and myo2-18 (encoding a mutant disrupted for Kar9p binding) cultures grown at 23°C or transferred to 34°C for 30 min.', 'hash': '38d1782540576d80f8d666536c3e8a83421cb02e6382497a35911cf9761dc4b0'}, {'image_id': 'zmk0151095280004', 'image_file_name': 'zmk0151095280004.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280004.jpg', 'caption': 'At least one formin and Myo2p are required for correct Kar9p polarity. (A and B) Effect of inactivation of both yeast formins on Kar9p polarized localization in synchronized budded cells. bnr1Δ or bnr1Δ bni1ts cells were released from a G1 block at 23°C, and samples were taken to monitor budding and progression of the spindle pathway (data not shown). (A) At 45 min after release from G1, budded cells were shifted to 34°C, and still images were acquired for scoring Kar9p localization onto short spindles after a 15-min incubation (nbnr1Δ = 249; nbnr1Δbni1ts = 75), 30 min (nbnr1Δ = 281; nbnr1Δbni1ts = 325), and 45 min (nbnr1Δ = 83; nbnr1Δbni1ts = 305). For reference, Kar9p localization in asynchronous cultures is shown (n > 226 cells). The bni1ts allele bni1-FH2#1 (Sagot et al., 2002) was introduced into 15D background as described in Supplemental Data. Single bnr1Δ or bni1Δ mutants exhibited correct Kar9p polarization, whereas combined inactivation of both formins increased Kar9p symmetry. (B) Representative images of cells from the experiment shown in A after a 30-min incubation at 34°C. Overlays show CFP-Tub1p (in magenta) and Kar9p-GFP3 (in green). Bar, 2 μm. Arrowheads point to cells with disrupted Kar9p polarity. (C) Distribution of Kar9p modes of localization in logarithmic cultures of MYO2 or myo2-16 cells grown at 23°C (nMYO2 = 152; nmyo2-16 = 99) or after 1-h shift to 34°C (nMYO2 = 184; nmyo2-16 = 275). Only budded cells were scored. After a temperature shift, the myo2-16 mutant exhibited a marked disruption of Kar9p polarized distribution.', 'hash': '18958aa2bedceb2fdd0b18c3079629486a5f2789053a5d6a203b55109f871aa3'}, {'image_id': 'zmk0151095280003', 'image_file_name': 'zmk0151095280003.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280003.jpg', 'caption': 'Effect of actin depolymerization by LatB on Kar9p-GFP3 localization. (A) After arrest in G1 by α factor, wild-type cells were released and allowed to proceed past bud emergence. At the point indicated by the arrow, the culture was divided for treatment with either 100 μM LatB or dimethyl sulfoxide (DMSO) as a control, and aliquots were drawn for scoring Kar9p polarity. Samples also were taken during the time course to monitor budding and progression of the spindle pathway (n = 300 cells). (B) Aliquots from the time course were analyzed for Kar9p distribution in short spindles after treatment for 15 min (nLatB = 268 cells; nDMSO = 204 cells) and 30 min (nLatB = 262 cells; nDMSO = 191 cells). More than 90% of cells exhibited Kar9p label at both poles after 30-min treatment with LatB. (C) Representative fields of cells treated with DMSO or LatB for 30 min from the experiment depicted in A. Treatment with LatB induced both spindle misalignment and symmetric localization of Kar9p (arrowheads). Bar, 2 μm.', 'hash': '10f9d852365b031ace116cfa649f79aea65d5cb1a650d76b03c87c4cecf7c7ad'}, {'image_id': 'zmk0151095280002', 'image_file_name': 'zmk0151095280002.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280002.jpg', 'caption': 'Kar9p-GFP3 symmetry is specifically caused by disruption of cell polarity irrespective of spindle orientation. (A) Distribution of cells according to spindle alignment and Kar9p-GFP3 (top) or Bfa1p-GFP (bottom) polarization in wild-type versus bud6Δ bni1Δ cells. The frequency of preanaphase spindle misalignment was 10% in wild-type cells (n = 400) and 52% in bud6Δ bni1Δ cells (n = 530). The frequency of misaligned anaphase spindles in bud6Δ bni1Δ cells was 16% (n = 275). No misaligned anaphase spindles were observed in wild-type cells (n = 270). Bfa1p-GFP symmetry strongly correlated with spindle mispositioning. By contrast, Kar9p-GFP3 localized symmetrically to a similar extent among both aligned and misaligned spindles. (B) Representative images for localization of Bfa1p-GFP in wild-type versus bud6Δ bni1Δ cells. Overlays of fluorescence images of Bfa1p-GFP (in green) and CFP-Tub1p (in magenta) are shown. Arrows point to the SPBbud. Arrowheads in images corresponding to bud6Δ bni1Δ cells point to symmetric label in misaligned short (top) and elongated (bottom) spindles. (C) Distribution of num1Δ cells according to spindle alignment and Kar9p-GFP3 (top) or Bfa1p-GFP (bottom) polarization. The frequency of spindle misalignment in the num1Δ mutant was 15% in preanaphase cells (n = 285) and 27% in anaphase cells (n = 151). num1Δ cells were not disrupted for Kar9p polarity irrespective of spindle alignment. By contrast, Bfa1p-GFP was symmetrically localized in misaligned spindles. (D) Representative images for localization of Kar9p-GFP3 or Bfa1p-GFP (shown in green) relative to CFP-Tub1p (shown in magenta) in num1Δ cells with misaligned spindles. An arrow points to Kar9p-GFP3 polarized label. Arrowheads point to Bfa1p-GFP symmetric label. Bar, 2 μm.', 'hash': '8f04202ddc62400629932590423c01562dbc7cd9ebcf7783acd8917bcc664c91'}, {'image_id': 'zmk0151095280005', 'image_file_name': 'zmk0151095280005.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280005.jpg', 'caption': 'Kar9p repolarization upon recovery from nocodazole treatment highlights the importance of intact MTs for Kar9p localization to the SPBbud. (A and B) Time course for repolarization of Kar9p versus Bfa1p after nocodazole washout in wild-type cells. Asynchronous wild type cells expressing Spc42p-CFP and either Kar9p-GFP3 or Bfa1p-GFP were incubated in 15 μg/ml nocodazole for 45 min. Cells were washed and then resuspended in fresh medium to resume cell cycle progression in the absence of the drug. Localization of Kar9p-GFP3 or Bfa1p-GFP (A) and formation of spindles (B) were scored at the indicated time points. Kar9p polarity was restored by 30 min. By contrast, Bfa1p presence on both spindle poles persisted throughout the time course. (C and D) Intact MTs are required for correct Kar9p polarization to the SPBbud irrespective of the spindle assembly checkpoint. Depolymerization of MTs in a mad2Δ mutant also led to Kar9p-GFP3 symmetry (C). Time course experiment for wild-type or mad2Δ cells expressing Kar9p-GFP3 and Spc42p-CFP as in A except that cells were incubated with nocodazole for only 30 min. Kar9p polarity (C) and formation of spindles in wild-type and mad2Δ cells (D) were scored at the indicated time points. (E) Kar9p-GFP3 recruitment increased after nocodazole washout. Wild-type cells were treated as described in A. Plot depicts Kar9p label intensity per cell along a time course for recovery after nocodazole washout. For reference, the accumulation of cells labeled at a single pole is also shown. Integrated intensity was measured in >50 cells by using digital images, and mean values are plotted. Error bars, SEM.', 'hash': '65a5160e2fd9210ea99f512ca40b65a814d040463491d9933d6cd4f82a5990e4'}]
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{'zmk0151095280001': ['As expected, Kar9p-GFP3 labeled both SPBs of wild-type cells in the early stages of spindle assembly (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, a and b, arrow and arrowhead), to then become polarized to the SPBA, a and b, arrow and arrowhead), to then become polarized to the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ac, arrows). Polarization favoring the SPBAc, arrows). Polarization favoring the SPBbud was maintained during anaphase (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ad, arrow). This trend was also apparent in Ad, arrow). This trend was also apparent in bni1Δ or bud6Δ single mutants, with a modest effect on <1-μm-long spindles observed in bud6Δ cells. By contrast, bud6Δ bni1Δ cells exhibited a marked loss of Kar9p-GFP3 polarization (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, e–m). As the spindle assembled, Kar9p-GFPA, e–m). As the spindle assembled, Kar9p-GFP3 remained associated with both SPBs in nearly 80% of cells containing preanaphase spindles (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>C). Excess symmetry in C). Excess symmetry in bud6Δ bni1Δ cells was not accompanied by bulk changes in Kar9p phosphorylation (data not shown). Remarkably, the ability to polarize Kar9p-GFP3 was entirely uncoupled from spindle alignment or SPBbud identity. Indeed, in addition to cells with misaligned spindles labeled at both poles (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, e and i), cells also exhibited correctly positioned spindles in which Kar9p-GFPA, e and i), cells also exhibited correctly positioned spindles in which Kar9p-GFP3 was nevertheless symmetric (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Af, arrow and arrowhead), asymmetric to favor the SPBAf, arrow and arrowhead), asymmetric to favor the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ag, arrow and arrowhead), asymmetric favoring the mother-bound pole or SPBAg, arrow and arrowhead), asymmetric favoring the mother-bound pole or SPBmother (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ah, arrowhead), or strongly polarized to the SPBAh, arrowhead), or strongly polarized to the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, j and k, arrows). Lack of correlation between spindle alignment and Kar9p-GFPA, j and k, arrows). Lack of correlation between spindle alignment and Kar9p-GFP3 symmetry persisted whether spindle elongation took place across the bud neck (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Al) or not (Al) or not (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Am). More than 90% of preanaphase wild-type cells with aligned spindles polarized Kar9p to the SPBAm). More than 90% of preanaphase wild-type cells with aligned spindles polarized Kar9p to the SPBbud (i.e., one pole labeled or strongly asymmetric), whereas this correlation was lost in bud6Δ bni1Δ cells (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>D). In the small proportion of preanaphase spindles that were misaligned in wild-type cells (10.7%), most favored one pole, indicating that misalignment per se did not increase symmetric recruitment of Kar9p (D). In the small proportion of preanaphase spindles that were misaligned in wild-type cells (10.7%), most favored one pole, indicating that misalignment per se did not increase symmetric recruitment of Kar9p (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>A). Similarly, misaligned preanaphase spindles in A). Similarly, misaligned preanaphase spindles in bud6Δ bni1Δ cells (51.3%) were not enriched for symmetry, emphasizing the lack of correlation between Kar9p polarization and spindle alignment in the mutant (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>A). This indicated that the disruption of cell polarity in the A). This indicated that the disruption of cell polarity in the bud6Δ bni1Δ mutant (Delgehyr et al., 2008) prevented an instructive mechanism that enforces progressive recruitment of Kar9p onto the SPBbud, in addition to compromising Kar9p function in delivery of aMT plus ends due to perturbation of actin integrity.', 'Two formin-dependent axes of cell polarity outline the organization of actin cables in yeast—one set from the bud tip (Bni1p dependent) and the other set from the bud neck (Bnr1p dependent). A bni1Δ mutant supported correct Kar9p polarization to the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>C), even though cell polarity and actin organization within the bud are severely compromised. Similarly, a C), even though cell polarity and actin organization within the bud are severely compromised. Similarly, a bnr1Δ mutant exhibited correct Kar9p polarization (<xref ref-type="fig" rid="zmk0151095280004">Figure 4</xref>, A and B) in spite of the reduction in actin cables in the mother cell (, A and B) in spite of the reduction in actin cables in the mother cell (Pruyne et al., 2004a). Yet, a bnr1Δ bni1ts mutant exhibited a marked increase in Kar9p symmetry upon shift of synchronized budded cells to 34°C (<xref ref-type="fig" rid="zmk0151095280004">Figure 4</xref>, A and B, arrowheads)., A and B, arrowheads).'], 'zmk0151095280002': ['Following these results, wild-type and bud6Δ bni1Δ cells were examined for their ability to support correct polarization of Bfa1p, a regulator of the MEN that favors the SPBbud (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009). In contrast to Kar9p, nearly 90% of preanaphase cells that contained aligned spindles showed a strong bias for Bfa1p-green fluorescent protein (GFP) at the SPBbud (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>A) in both wild-type and A) in both wild-type and bud6Δ bni1Δ cells. Moreover, in 90% of anaphase cells containing elongated spindles across the bud neck, Bfa1p-GFP strongly favored the SPBbud, in sharp contrast to Kar9p-GFP3 symmetric localization in the mutant at the same stage. Yet, consistent with the importance of asymmetric aMT–cortex interactions in enforcing localization of Bfa1p to the SPBbud in response to alignment (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009), 50% of bud6Δ bni1Δ cells containing misaligned preanaphase spindles, typically away from the bud neck, showed symmetric localization of Bfa1p-GFP (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>, A and B, arrowheads). Misaligned anaphase spindles of , A and B, arrowheads). Misaligned anaphase spindles of bud6Δ bni1Δ cells (16% of all anaphases in the mutant) were also markedly enriched for Bfa1p symmetry. In conclusion, bud6Δ bni1Δ cells were impaired in polarizing Kar9p to the SPBbud (even past anaphase), whereas fully proficient to regulate the recruitment of Bfa1p-GFP upon spindle alignment. Finally, the fact that Kar9p-GFP3 symmetry did not relate to spindle misalignment but specifically to the bud6Δ bni1Δ mutations was further confirmed by the contrasting behavior of Kar9p versus Bfa1p in a num1Δ mutant (in which spindle orientation is impaired by the inactivation of the cortical anchor for dynein). Indeed, a num1Δ mutation did not affect Kar9p polarity irrespective of spindle alignment (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>, C and D, arrow). By contrast, misaligned spindles in the same mutant were symmetrically decorated by Bfa1p-GFP (, C and D, arrow). By contrast, misaligned spindles in the same mutant were symmetrically decorated by Bfa1p-GFP (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>, C and D, arrowhead). In conclusion, Kar9p and Bfa1p polarized localization in response to cell polarity may arise by distinct mechanisms., C and D, arrowhead). In conclusion, Kar9p and Bfa1p polarized localization in response to cell polarity may arise by distinct mechanisms.'], 'zmk0151095280003': ['The impact of bud6Δ and bni1Δ mutations on Kar9p distribution suggested the importance of actin organization for polarizing Kar9p. This was directly probed by treating otherwise wild-type cells with the actin-depolymerizing drug latrunculin (Lat). A synchronous cell population was allowed to proceed past bud emergence and was then treated with 100 μM LatB (<xref ref-type="fig" rid="zmk0151095280003">Figure 3</xref>A, arrow) to selectively induce actin cable depolymerization (A, arrow) to selectively induce actin cable depolymerization (Irazoqui et al., 2005). More than 90% of budded cells containing a short spindle exhibited Kar9p-GFP3 recruitment to both poles within 30 min (<xref ref-type="fig" rid="zmk0151095280003">Figure 3</xref>, B and C, arrowheads). The effect of LatB was reversible (see below); yet, it randomized the pattern of SPB inheritance (data not shown). Treatment with 200 μM LatA, additionally disrupting actin patches, also increased Kar9p symmetry (Supplemental Figure S2A)., B and C, arrowheads). The effect of LatB was reversible (see below); yet, it randomized the pattern of SPB inheritance (data not shown). Treatment with 200 μM LatA, additionally disrupting actin patches, also increased Kar9p symmetry (Supplemental Figure S2A).'], 'zmk0151095280004': ['To further assign elements participating in a mechanism by which Kar9p may single out the SPBbud dependent upon actin cables, the possible roles of the yeast type V myosins Myo2p and the nonessential Myo4p/She1p (Pruyne et al., 2004b) were explored. A shift to 34°C of an asynchronous culture of a myo2-16 mutant, which carries a conditional-lethal allele disrupting the function of the Myo2p cargo domain without directly perturbing actin cable organization (Schott et al., 1999), led to loss of Kar9p polarity (<xref ref-type="fig" rid="zmk0151095280004">Figure 4</xref>C). By contrast, a C). By contrast, a myo4Δ strain was unaffected (data not shown). Thus, Kar9p polarization required intact actin cables and specifically Myo2p-dependent transport.'], 'zmk0151095280005': ['We therefore asked how symmetry breaking as the spindle reforms from equivalent SPBs relates to Kar9p polarity. To this end, Kar9p localization was monitored (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>A) alongside reformation of the mitotic spindle (A) alongside reformation of the mitotic spindle (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>B), during recovery from nocodazole treatment. Wild-type cells expressing Spc42p-CFP (a marker for the SPB) and either Kar9p-GFPB), during recovery from nocodazole treatment. Wild-type cells expressing Spc42p-CFP (a marker for the SPB) and either Kar9p-GFP3 or Bfa1p-GFP were released from a treatment with 15 μg/ml nocodazole to follow their recovery. Cells in which SPBs had separated were scored for the extent of polarized localization (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>A). Kar9p initially localized to both SPBs and became polarized within 30 min from the nocodazole washout. By contrast, polarization of Bfa1p, which depends on both correct polarized aMT–cortex interactions and spindle alignment, proceeded more slowly.A). Kar9p initially localized to both SPBs and became polarized within 30 min from the nocodazole washout. By contrast, polarization of Bfa1p, which depends on both correct polarized aMT–cortex interactions and spindle alignment, proceeded more slowly.', 'Nocodazole treatment could evoke the spindle assembly checkpoint (Musacchio and Salmon, 2007). However, a mad2Δ strain also exhibited symmetric Kar9p localization upon nocodazole treatment (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>, C and D), indicating that loss of Kar9p polarity resulted from disrupting MTs without involving this checkpoint., C and D), indicating that loss of Kar9p polarity resulted from disrupting MTs without involving this checkpoint.', 'Kar9p label intensity was relatively low in the presence of nocodazole, ∼15% relative to the maximal label present in asynchronous cells (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>E). After nocodazole washout, the intensity of the label per cell increased, pointing to a contribution of aMTs to Kar9p loading. Kar9p repolarization followed, showing that polarized label stemmed from a net increase in the amount of Kar9p associated with one SPB. The extent of this differential increase could not be measured with statistical significance due to the variability in the label among cells for individual SPBs and associated aMTs in the later part of the time course.E). After nocodazole washout, the intensity of the label per cell increased, pointing to a contribution of aMTs to Kar9p loading. Kar9p repolarization followed, showing that polarized label stemmed from a net increase in the amount of Kar9p associated with one SPB. The extent of this differential increase could not be measured with statistical significance due to the variability in the label among cells for individual SPBs and associated aMTs in the later part of the time course.', 'Efficient Kar9p repolarization to the SPBbud requires polarity determinants and actin cables. (A) Time course analysis of Kar9p repolarization after treatment with nocodazole in the indicated strains was carried out as described in <xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>. For simplicity, the “one pole” category is depicted in the plot. For full details on the time course see Supplemental Figure S3A. Cell polarity mutants were delayed in Kar9p repolarization. (B) LatB prevented Kar9p repolarization after nocodazole washout. Time course analysis in wild-type cells was carried out as described in . For simplicity, the “one pole” category is depicted in the plot. For full details on the time course see Supplemental Figure S3A. Cell polarity mutants were delayed in Kar9p repolarization. (B) LatB prevented Kar9p repolarization after nocodazole washout. Time course analysis in wild-type cells was carried out as described in <xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref> except that after removal of nocodazole, part of the culture was treated with either dimethyl sulfoxide (DMSO) or 100 μM LatB for the indicated times, and aliquots were drawn for scoring (n > 128). In the presence of LatB, symmetry persisted but unequal loading to the SPBs increased over time. (C and D) Time lapse analysis of Kar9p behavior during recovery from nocodazole treatment in a wild-type cell. (C) Unseparated SPBs became repositioned near the bud neck (0–10 min, arrows). At onset of SPB separation (5–10 min) Kar9p preferentially marked one SPB (arrow vs. arrowhead). Bias persisted during alignment (10–20 min). Numbers indicate time elapsed in minutes. Bars, 2 μm. (D) Label intensities for Spc42p-CFP at SPBs (SPB1 is indicated by the arrow in C) and Kar9p-GFP except that after removal of nocodazole, part of the culture was treated with either dimethyl sulfoxide (DMSO) or 100 μM LatB for the indicated times, and aliquots were drawn for scoring (n > 128). In the presence of LatB, symmetry persisted but unequal loading to the SPBs increased over time. (C and D) Time lapse analysis of Kar9p behavior during recovery from nocodazole treatment in a wild-type cell. (C) Unseparated SPBs became repositioned near the bud neck (0–10 min, arrows). At onset of SPB separation (5–10 min) Kar9p preferentially marked one SPB (arrow vs. arrowhead). Bias persisted during alignment (10–20 min). Numbers indicate time elapsed in minutes. Bars, 2 μm. (D) Label intensities for Spc42p-CFP at SPBs (SPB1 is indicated by the arrow in C) and Kar9p-GFP3 associated with each pole during the time lapse depicted in C. When SPB separation began at 5 min, Kar9p labeled SPBs unequally. At 10 min, SPBs are clearly separated and Kar9p label increased at SPB1. Further increase occurred by 20 min. (E) Summary of time lapse analysis for symmetry breaking upon recovery from nocodazole treatment in wild-type versus bud6Δ cells. In wild-type cells, 45.8% cells recorded (n = 24 cells) exhibited Kar9p symmetric label at onset of SPB separation that became polarized within 10 min. In bud6Δ cells, 69.6% (n = 24 cells recorded) exhibited symmetric label that took longer to repolarize. For further representative time lapse sequences see Supplemental Figure S3C.'], 'zmk0151095280006': ['Recovery from nocodazole treatment was then monitored in cell polarity mutants. Relative to wild-type cells, bud6Δ cells exhibited a delay in repolarization of Kar9p but eventually reached a level comparable with that observed in the asynchronous culture (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>A and Supplemental Figure S4A). Failure to break symmetry led to spindle transits into the bud (data not shown). A A and Supplemental Figure S4A). Failure to break symmetry led to spindle transits into the bud (data not shown). A bud6Δ bni1Δ mutant exhibited a further delay relative to a bud6Δ mutant, whereas a bni1Δ mutant initiated recovery like wild-type cells but leveled off to an intermediate value. Finally, symmetry breaking in wild-type cells after nocodazole washout was prevented by LatB (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>B). The percentage of cells exhibiting label at both poles remained constant, yet absolute symmetry progressively decreased, demonstrating that increased Kar9p accumulation in the presence of aMTs permitted further stochastic variation in Kar9p loading among SPBs over time. This may explain why absolute symmetry may not be achievable following a 30-min LatB treatment that renders >90% of cells exhibiting label on both SPBs (B). The percentage of cells exhibiting label at both poles remained constant, yet absolute symmetry progressively decreased, demonstrating that increased Kar9p accumulation in the presence of aMTs permitted further stochastic variation in Kar9p loading among SPBs over time. This may explain why absolute symmetry may not be achievable following a 30-min LatB treatment that renders >90% of cells exhibiting label on both SPBs (<xref ref-type="fig" rid="zmk0151095280003">Figure 3</xref>).).', 'For further validation, symmetry breaking was also evaluated in single cells by time-lapse recordings at relatively low time resolution. This made it possible to use dual-color imaging to score the approximate time elapsed from the separation of SPBs marked symmetrically until Kar9p polarized distribution became established (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>, C–E, and Supplemental Figure S4C). Although it was not technically possible to fully correlate Kar9p behavior and MT–cortex interactions, the general trend was that, during recovery, SPBs of wild-type cells moved toward the bud neck and often before SPB separation, as Kar9p-mediated delivery of aMT plus ends was restored. This was followed by SPB separation in the presence of polarized Kar9p (, C–E, and Supplemental Figure S4C). Although it was not technically possible to fully correlate Kar9p behavior and MT–cortex interactions, the general trend was that, during recovery, SPBs of wild-type cells moved toward the bud neck and often before SPB separation, as Kar9p-mediated delivery of aMT plus ends was restored. This was followed by SPB separation in the presence of polarized Kar9p (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>C, 10 min, arrow and arrowhead; and D) in 54.2% of cells recorded (n = 24 cells). As the spindle became aligned, Kar9p label on the newly designated SPBC, 10 min, arrow and arrowhead; and D) in 54.2% of cells recorded (n = 24 cells). As the spindle became aligned, Kar9p label on the newly designated SPBbud intensified (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>C, 15–20 min, arrows; and D). The observed increase in Kar9p recruitment at the SPBC, 15–20 min, arrows; and D). The observed increase in Kar9p recruitment at the SPBbud validated the results of the population analysis depicted in <xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>E. In cases in which Kar9p was still symmetric when SPB separation took place, Kar9p polarization seemed to coincide with the reorientation of the spindle (Supplemental Figure S4C). In 82% of cells label was repolarized within 10 min from SPB separation (E. In cases in which Kar9p was still symmetric when SPB separation took place, Kar9p polarization seemed to coincide with the reorientation of the spindle (Supplemental Figure S4C). In 82% of cells label was repolarized within 10 min from SPB separation (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>E). By contrast, a E). By contrast, a bud6Δ mutant exhibited a lower frequency of asymmetric label at the time of SPB separation (30.4% of cells recorded; n = 23 cells). Moreover, 62.5% of cells exhibiting symmetric label took longer than 10 min to break symmetry (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>E) with SPB separation often preceding complete repositioning near the bud neck as cells recovered (Supplemental Figure S4C). In either case, symmetry breaking was never observed without initial SPB movement toward the bud (n > 23 cells), suggesting that the initiation of transports was a key factor in rebuilding Kar9p polarity.E) with SPB separation often preceding complete repositioning near the bud neck as cells recovered (Supplemental Figure S4C). In either case, symmetry breaking was never observed without initial SPB movement toward the bud (n > 23 cells), suggesting that the initiation of transports was a key factor in rebuilding Kar9p polarity.', 'The requirement for actin and cell polarity for Kar9p polarization could have suggested that Kar9p itself may be subject to such control. Yet, the phenotype of bud6Δ bni1Δ cells, Kar9p behavior during symmetry breaking and the allele-specific induction of Kar9p symmetry by myo2 mutants are inconsistent with this scenario. Indeed, Kar9p symmetry was not increased in response to misalignment per se. Furthermore, symmetry breaking could take place within the mother cell, even if SPBs were far from the bud neck (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>; data not shown). Thus, Kar9p could become polarized even if both SPBs occupied, and interacted with, the mother compartment, in sharp contrast with the differential dynamic association of Bfa1p to mother and daughter-bound SPBs induced by both asymmetric interactions and/or access of SPBs to their intended compartments (; data not shown). Thus, Kar9p could become polarized even if both SPBs occupied, and interacted with, the mother compartment, in sharp contrast with the differential dynamic association of Bfa1p to mother and daughter-bound SPBs induced by both asymmetric interactions and/or access of SPBs to their intended compartments (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009). Indeed, one axis of polarity outlined by actin cables ending at the bud neck in a bni1Δ mutant proved sufficient to sustain Kar9p polarization despite the impairment in spindle alignment arising from lack of actin cables in the bud (Pruyne et al., 2004b; Delgehyr et al., 2008). Finally, polarization best correlated with Kar9p-mediated transports eliciting SPB movement.'], 'zmk0151095280007': ['The effect of LatB was indeed reversible, because Kar9p-decorated aMTs emanating from each spindle pole began the characteristic angular displacements relative to the polarity-axis reflecting delivery of aMT plus ends toward the bud (Liakopoulos et al., 2003; Huisman et al., 2004). The concomitant SPB movement led to a seesaw-like behavior of the spindle. During alternating transits, the load of Kar9p onto both SPBs persisted (<xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, arrows). Over time, delivery of plus ends of aMTs emanating from one particular SPB gained momentum and polarity became established (A, arrows). Over time, delivery of plus ends of aMTs emanating from one particular SPB gained momentum and polarity became established (<xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, 28–31 min, arrowheads), suggesting that cycles of aMT delivery enforced the localization of Kar9p to a particular SPB (79%; n = 249 aMTs delivered toward the bud). Enhanced label at the SPB was partly contributed by Kar9p returning to the SPB on a depolymerizing aMT after a transport event (e.g., A, 28–31 min, arrowheads), suggesting that cycles of aMT delivery enforced the localization of Kar9p to a particular SPB (79%; n = 249 aMTs delivered toward the bud). Enhanced label at the SPB was partly contributed by Kar9p returning to the SPB on a depolymerizing aMT after a transport event (e.g., <xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, 4–5 min, yellow arrow; 6–7 min, white arrow) in agreement with previous observations at higher temporal resolution (A, 4–5 min, yellow arrow; 6–7 min, white arrow) in agreement with previous observations at higher temporal resolution (Liakopoulos et al., 2003; Huisman et al., 2004; Cuschieri et al., 2006). Even when asymmetric, the residual presence of relatively low amounts of Kar9p at one SPB was sufficient to engage that pole in aMT mediated transports that were followed by an increase in Kar9p loading to the same pole (e.g., <xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, 0–5 min and 14–18 min, yellow arrows). Due to lack of temporal resolution, however, it was not possible to estimate a threshold in Kar9p recruitment below which delivery of aMT plus ends along actin cables would cease. By contrast, Kar9p polarity was quickly restored (<10 min; data not shown) in cells in which symmetrically labeled spindles were already inserted at the bud neck at the start of the time lapse presumably posing a constraint to Kar9p-based movement by the mother-bound pole.A, 0–5 min and 14–18 min, yellow arrows). Due to lack of temporal resolution, however, it was not possible to estimate a threshold in Kar9p recruitment below which delivery of aMT plus ends along actin cables would cease. By contrast, Kar9p polarity was quickly restored (<10 min; data not shown) in cells in which symmetrically labeled spindles were already inserted at the bud neck at the start of the time lapse presumably posing a constraint to Kar9p-based movement by the mother-bound pole.', 'Thus, we asked whether actin cables may promote, at least in part, Kar9p polarization by supporting a bias in Kar9p recruitment to the SPB engaged in Myo2p-dependent delivery of aMT plus ends. Among a collection of temperature-sensitive alleles of MYO2 that disrupt cargo domain functions in vesicular trafficking and polarized growth (Schott et al., 1999), myo2-18 also disrupted the corresponding interaction between Kar9p and the myosin tail, whereas myo2-17 did not (Yin et al., 2000). In support of the importance of Myo2p for a mechanism that promotes asymmetric loading in response to delivery of Kar9p-decorated aMTs along actin cables, myo2-18 led to a marked decrease in cells labeled at a single SPB relative to myo2-17 after a shift to a semipermissive temperature (<xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>B). By contrast, the same treatment disrupted Sec4p-GFP localization in both mutants (B). By contrast, the same treatment disrupted Sec4p-GFP localization in both mutants (myo2-18, 66.5% ± 4.9; myo2-17 65.5% ± 0.7; MYO2, 13%). Thus, the role of Myo2p in promoting Kar9p polarity was directly linked to the ability of Kar9p to act as cargo in Myo2p-based transport.'], 'zmk0151095280008': ['We therefore propose that the impact of actin integrity on Kar9p polarity is a direct consequence of the requirement of Myo2p-dependent transport for inducing cycles of delivery of aMT plus ends that stimulate loading of Kar9p to the pole engaged, thus generating a feedback loop that can sustain further delivery of aMT plus ends from this pole (<xref ref-type="fig" rid="zmk0151095280008">Figure 8</xref>A). Accordingly, A). Accordingly, bud6Δ or bud6Δ bni1Δ cells would be delayed in symmetry breaking to the extent in which delivery of aMT plus ends is compromised by the perturbation in actin cable organization (Amberg et al., 1997; Pruyne et al., 2004a; Delgehyr et al., 2008). Kar9p can be recruited at the SPB from a cytoplasmic pool, and it may not be readily exchangeable, because recovery after photobleaching at the SPB under otherwise unperturbed conditions may exceed 4 min (Liakopoulos et al., 2003). In addition, once reaching aMT plus ends, Kar9p can return to the SPB along shortening aMTs (Liakopoulos et al., 2003; Huisman et al., 2004; Cuschieri et al., 2006). This recycling on aMTs after disengagement from Myo2p may contribute toward the accumulation of Kar9p upon cycles of delivery of aMT plus ends, as suggested by our time-lapse analysis. Consistent with this notion, the abnormally persistent interaction between Kar9p and Myo2p provoked by a tub4-Δdsyl mutation results in both excessive aMT plus end dwelling at the bud cell cortex and depletion of Kar9p label at the SPB (Cuschieri et al., 2006). Yet, it was not technically possible to assess the respective contributions of both pools of Kar9p (recruitment vs. returning to the SPB) to this recycling by photobleaching experiments and dual-color imaging, in the context of dynamic delivery of aMT plus ends.', 'Kar9p begins to single out the SPBbud coincident with the establishment of spindle polarity, thus enforcing delivery of aMT plus ends from this pole to the bud after SPB separation (Huisman et al., 2004; Maekawa and Schiebel, 2004). Early in the cell cycle, Kar9p may associate with both side-by-side SPBs already tethered to the growing bud in response to cortical cues. However, stimulation of recruitment through feedback can only engage the old SPB through its existing aMTs (<xref ref-type="fig" rid="zmk0151095280008">Figure 8</xref>B). Cycles of delivery of aMT plus ends would continue over time, encouraged by this feedback. After SPB separation, two mechanisms conspire to confine the new SPB to the mother cell. First, aMTs now formed by the new SPB are restricted from access to the bud (B). Cycles of delivery of aMT plus ends would continue over time, encouraged by this feedback. After SPB separation, two mechanisms conspire to confine the new SPB to the mother cell. First, aMTs now formed by the new SPB are restricted from access to the bud (Segal et al., 2000a; Delgehyr et al., 2008). Second, Kar9p recruitment to this pole would fade, because a feedback loop cannot be established, because the history of the two SPBs would have built a pronounced bias in favor of the old SPB, now the SPBbud. Treatment with either nocodazole or LatB renders the SPBs equal to recruit Kar9p. Yet, upon recovery from either drug treatment, cell polarity and actin cables can promote symmetry breaking and repolarization of Kar9p to mark one SPB to become the SPBbud.']}
|
Actin-mediated Delivery of Astral Microtubules Instructs Kar9p Asymmetric Loading to the Bud-Ward Spindle Pole
| null |
Mol Biol Cell
|
1280646000
|
Functional analysis of cytoplasmic dynein in Caenorhabditis elegans has revealed a wide range of cellular functions for this minus-end-directed motor protein. Dynein transports a variety of cargos to diverse cellular locations, and thus cargo selection and destination are likely regulated by accessory proteins. The microtubule-associated proteins LIS-1 and dynein interact, but the nature of this interaction remains poorly understood. Here we show that both LIS-1 and the dynein heavy-chain DHC-1 are required for integrity of the actin cytoskeleton in C. elegans. Although both dhc-1(or195ts) and lis-1 loss-of-function disrupt the actin cytoskeleton and produce embryonic lethality, a double mutant suppresses these defects. A targeted RNA interference screen revealed that knockdown of other actin regulators, including actin-capping protein genes and prefoldin subunit genes, suppresses dhc-1(or195ts)-induced lethality. We propose that release or relocation of the mutant dynein complex mediates this suppression of dhc-1(or195ts)-induced phenotypes. These results reveal an unexpected direct or indirect interaction between the actin cytoskeleton and dynein activity.
|
[
"Actins",
"Alleles",
"Animals",
"Caenorhabditis elegans",
"Caenorhabditis elegans Proteins",
"Cytoplasmic Dyneins",
"Cytoskeleton",
"Depsipeptides",
"Dyneins",
"Embryo, Nonmammalian",
"Gene Knockdown Techniques",
"Genes, Helminth",
"Genes, Suppressor",
"Gonads",
"Microtubule-Associated Proteins",
"Microtubules",
"Mutation",
"Pachytene Stage",
"Protein Transport",
"RNA Interference",
"Suppression, Genetic"
] |
other
|
PMC2912354
| null |
35
|
[
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Mol Biol Cell. 2010 Aug 1; 21(15):2685-2695
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Kar9p-GFP3 symmetry is specifically caused by disruption of cell polarity irrespective of spindle orientation. (A) Distribution of cells according to spindle alignment and Kar9p-GFP3 (top) or Bfa1p-GFP (bottom) polarization in wild-type versus bud6Δ bni1Δ cells. The frequency of preanaphase spindle misalignment was 10% in wild-type cells (n = 400) and 52% in bud6Δ bni1Δ cells (n = 530). The frequency of misaligned anaphase spindles in bud6Δ bni1Δ cells was 16% (n = 275). No misaligned anaphase spindles were observed in wild-type cells (n = 270). Bfa1p-GFP symmetry strongly correlated with spindle mispositioning. By contrast, Kar9p-GFP3 localized symmetrically to a similar extent among both aligned and misaligned spindles. (B) Representative images for localization of Bfa1p-GFP in wild-type versus bud6Δ bni1Δ cells. Overlays of fluorescence images of Bfa1p-GFP (in green) and CFP-Tub1p (in magenta) are shown. Arrows point to the SPBbud. Arrowheads in images corresponding to bud6Δ bni1Δ cells point to symmetric label in misaligned short (top) and elongated (bottom) spindles. (C) Distribution of num1Δ cells according to spindle alignment and Kar9p-GFP3 (top) or Bfa1p-GFP (bottom) polarization. The frequency of spindle misalignment in the num1Δ mutant was 15% in preanaphase cells (n = 285) and 27% in anaphase cells (n = 151). num1Δ cells were not disrupted for Kar9p polarity irrespective of spindle alignment. By contrast, Bfa1p-GFP was symmetrically localized in misaligned spindles. (D) Representative images for localization of Kar9p-GFP3 or Bfa1p-GFP (shown in green) relative to CFP-Tub1p (shown in magenta) in num1Δ cells with misaligned spindles. An arrow points to Kar9p-GFP3 polarized label. Arrowheads point to Bfa1p-GFP symmetric label. Bar, 2 μm.
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multiple panels: images & plots
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[
"Microscopy",
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"Immuno Assays"
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[{'image_id': 'zmk0151095280006', 'image_file_name': 'zmk0151095280006.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280006.jpg', 'caption': 'Efficient Kar9p repolarization to the SPBbud requires polarity determinants and actin cables. (A) Time course analysis of Kar9p repolarization after treatment with nocodazole in the indicated strains was carried out as described in Figure 5. For simplicity, the “one pole” category is depicted in the plot. For full details on the time course see Supplemental Figure S3A. Cell polarity mutants were delayed in Kar9p repolarization. (B) LatB prevented Kar9p repolarization after nocodazole washout. Time course analysis in wild-type cells was carried out as described in Figure 5 except that after removal of nocodazole, part of the culture was treated with either dimethyl sulfoxide (DMSO) or 100 μM LatB for the indicated times, and aliquots were drawn for scoring (n > 128). In the presence of LatB, symmetry persisted but unequal loading to the SPBs increased over time. (C and D) Time lapse analysis of Kar9p behavior during recovery from nocodazole treatment in a wild-type cell. (C) Unseparated SPBs became repositioned near the bud neck (0–10 min, arrows). At onset of SPB separation (5–10 min) Kar9p preferentially marked one SPB (arrow vs. arrowhead). Bias persisted during alignment (10–20 min). Numbers indicate time elapsed in minutes. Bars, 2 μm. (D) Label intensities for Spc42p-CFP at SPBs (SPB1 is indicated by the arrow in C) and Kar9p-GFP3 associated with each pole during the time lapse depicted in C. When SPB separation began at 5 min, Kar9p labeled SPBs unequally. At 10 min, SPBs are clearly separated and Kar9p label increased at SPB1. Further increase occurred by 20 min. (E) Summary of time lapse analysis for symmetry breaking upon recovery from nocodazole treatment in wild-type versus bud6Δ cells. In wild-type cells, 45.8% cells recorded (n = 24 cells) exhibited Kar9p symmetric label at onset of SPB separation that became polarized within 10 min. In bud6Δ cells, 69.6% (n = 24 cells recorded) exhibited symmetric label that took longer to repolarize. For further representative time lapse sequences see Supplemental Figure S3C.', 'hash': '79855d8002981f2f828d46122d6e0aea1e0e3005236bce90c8c9360d0fc5ed37'}, {'image_id': 'zmk0151095280001', 'image_file_name': 'zmk0151095280001.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280001.jpg', 'caption': 'Kar9p polarization to the SPBbud is disrupted in a bud6Δ bni1Δ mutant. (A) Representative images of wild-type (a–d) and bud6Δ bni1Δ cells (e–m) expressing Kar9p-GFP3 (in green) and CFP-Tub1p (in magenta), showing the extent of polarization of Kar9p-GFP3 along the spindle pathway. Wild-type cells exhibited Kar9p-GFP3 label on both poles at onset of spindle assembly (a and b, arrows and arrowheads). Label became polarized to the SPBbud in preanaphase spindles (c, arrows) and continued to favor the SPBbud in anaphase (d, arrow). bud6Δ bni1Δ cells did not coordinate polarization of Kar9p-GFP3 with spindle alignment. (e) Two cells with misaligned spindles marked at both poles by Kar9-GFP3. (f) A cell with a short spindle carrying symmetric Kar9-GFP3 label (arrow and arrowhead) despite spindle alignment. (g) A cell with a correctly aligned spindle and Kar9p-GFP3 label favoring the SPBbud (arrow). (h) Kar9p-GFP3 preferentially marks the SPBmother (arrowhead) despite spindle alignment. (i) Misaligned spindle symmetrically labeled by Kar9-GFP3. (j and k) Kar9p-GFP3 label is associated mainly with the SPBbud (arrows). (l) Two cells with elongated spindle exhibit Kar9p-GFP3 label associated with both poles despite alignment. (m) A cell containing a misaligned anaphase spindle with Kar9p-GFP3 present at both poles. In conclusion, loss of polarity persisted throughout anaphase and seemed unrelated to alignment or SPB identity. (B) Categories of Kar9p localization used in this study: one pole (a), asymmetric (b), partially symmetric (c), and symmetric (d). (C) Distribution of asynchronous cells according to their labeling by Kar9p-GFP3 as depicted in B. Cells with <1-μm-long spindles (n > 282 cells); 1- to 2.5-μm-long spindles (n > 290 cells) or elongated spindles (n > 270 cells) in asynchronous cultures were scored. (D) Distribution of preanaphase cells with correctly aligned spindles according to the relative bias of Kar9p-GFP3 toward the SPBbud or SPBmother, n > 390. The frequency of preanaphase spindle misalignment was 10.7% in wild-type cells, 25% in bud6Δ cells, 40% in bni1Δ cells, and 51.3% in bud6Δ bni1Δ cells. Only in bud6Δ bni1Δ cells Kar9p-GFP3 no longer favored the SPBbud. For a detailed breakdown of all categories scored, see Supplemental Figure S1. Bars, 2 μm.', 'hash': 'b5a15bb236f650914daaf8c43ed93d0c1fd9255504b6ed07677ee2dba17dea13'}, {'image_id': 'zmk0151095280008', 'image_file_name': 'zmk0151095280008.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280008.jpg', 'caption': 'A model for Kar9p polarization driven by actin cables, Myo2p, and aMTs. (A) Nocodazole treatment erases differential SPB history and allows Kar9p symmetric recruitment to a basal level. After removal of the drug, Kar9p recruitment increases after regrowth of aMTs. aMTs from one SPB may stochastically engage in Kar9p-mediated deliveries. These interactions can rapidly evoke a feedback loop that helps symmetry breaking, before (solid arrow) or after (dashed arrows) SPB separation. In a bud6Δ mutant, the reduction in actin cables may lead to a delay in SPB repositioning and symmetry breaking. (B) In an unperturbed wild-type cell, spindle polarity is primed by aMTs from the old SPB tethering the side-by-side SPBs to the incipient bud. The old SPB also engages in Kar9p-mediated transport through its existing aMTs. The new SPB can also recruit Kar9p to a basal level but lacks aMTs. A bias for Kar9p recruitment to the old SPB can become established before aMTs form at the new SPB. This bias prevails after SPB separation leading to the loss of Kar9p from the new SPB.', 'hash': 'e84f722581755afb43811f474477269f4aae9fbec828942d6727585b8cf51391'}, {'image_id': 'zmk0151095280007', 'image_file_name': 'zmk0151095280007.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280007.jpg', 'caption': 'Kar9p accumulation bias may be enforced by sustained cycles of aMT delivery along actin cables. (A) Kar9p behavior after recovery from treatment with LatB. Wild-type cells were synchronized and released from G1 to allow formation of a bud before treatment with LatB. Overlays of fluorescence images for Kar9p (in green) and Tub1p (in magenta) are shown. Linescans for fluorescence intensity depict Kar9p-GFP3 distribution along an aMT (with the spindle pole close to the origin and the plus end always to the right; distance in micrometers) for the indicated frames. Seesaw-like behavior of the spindle accompanied Kar9p-mediated transport from alternating poles because aMTs decorated by Kar9p displayed the characteristic angular movements. Initially, Kar9p was asymmetric, but a cycle of transport engaging the right pole (yellow arrows; 0–5 min) led to intensified label at that pole as the aMT depolymerized (5 min). Label decreased as the left pole (white arrows) engaged in deliveries (5–7 min). A second cycle from the right pole (14–18 min, yellow arrows) triggered an increase in Kar9p label at this pole. Both poles continued to move in response to alternating transits, although the left pole was favored, restoring the bias. Progressively, label disappeared from the right pole (28–31 min) and persisted on the left pole and further transports began to align the spindle (29–31 min, arrowheads). Numbers indicate time elapsed in minutes. Bars, 2 μm. (B) Inactivation of a myo2ts allele disrupting Kar9p binding to the cargo domain of Myo2p increased Kar9p symmetry. Plot for distribution of cells with short spindles labeled at one pole by Kar9p in asynchronous MYO2, myo2-17 (encoding a cargo domain mutant with defects in polarized secretion, yet able to bind Kar9p) and myo2-18 (encoding a mutant disrupted for Kar9p binding) cultures grown at 23°C or transferred to 34°C for 30 min.', 'hash': '38d1782540576d80f8d666536c3e8a83421cb02e6382497a35911cf9761dc4b0'}, {'image_id': 'zmk0151095280004', 'image_file_name': 'zmk0151095280004.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280004.jpg', 'caption': 'At least one formin and Myo2p are required for correct Kar9p polarity. (A and B) Effect of inactivation of both yeast formins on Kar9p polarized localization in synchronized budded cells. bnr1Δ or bnr1Δ bni1ts cells were released from a G1 block at 23°C, and samples were taken to monitor budding and progression of the spindle pathway (data not shown). (A) At 45 min after release from G1, budded cells were shifted to 34°C, and still images were acquired for scoring Kar9p localization onto short spindles after a 15-min incubation (nbnr1Δ = 249; nbnr1Δbni1ts = 75), 30 min (nbnr1Δ = 281; nbnr1Δbni1ts = 325), and 45 min (nbnr1Δ = 83; nbnr1Δbni1ts = 305). For reference, Kar9p localization in asynchronous cultures is shown (n > 226 cells). The bni1ts allele bni1-FH2#1 (Sagot et al., 2002) was introduced into 15D background as described in Supplemental Data. Single bnr1Δ or bni1Δ mutants exhibited correct Kar9p polarization, whereas combined inactivation of both formins increased Kar9p symmetry. (B) Representative images of cells from the experiment shown in A after a 30-min incubation at 34°C. Overlays show CFP-Tub1p (in magenta) and Kar9p-GFP3 (in green). Bar, 2 μm. Arrowheads point to cells with disrupted Kar9p polarity. (C) Distribution of Kar9p modes of localization in logarithmic cultures of MYO2 or myo2-16 cells grown at 23°C (nMYO2 = 152; nmyo2-16 = 99) or after 1-h shift to 34°C (nMYO2 = 184; nmyo2-16 = 275). Only budded cells were scored. After a temperature shift, the myo2-16 mutant exhibited a marked disruption of Kar9p polarized distribution.', 'hash': '18958aa2bedceb2fdd0b18c3079629486a5f2789053a5d6a203b55109f871aa3'}, {'image_id': 'zmk0151095280003', 'image_file_name': 'zmk0151095280003.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280003.jpg', 'caption': 'Effect of actin depolymerization by LatB on Kar9p-GFP3 localization. (A) After arrest in G1 by α factor, wild-type cells were released and allowed to proceed past bud emergence. At the point indicated by the arrow, the culture was divided for treatment with either 100 μM LatB or dimethyl sulfoxide (DMSO) as a control, and aliquots were drawn for scoring Kar9p polarity. Samples also were taken during the time course to monitor budding and progression of the spindle pathway (n = 300 cells). (B) Aliquots from the time course were analyzed for Kar9p distribution in short spindles after treatment for 15 min (nLatB = 268 cells; nDMSO = 204 cells) and 30 min (nLatB = 262 cells; nDMSO = 191 cells). More than 90% of cells exhibited Kar9p label at both poles after 30-min treatment with LatB. (C) Representative fields of cells treated with DMSO or LatB for 30 min from the experiment depicted in A. Treatment with LatB induced both spindle misalignment and symmetric localization of Kar9p (arrowheads). Bar, 2 μm.', 'hash': '10f9d852365b031ace116cfa649f79aea65d5cb1a650d76b03c87c4cecf7c7ad'}, {'image_id': 'zmk0151095280002', 'image_file_name': 'zmk0151095280002.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280002.jpg', 'caption': 'Kar9p-GFP3 symmetry is specifically caused by disruption of cell polarity irrespective of spindle orientation. (A) Distribution of cells according to spindle alignment and Kar9p-GFP3 (top) or Bfa1p-GFP (bottom) polarization in wild-type versus bud6Δ bni1Δ cells. The frequency of preanaphase spindle misalignment was 10% in wild-type cells (n = 400) and 52% in bud6Δ bni1Δ cells (n = 530). The frequency of misaligned anaphase spindles in bud6Δ bni1Δ cells was 16% (n = 275). No misaligned anaphase spindles were observed in wild-type cells (n = 270). Bfa1p-GFP symmetry strongly correlated with spindle mispositioning. By contrast, Kar9p-GFP3 localized symmetrically to a similar extent among both aligned and misaligned spindles. (B) Representative images for localization of Bfa1p-GFP in wild-type versus bud6Δ bni1Δ cells. Overlays of fluorescence images of Bfa1p-GFP (in green) and CFP-Tub1p (in magenta) are shown. Arrows point to the SPBbud. Arrowheads in images corresponding to bud6Δ bni1Δ cells point to symmetric label in misaligned short (top) and elongated (bottom) spindles. (C) Distribution of num1Δ cells according to spindle alignment and Kar9p-GFP3 (top) or Bfa1p-GFP (bottom) polarization. The frequency of spindle misalignment in the num1Δ mutant was 15% in preanaphase cells (n = 285) and 27% in anaphase cells (n = 151). num1Δ cells were not disrupted for Kar9p polarity irrespective of spindle alignment. By contrast, Bfa1p-GFP was symmetrically localized in misaligned spindles. (D) Representative images for localization of Kar9p-GFP3 or Bfa1p-GFP (shown in green) relative to CFP-Tub1p (shown in magenta) in num1Δ cells with misaligned spindles. An arrow points to Kar9p-GFP3 polarized label. Arrowheads point to Bfa1p-GFP symmetric label. Bar, 2 μm.', 'hash': '8f04202ddc62400629932590423c01562dbc7cd9ebcf7783acd8917bcc664c91'}, {'image_id': 'zmk0151095280005', 'image_file_name': 'zmk0151095280005.jpg', 'image_path': '../data/media_files/PMC2912354/zmk0151095280005.jpg', 'caption': 'Kar9p repolarization upon recovery from nocodazole treatment highlights the importance of intact MTs for Kar9p localization to the SPBbud. (A and B) Time course for repolarization of Kar9p versus Bfa1p after nocodazole washout in wild-type cells. Asynchronous wild type cells expressing Spc42p-CFP and either Kar9p-GFP3 or Bfa1p-GFP were incubated in 15 μg/ml nocodazole for 45 min. Cells were washed and then resuspended in fresh medium to resume cell cycle progression in the absence of the drug. Localization of Kar9p-GFP3 or Bfa1p-GFP (A) and formation of spindles (B) were scored at the indicated time points. Kar9p polarity was restored by 30 min. By contrast, Bfa1p presence on both spindle poles persisted throughout the time course. (C and D) Intact MTs are required for correct Kar9p polarization to the SPBbud irrespective of the spindle assembly checkpoint. Depolymerization of MTs in a mad2Δ mutant also led to Kar9p-GFP3 symmetry (C). Time course experiment for wild-type or mad2Δ cells expressing Kar9p-GFP3 and Spc42p-CFP as in A except that cells were incubated with nocodazole for only 30 min. Kar9p polarity (C) and formation of spindles in wild-type and mad2Δ cells (D) were scored at the indicated time points. (E) Kar9p-GFP3 recruitment increased after nocodazole washout. Wild-type cells were treated as described in A. Plot depicts Kar9p label intensity per cell along a time course for recovery after nocodazole washout. For reference, the accumulation of cells labeled at a single pole is also shown. Integrated intensity was measured in >50 cells by using digital images, and mean values are plotted. Error bars, SEM.', 'hash': '65a5160e2fd9210ea99f512ca40b65a814d040463491d9933d6cd4f82a5990e4'}]
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{'zmk0151095280001': ['As expected, Kar9p-GFP3 labeled both SPBs of wild-type cells in the early stages of spindle assembly (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, a and b, arrow and arrowhead), to then become polarized to the SPBA, a and b, arrow and arrowhead), to then become polarized to the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ac, arrows). Polarization favoring the SPBAc, arrows). Polarization favoring the SPBbud was maintained during anaphase (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ad, arrow). This trend was also apparent in Ad, arrow). This trend was also apparent in bni1Δ or bud6Δ single mutants, with a modest effect on <1-μm-long spindles observed in bud6Δ cells. By contrast, bud6Δ bni1Δ cells exhibited a marked loss of Kar9p-GFP3 polarization (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, e–m). As the spindle assembled, Kar9p-GFPA, e–m). As the spindle assembled, Kar9p-GFP3 remained associated with both SPBs in nearly 80% of cells containing preanaphase spindles (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>C). Excess symmetry in C). Excess symmetry in bud6Δ bni1Δ cells was not accompanied by bulk changes in Kar9p phosphorylation (data not shown). Remarkably, the ability to polarize Kar9p-GFP3 was entirely uncoupled from spindle alignment or SPBbud identity. Indeed, in addition to cells with misaligned spindles labeled at both poles (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, e and i), cells also exhibited correctly positioned spindles in which Kar9p-GFPA, e and i), cells also exhibited correctly positioned spindles in which Kar9p-GFP3 was nevertheless symmetric (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Af, arrow and arrowhead), asymmetric to favor the SPBAf, arrow and arrowhead), asymmetric to favor the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ag, arrow and arrowhead), asymmetric favoring the mother-bound pole or SPBAg, arrow and arrowhead), asymmetric favoring the mother-bound pole or SPBmother (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Ah, arrowhead), or strongly polarized to the SPBAh, arrowhead), or strongly polarized to the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>A, j and k, arrows). Lack of correlation between spindle alignment and Kar9p-GFPA, j and k, arrows). Lack of correlation between spindle alignment and Kar9p-GFP3 symmetry persisted whether spindle elongation took place across the bud neck (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Al) or not (Al) or not (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>Am). More than 90% of preanaphase wild-type cells with aligned spindles polarized Kar9p to the SPBAm). More than 90% of preanaphase wild-type cells with aligned spindles polarized Kar9p to the SPBbud (i.e., one pole labeled or strongly asymmetric), whereas this correlation was lost in bud6Δ bni1Δ cells (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>D). In the small proportion of preanaphase spindles that were misaligned in wild-type cells (10.7%), most favored one pole, indicating that misalignment per se did not increase symmetric recruitment of Kar9p (D). In the small proportion of preanaphase spindles that were misaligned in wild-type cells (10.7%), most favored one pole, indicating that misalignment per se did not increase symmetric recruitment of Kar9p (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>A). Similarly, misaligned preanaphase spindles in A). Similarly, misaligned preanaphase spindles in bud6Δ bni1Δ cells (51.3%) were not enriched for symmetry, emphasizing the lack of correlation between Kar9p polarization and spindle alignment in the mutant (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>A). This indicated that the disruption of cell polarity in the A). This indicated that the disruption of cell polarity in the bud6Δ bni1Δ mutant (Delgehyr et al., 2008) prevented an instructive mechanism that enforces progressive recruitment of Kar9p onto the SPBbud, in addition to compromising Kar9p function in delivery of aMT plus ends due to perturbation of actin integrity.', 'Two formin-dependent axes of cell polarity outline the organization of actin cables in yeast—one set from the bud tip (Bni1p dependent) and the other set from the bud neck (Bnr1p dependent). A bni1Δ mutant supported correct Kar9p polarization to the SPBbud (<xref ref-type="fig" rid="zmk0151095280001">Figure 1</xref>C), even though cell polarity and actin organization within the bud are severely compromised. Similarly, a C), even though cell polarity and actin organization within the bud are severely compromised. Similarly, a bnr1Δ mutant exhibited correct Kar9p polarization (<xref ref-type="fig" rid="zmk0151095280004">Figure 4</xref>, A and B) in spite of the reduction in actin cables in the mother cell (, A and B) in spite of the reduction in actin cables in the mother cell (Pruyne et al., 2004a). Yet, a bnr1Δ bni1ts mutant exhibited a marked increase in Kar9p symmetry upon shift of synchronized budded cells to 34°C (<xref ref-type="fig" rid="zmk0151095280004">Figure 4</xref>, A and B, arrowheads)., A and B, arrowheads).'], 'zmk0151095280002': ['Following these results, wild-type and bud6Δ bni1Δ cells were examined for their ability to support correct polarization of Bfa1p, a regulator of the MEN that favors the SPBbud (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009). In contrast to Kar9p, nearly 90% of preanaphase cells that contained aligned spindles showed a strong bias for Bfa1p-green fluorescent protein (GFP) at the SPBbud (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>A) in both wild-type and A) in both wild-type and bud6Δ bni1Δ cells. Moreover, in 90% of anaphase cells containing elongated spindles across the bud neck, Bfa1p-GFP strongly favored the SPBbud, in sharp contrast to Kar9p-GFP3 symmetric localization in the mutant at the same stage. Yet, consistent with the importance of asymmetric aMT–cortex interactions in enforcing localization of Bfa1p to the SPBbud in response to alignment (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009), 50% of bud6Δ bni1Δ cells containing misaligned preanaphase spindles, typically away from the bud neck, showed symmetric localization of Bfa1p-GFP (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>, A and B, arrowheads). Misaligned anaphase spindles of , A and B, arrowheads). Misaligned anaphase spindles of bud6Δ bni1Δ cells (16% of all anaphases in the mutant) were also markedly enriched for Bfa1p symmetry. In conclusion, bud6Δ bni1Δ cells were impaired in polarizing Kar9p to the SPBbud (even past anaphase), whereas fully proficient to regulate the recruitment of Bfa1p-GFP upon spindle alignment. Finally, the fact that Kar9p-GFP3 symmetry did not relate to spindle misalignment but specifically to the bud6Δ bni1Δ mutations was further confirmed by the contrasting behavior of Kar9p versus Bfa1p in a num1Δ mutant (in which spindle orientation is impaired by the inactivation of the cortical anchor for dynein). Indeed, a num1Δ mutation did not affect Kar9p polarity irrespective of spindle alignment (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>, C and D, arrow). By contrast, misaligned spindles in the same mutant were symmetrically decorated by Bfa1p-GFP (, C and D, arrow). By contrast, misaligned spindles in the same mutant were symmetrically decorated by Bfa1p-GFP (<xref ref-type="fig" rid="zmk0151095280002">Figure 2</xref>, C and D, arrowhead). In conclusion, Kar9p and Bfa1p polarized localization in response to cell polarity may arise by distinct mechanisms., C and D, arrowhead). In conclusion, Kar9p and Bfa1p polarized localization in response to cell polarity may arise by distinct mechanisms.'], 'zmk0151095280003': ['The impact of bud6Δ and bni1Δ mutations on Kar9p distribution suggested the importance of actin organization for polarizing Kar9p. This was directly probed by treating otherwise wild-type cells with the actin-depolymerizing drug latrunculin (Lat). A synchronous cell population was allowed to proceed past bud emergence and was then treated with 100 μM LatB (<xref ref-type="fig" rid="zmk0151095280003">Figure 3</xref>A, arrow) to selectively induce actin cable depolymerization (A, arrow) to selectively induce actin cable depolymerization (Irazoqui et al., 2005). More than 90% of budded cells containing a short spindle exhibited Kar9p-GFP3 recruitment to both poles within 30 min (<xref ref-type="fig" rid="zmk0151095280003">Figure 3</xref>, B and C, arrowheads). The effect of LatB was reversible (see below); yet, it randomized the pattern of SPB inheritance (data not shown). Treatment with 200 μM LatA, additionally disrupting actin patches, also increased Kar9p symmetry (Supplemental Figure S2A)., B and C, arrowheads). The effect of LatB was reversible (see below); yet, it randomized the pattern of SPB inheritance (data not shown). Treatment with 200 μM LatA, additionally disrupting actin patches, also increased Kar9p symmetry (Supplemental Figure S2A).'], 'zmk0151095280004': ['To further assign elements participating in a mechanism by which Kar9p may single out the SPBbud dependent upon actin cables, the possible roles of the yeast type V myosins Myo2p and the nonessential Myo4p/She1p (Pruyne et al., 2004b) were explored. A shift to 34°C of an asynchronous culture of a myo2-16 mutant, which carries a conditional-lethal allele disrupting the function of the Myo2p cargo domain without directly perturbing actin cable organization (Schott et al., 1999), led to loss of Kar9p polarity (<xref ref-type="fig" rid="zmk0151095280004">Figure 4</xref>C). By contrast, a C). By contrast, a myo4Δ strain was unaffected (data not shown). Thus, Kar9p polarization required intact actin cables and specifically Myo2p-dependent transport.'], 'zmk0151095280005': ['We therefore asked how symmetry breaking as the spindle reforms from equivalent SPBs relates to Kar9p polarity. To this end, Kar9p localization was monitored (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>A) alongside reformation of the mitotic spindle (A) alongside reformation of the mitotic spindle (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>B), during recovery from nocodazole treatment. Wild-type cells expressing Spc42p-CFP (a marker for the SPB) and either Kar9p-GFPB), during recovery from nocodazole treatment. Wild-type cells expressing Spc42p-CFP (a marker for the SPB) and either Kar9p-GFP3 or Bfa1p-GFP were released from a treatment with 15 μg/ml nocodazole to follow their recovery. Cells in which SPBs had separated were scored for the extent of polarized localization (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>A). Kar9p initially localized to both SPBs and became polarized within 30 min from the nocodazole washout. By contrast, polarization of Bfa1p, which depends on both correct polarized aMT–cortex interactions and spindle alignment, proceeded more slowly.A). Kar9p initially localized to both SPBs and became polarized within 30 min from the nocodazole washout. By contrast, polarization of Bfa1p, which depends on both correct polarized aMT–cortex interactions and spindle alignment, proceeded more slowly.', 'Nocodazole treatment could evoke the spindle assembly checkpoint (Musacchio and Salmon, 2007). However, a mad2Δ strain also exhibited symmetric Kar9p localization upon nocodazole treatment (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>, C and D), indicating that loss of Kar9p polarity resulted from disrupting MTs without involving this checkpoint., C and D), indicating that loss of Kar9p polarity resulted from disrupting MTs without involving this checkpoint.', 'Kar9p label intensity was relatively low in the presence of nocodazole, ∼15% relative to the maximal label present in asynchronous cells (<xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>E). After nocodazole washout, the intensity of the label per cell increased, pointing to a contribution of aMTs to Kar9p loading. Kar9p repolarization followed, showing that polarized label stemmed from a net increase in the amount of Kar9p associated with one SPB. The extent of this differential increase could not be measured with statistical significance due to the variability in the label among cells for individual SPBs and associated aMTs in the later part of the time course.E). After nocodazole washout, the intensity of the label per cell increased, pointing to a contribution of aMTs to Kar9p loading. Kar9p repolarization followed, showing that polarized label stemmed from a net increase in the amount of Kar9p associated with one SPB. The extent of this differential increase could not be measured with statistical significance due to the variability in the label among cells for individual SPBs and associated aMTs in the later part of the time course.', 'Efficient Kar9p repolarization to the SPBbud requires polarity determinants and actin cables. (A) Time course analysis of Kar9p repolarization after treatment with nocodazole in the indicated strains was carried out as described in <xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>. For simplicity, the “one pole” category is depicted in the plot. For full details on the time course see Supplemental Figure S3A. Cell polarity mutants were delayed in Kar9p repolarization. (B) LatB prevented Kar9p repolarization after nocodazole washout. Time course analysis in wild-type cells was carried out as described in . For simplicity, the “one pole” category is depicted in the plot. For full details on the time course see Supplemental Figure S3A. Cell polarity mutants were delayed in Kar9p repolarization. (B) LatB prevented Kar9p repolarization after nocodazole washout. Time course analysis in wild-type cells was carried out as described in <xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref> except that after removal of nocodazole, part of the culture was treated with either dimethyl sulfoxide (DMSO) or 100 μM LatB for the indicated times, and aliquots were drawn for scoring (n > 128). In the presence of LatB, symmetry persisted but unequal loading to the SPBs increased over time. (C and D) Time lapse analysis of Kar9p behavior during recovery from nocodazole treatment in a wild-type cell. (C) Unseparated SPBs became repositioned near the bud neck (0–10 min, arrows). At onset of SPB separation (5–10 min) Kar9p preferentially marked one SPB (arrow vs. arrowhead). Bias persisted during alignment (10–20 min). Numbers indicate time elapsed in minutes. Bars, 2 μm. (D) Label intensities for Spc42p-CFP at SPBs (SPB1 is indicated by the arrow in C) and Kar9p-GFP except that after removal of nocodazole, part of the culture was treated with either dimethyl sulfoxide (DMSO) or 100 μM LatB for the indicated times, and aliquots were drawn for scoring (n > 128). In the presence of LatB, symmetry persisted but unequal loading to the SPBs increased over time. (C and D) Time lapse analysis of Kar9p behavior during recovery from nocodazole treatment in a wild-type cell. (C) Unseparated SPBs became repositioned near the bud neck (0–10 min, arrows). At onset of SPB separation (5–10 min) Kar9p preferentially marked one SPB (arrow vs. arrowhead). Bias persisted during alignment (10–20 min). Numbers indicate time elapsed in minutes. Bars, 2 μm. (D) Label intensities for Spc42p-CFP at SPBs (SPB1 is indicated by the arrow in C) and Kar9p-GFP3 associated with each pole during the time lapse depicted in C. When SPB separation began at 5 min, Kar9p labeled SPBs unequally. At 10 min, SPBs are clearly separated and Kar9p label increased at SPB1. Further increase occurred by 20 min. (E) Summary of time lapse analysis for symmetry breaking upon recovery from nocodazole treatment in wild-type versus bud6Δ cells. In wild-type cells, 45.8% cells recorded (n = 24 cells) exhibited Kar9p symmetric label at onset of SPB separation that became polarized within 10 min. In bud6Δ cells, 69.6% (n = 24 cells recorded) exhibited symmetric label that took longer to repolarize. For further representative time lapse sequences see Supplemental Figure S3C.'], 'zmk0151095280006': ['Recovery from nocodazole treatment was then monitored in cell polarity mutants. Relative to wild-type cells, bud6Δ cells exhibited a delay in repolarization of Kar9p but eventually reached a level comparable with that observed in the asynchronous culture (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>A and Supplemental Figure S4A). Failure to break symmetry led to spindle transits into the bud (data not shown). A A and Supplemental Figure S4A). Failure to break symmetry led to spindle transits into the bud (data not shown). A bud6Δ bni1Δ mutant exhibited a further delay relative to a bud6Δ mutant, whereas a bni1Δ mutant initiated recovery like wild-type cells but leveled off to an intermediate value. Finally, symmetry breaking in wild-type cells after nocodazole washout was prevented by LatB (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>B). The percentage of cells exhibiting label at both poles remained constant, yet absolute symmetry progressively decreased, demonstrating that increased Kar9p accumulation in the presence of aMTs permitted further stochastic variation in Kar9p loading among SPBs over time. This may explain why absolute symmetry may not be achievable following a 30-min LatB treatment that renders >90% of cells exhibiting label on both SPBs (B). The percentage of cells exhibiting label at both poles remained constant, yet absolute symmetry progressively decreased, demonstrating that increased Kar9p accumulation in the presence of aMTs permitted further stochastic variation in Kar9p loading among SPBs over time. This may explain why absolute symmetry may not be achievable following a 30-min LatB treatment that renders >90% of cells exhibiting label on both SPBs (<xref ref-type="fig" rid="zmk0151095280003">Figure 3</xref>).).', 'For further validation, symmetry breaking was also evaluated in single cells by time-lapse recordings at relatively low time resolution. This made it possible to use dual-color imaging to score the approximate time elapsed from the separation of SPBs marked symmetrically until Kar9p polarized distribution became established (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>, C–E, and Supplemental Figure S4C). Although it was not technically possible to fully correlate Kar9p behavior and MT–cortex interactions, the general trend was that, during recovery, SPBs of wild-type cells moved toward the bud neck and often before SPB separation, as Kar9p-mediated delivery of aMT plus ends was restored. This was followed by SPB separation in the presence of polarized Kar9p (, C–E, and Supplemental Figure S4C). Although it was not technically possible to fully correlate Kar9p behavior and MT–cortex interactions, the general trend was that, during recovery, SPBs of wild-type cells moved toward the bud neck and often before SPB separation, as Kar9p-mediated delivery of aMT plus ends was restored. This was followed by SPB separation in the presence of polarized Kar9p (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>C, 10 min, arrow and arrowhead; and D) in 54.2% of cells recorded (n = 24 cells). As the spindle became aligned, Kar9p label on the newly designated SPBC, 10 min, arrow and arrowhead; and D) in 54.2% of cells recorded (n = 24 cells). As the spindle became aligned, Kar9p label on the newly designated SPBbud intensified (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>C, 15–20 min, arrows; and D). The observed increase in Kar9p recruitment at the SPBC, 15–20 min, arrows; and D). The observed increase in Kar9p recruitment at the SPBbud validated the results of the population analysis depicted in <xref ref-type="fig" rid="zmk0151095280005">Figure 5</xref>E. In cases in which Kar9p was still symmetric when SPB separation took place, Kar9p polarization seemed to coincide with the reorientation of the spindle (Supplemental Figure S4C). In 82% of cells label was repolarized within 10 min from SPB separation (E. In cases in which Kar9p was still symmetric when SPB separation took place, Kar9p polarization seemed to coincide with the reorientation of the spindle (Supplemental Figure S4C). In 82% of cells label was repolarized within 10 min from SPB separation (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>E). By contrast, a E). By contrast, a bud6Δ mutant exhibited a lower frequency of asymmetric label at the time of SPB separation (30.4% of cells recorded; n = 23 cells). Moreover, 62.5% of cells exhibiting symmetric label took longer than 10 min to break symmetry (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>E) with SPB separation often preceding complete repositioning near the bud neck as cells recovered (Supplemental Figure S4C). In either case, symmetry breaking was never observed without initial SPB movement toward the bud (n > 23 cells), suggesting that the initiation of transports was a key factor in rebuilding Kar9p polarity.E) with SPB separation often preceding complete repositioning near the bud neck as cells recovered (Supplemental Figure S4C). In either case, symmetry breaking was never observed without initial SPB movement toward the bud (n > 23 cells), suggesting that the initiation of transports was a key factor in rebuilding Kar9p polarity.', 'The requirement for actin and cell polarity for Kar9p polarization could have suggested that Kar9p itself may be subject to such control. Yet, the phenotype of bud6Δ bni1Δ cells, Kar9p behavior during symmetry breaking and the allele-specific induction of Kar9p symmetry by myo2 mutants are inconsistent with this scenario. Indeed, Kar9p symmetry was not increased in response to misalignment per se. Furthermore, symmetry breaking could take place within the mother cell, even if SPBs were far from the bud neck (<xref ref-type="fig" rid="zmk0151095280006">Figure 6</xref>; data not shown). Thus, Kar9p could become polarized even if both SPBs occupied, and interacted with, the mother compartment, in sharp contrast with the differential dynamic association of Bfa1p to mother and daughter-bound SPBs induced by both asymmetric interactions and/or access of SPBs to their intended compartments (; data not shown). Thus, Kar9p could become polarized even if both SPBs occupied, and interacted with, the mother compartment, in sharp contrast with the differential dynamic association of Bfa1p to mother and daughter-bound SPBs induced by both asymmetric interactions and/or access of SPBs to their intended compartments (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009). Indeed, one axis of polarity outlined by actin cables ending at the bud neck in a bni1Δ mutant proved sufficient to sustain Kar9p polarization despite the impairment in spindle alignment arising from lack of actin cables in the bud (Pruyne et al., 2004b; Delgehyr et al., 2008). Finally, polarization best correlated with Kar9p-mediated transports eliciting SPB movement.'], 'zmk0151095280007': ['The effect of LatB was indeed reversible, because Kar9p-decorated aMTs emanating from each spindle pole began the characteristic angular displacements relative to the polarity-axis reflecting delivery of aMT plus ends toward the bud (Liakopoulos et al., 2003; Huisman et al., 2004). The concomitant SPB movement led to a seesaw-like behavior of the spindle. During alternating transits, the load of Kar9p onto both SPBs persisted (<xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, arrows). Over time, delivery of plus ends of aMTs emanating from one particular SPB gained momentum and polarity became established (A, arrows). Over time, delivery of plus ends of aMTs emanating from one particular SPB gained momentum and polarity became established (<xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, 28–31 min, arrowheads), suggesting that cycles of aMT delivery enforced the localization of Kar9p to a particular SPB (79%; n = 249 aMTs delivered toward the bud). Enhanced label at the SPB was partly contributed by Kar9p returning to the SPB on a depolymerizing aMT after a transport event (e.g., A, 28–31 min, arrowheads), suggesting that cycles of aMT delivery enforced the localization of Kar9p to a particular SPB (79%; n = 249 aMTs delivered toward the bud). Enhanced label at the SPB was partly contributed by Kar9p returning to the SPB on a depolymerizing aMT after a transport event (e.g., <xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, 4–5 min, yellow arrow; 6–7 min, white arrow) in agreement with previous observations at higher temporal resolution (A, 4–5 min, yellow arrow; 6–7 min, white arrow) in agreement with previous observations at higher temporal resolution (Liakopoulos et al., 2003; Huisman et al., 2004; Cuschieri et al., 2006). Even when asymmetric, the residual presence of relatively low amounts of Kar9p at one SPB was sufficient to engage that pole in aMT mediated transports that were followed by an increase in Kar9p loading to the same pole (e.g., <xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>A, 0–5 min and 14–18 min, yellow arrows). Due to lack of temporal resolution, however, it was not possible to estimate a threshold in Kar9p recruitment below which delivery of aMT plus ends along actin cables would cease. By contrast, Kar9p polarity was quickly restored (<10 min; data not shown) in cells in which symmetrically labeled spindles were already inserted at the bud neck at the start of the time lapse presumably posing a constraint to Kar9p-based movement by the mother-bound pole.A, 0–5 min and 14–18 min, yellow arrows). Due to lack of temporal resolution, however, it was not possible to estimate a threshold in Kar9p recruitment below which delivery of aMT plus ends along actin cables would cease. By contrast, Kar9p polarity was quickly restored (<10 min; data not shown) in cells in which symmetrically labeled spindles were already inserted at the bud neck at the start of the time lapse presumably posing a constraint to Kar9p-based movement by the mother-bound pole.', 'Thus, we asked whether actin cables may promote, at least in part, Kar9p polarization by supporting a bias in Kar9p recruitment to the SPB engaged in Myo2p-dependent delivery of aMT plus ends. Among a collection of temperature-sensitive alleles of MYO2 that disrupt cargo domain functions in vesicular trafficking and polarized growth (Schott et al., 1999), myo2-18 also disrupted the corresponding interaction between Kar9p and the myosin tail, whereas myo2-17 did not (Yin et al., 2000). In support of the importance of Myo2p for a mechanism that promotes asymmetric loading in response to delivery of Kar9p-decorated aMTs along actin cables, myo2-18 led to a marked decrease in cells labeled at a single SPB relative to myo2-17 after a shift to a semipermissive temperature (<xref ref-type="fig" rid="zmk0151095280007">Figure 7</xref>B). By contrast, the same treatment disrupted Sec4p-GFP localization in both mutants (B). By contrast, the same treatment disrupted Sec4p-GFP localization in both mutants (myo2-18, 66.5% ± 4.9; myo2-17 65.5% ± 0.7; MYO2, 13%). Thus, the role of Myo2p in promoting Kar9p polarity was directly linked to the ability of Kar9p to act as cargo in Myo2p-based transport.'], 'zmk0151095280008': ['We therefore propose that the impact of actin integrity on Kar9p polarity is a direct consequence of the requirement of Myo2p-dependent transport for inducing cycles of delivery of aMT plus ends that stimulate loading of Kar9p to the pole engaged, thus generating a feedback loop that can sustain further delivery of aMT plus ends from this pole (<xref ref-type="fig" rid="zmk0151095280008">Figure 8</xref>A). Accordingly, A). Accordingly, bud6Δ or bud6Δ bni1Δ cells would be delayed in symmetry breaking to the extent in which delivery of aMT plus ends is compromised by the perturbation in actin cable organization (Amberg et al., 1997; Pruyne et al., 2004a; Delgehyr et al., 2008). Kar9p can be recruited at the SPB from a cytoplasmic pool, and it may not be readily exchangeable, because recovery after photobleaching at the SPB under otherwise unperturbed conditions may exceed 4 min (Liakopoulos et al., 2003). In addition, once reaching aMT plus ends, Kar9p can return to the SPB along shortening aMTs (Liakopoulos et al., 2003; Huisman et al., 2004; Cuschieri et al., 2006). This recycling on aMTs after disengagement from Myo2p may contribute toward the accumulation of Kar9p upon cycles of delivery of aMT plus ends, as suggested by our time-lapse analysis. Consistent with this notion, the abnormally persistent interaction between Kar9p and Myo2p provoked by a tub4-Δdsyl mutation results in both excessive aMT plus end dwelling at the bud cell cortex and depletion of Kar9p label at the SPB (Cuschieri et al., 2006). Yet, it was not technically possible to assess the respective contributions of both pools of Kar9p (recruitment vs. returning to the SPB) to this recycling by photobleaching experiments and dual-color imaging, in the context of dynamic delivery of aMT plus ends.', 'Kar9p begins to single out the SPBbud coincident with the establishment of spindle polarity, thus enforcing delivery of aMT plus ends from this pole to the bud after SPB separation (Huisman et al., 2004; Maekawa and Schiebel, 2004). Early in the cell cycle, Kar9p may associate with both side-by-side SPBs already tethered to the growing bud in response to cortical cues. However, stimulation of recruitment through feedback can only engage the old SPB through its existing aMTs (<xref ref-type="fig" rid="zmk0151095280008">Figure 8</xref>B). Cycles of delivery of aMT plus ends would continue over time, encouraged by this feedback. After SPB separation, two mechanisms conspire to confine the new SPB to the mother cell. First, aMTs now formed by the new SPB are restricted from access to the bud (B). Cycles of delivery of aMT plus ends would continue over time, encouraged by this feedback. After SPB separation, two mechanisms conspire to confine the new SPB to the mother cell. First, aMTs now formed by the new SPB are restricted from access to the bud (Segal et al., 2000a; Delgehyr et al., 2008). Second, Kar9p recruitment to this pole would fade, because a feedback loop cannot be established, because the history of the two SPBs would have built a pronounced bias in favor of the old SPB, now the SPBbud. Treatment with either nocodazole or LatB renders the SPBs equal to recruit Kar9p. Yet, upon recovery from either drug treatment, cell polarity and actin cables can promote symmetry breaking and repolarization of Kar9p to mark one SPB to become the SPBbud.']}
|
Actin-mediated Delivery of Astral Microtubules Instructs Kar9p Asymmetric Loading to the Bud-Ward Spindle Pole
| null |
Mol Biol Cell
|
1280646000
|
Functional analysis of cytoplasmic dynein in Caenorhabditis elegans has revealed a wide range of cellular functions for this minus-end-directed motor protein. Dynein transports a variety of cargos to diverse cellular locations, and thus cargo selection and destination are likely regulated by accessory proteins. The microtubule-associated proteins LIS-1 and dynein interact, but the nature of this interaction remains poorly understood. Here we show that both LIS-1 and the dynein heavy-chain DHC-1 are required for integrity of the actin cytoskeleton in C. elegans. Although both dhc-1(or195ts) and lis-1 loss-of-function disrupt the actin cytoskeleton and produce embryonic lethality, a double mutant suppresses these defects. A targeted RNA interference screen revealed that knockdown of other actin regulators, including actin-capping protein genes and prefoldin subunit genes, suppresses dhc-1(or195ts)-induced lethality. We propose that release or relocation of the mutant dynein complex mediates this suppression of dhc-1(or195ts)-induced phenotypes. These results reveal an unexpected direct or indirect interaction between the actin cytoskeleton and dynein activity.
|
[
"Actins",
"Alleles",
"Animals",
"Caenorhabditis elegans",
"Caenorhabditis elegans Proteins",
"Cytoplasmic Dyneins",
"Cytoskeleton",
"Depsipeptides",
"Dyneins",
"Embryo, Nonmammalian",
"Gene Knockdown Techniques",
"Genes, Helminth",
"Genes, Suppressor",
"Gonads",
"Microtubule-Associated Proteins",
"Microtubules",
"Mutation",
"Pachytene Stage",
"Protein Transport",
"RNA Interference",
"Suppression, Genetic"
] |
other
|
PMC2912354
| null |
35
|
[
"{'Citation': 'Bubb M. R., Senderowicz A. M., Sausville E. A., Duncan K. L., Korn E. D. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J. Biol. Chem. 1994;269:14869–14871.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '8195116'}}}",
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"{'Citation': 'Zhuang L., Zhang J., Xiang X. Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans. Genetics. 2007;175:1185–1196.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC1840067'}, {'@IdType': 'pubmed', '#text': '17237507'}]}}"
] |
Mol Biol Cell. 2010 Aug 1; 21(15):2685-2695
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NO-CC CODE
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Evaluation
of SNAP-tag activity in cellulo. (A)
Scheme depicting in cellulo labeling of SNAP-tag
conjugate proteins with SNAP-Cell© 505-Star. Saos-2 cells were
incubated with 3 μM of each unlabeled SNAP-tag conjugate for
30 min followed by treatment with 1 μM SNAP-Cell© 505-Star
for 45 min. Intracellular fluorescence was assessed using confocal
microscopy and flow cytometry. (B) Confocal microscopy images of live
cells treated with each indicated SNAP-tag conjugate and SNAP-Cell©
505-Star as described above. Scale bar = 20 μm. (C) Flow cytometry
bar plots illustrating relative levels of total intracellular fluorescence
resulting from treatment with each SNAP-tag conjugate followed by
SNAP-Cell© 505-Star. MFI values represent the average median
fluorescence intensity of cells determined from three individual replicates
(10 000 cells each). MFI values corresponding to each SNAP-tag
conjugate were compared to cells incubated with blank media (no added
conjugate) followed by SNAP-Cell© 505-Star. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05; one-way
ANOVA followed by post hoc Dunnett’s test.
|
oc-2018-00446g_0006
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2
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63d007976df663fb2259a0b75041165f49d56b2e02fd66fd63c4d6e53a8f5267
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oc-2018-00446g_0006.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
620,
1027
] |
[{'image_id': 'oc-2018-00446g_m005', 'image_file_name': 'oc-2018-00446g_m005.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_m005.jpg', 'caption': 'No caption found', 'hash': '4a2837a6155c5eea5fb6369717b1bd500b377fc8204f1830d63f894c2d4c2d74'}, {'image_id': 'oc-2018-00446g_m002', 'image_file_name': 'oc-2018-00446g_m002.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_m002.jpg', 'caption': 'No caption found', 'hash': 'd3a9b5ed4a36e90d5d5b23d63449793c97c1b84948421a668853d24b5e6efd09'}, {'image_id': 'oc-2018-00446g_0007', 'image_file_name': 'oc-2018-00446g_0007.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0007.jpg', 'caption': 'Assessment of ZF5.3-mediated delivery\nof APEX2-Rho. (A) Confocal\nmicroscopy images of live Saos-2 cells treated with 1 μM of\neach APEX2 conjugate for 30 min with the indicated Rho-tagged SNAP-tag\nconjugate. Scale bar = 20 μm. (B) Flow cytometry bar plots illustrating\nthe relative uptake of each Rho-tagged APEX2 conjugate after 30 min\nincubation and treatment with trypsin. MFI values represent the average\nmedian fluorescence intensity of cells determined from three individual\nreplicates (10\u202f000 cells each). Error bars represent the standard\nerror of the mean. MFI values corresponding to each SNAP-tag conjugate\nwere statistically compared to nontreated cells. (C) Representative in cellulo FCS traces (left) corresponding to each indicated\nRho-tagged APEX2 conjugate displaying the diffusion time (τD) as well as the anomalous coefficient (a) associated with\neach representative trace and scatter plot representation (right)\nof intracellular concentrations of Rho-tagged APEX2 conjugates determined\nfrom respective autocorrelation fits. ****p ≤\n0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05; one-way ANOVA\nfollowed by post hoc Dunnett’s test.', 'hash': 'f64b56873192446b183e0863efe327504fa3a0171e910030b7437dbccef509f6'}, {'image_id': 'oc-2018-00446g_0009', 'image_file_name': 'oc-2018-00446g_0009.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0009.jpg', 'caption': 'No caption found', 'hash': '62be106a35a222a1f69220f7e54f8bcef9784ad7c44d4ee47dc7788fc1c064f9'}, {'image_id': 'oc-2018-00446g_0008', 'image_file_name': 'oc-2018-00446g_0008.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0008.jpg', 'caption': 'Evaluation\nof APEX2 activity in cellulo by flow\ncytometry and confocal microscopy using Amplex UltraRed. (A) Scheme\ndepicting assay for assessing APEX2 activity in cellulo with Amplex UltraRed. Saos-2 cells were incubated with 500 nM of\neach APEX2 conjugate for 30 min followed by treatment with 10 μM\nAmplex UltraRed and 1 mM H2O2 for 1 min. Intracellular\nfluorescence was assessed using confocal microscopy and flow cytometry.\n(B) Flow cytometry bar plots representing the levels of intracellular\nAmplex UltraRed fluorescence. MFI values represent the average median\nfluorescence intensity of cells determined from three individual replicates\n(10,000 cells each). Error bars represent the standard error of the\nmean. (C) Confocal microscopy images of live cells treated with 500\nnM of each indicated APEX2 conjugate followed by 10 μM Amplex\nUltraRed, and 1 mM H2O2 for 1 min. Scale bar\n= 20 μm.', 'hash': 'f90c6b869cccfebf0dcd30fa1d881d8696c96eead62b23d0f1351c5b764c282f'}, {'image_id': 'oc-2018-00446g_0006', 'image_file_name': 'oc-2018-00446g_0006.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0006.jpg', 'caption': 'Evaluation\nof SNAP-tag activity in cellulo. (A)\nScheme depicting in cellulo labeling of SNAP-tag\nconjugate proteins with SNAP-Cell© 505-Star. Saos-2 cells were\nincubated with 3 μM of each unlabeled SNAP-tag conjugate for\n30 min followed by treatment with 1 μM SNAP-Cell© 505-Star\nfor 45 min. Intracellular fluorescence was assessed using confocal\nmicroscopy and flow cytometry. (B) Confocal microscopy images of live\ncells treated with each indicated SNAP-tag conjugate and SNAP-Cell©\n505-Star as described above. Scale bar = 20 μm. (C) Flow cytometry\nbar plots illustrating relative levels of total intracellular fluorescence\nresulting from treatment with each SNAP-tag conjugate followed by\nSNAP-Cell© 505-Star. MFI values represent the average median\nfluorescence intensity of cells determined from three individual replicates\n(10\u202f000 cells each). MFI values corresponding to each SNAP-tag\nconjugate were compared to cells incubated with blank media (no added\nconjugate) followed by SNAP-Cell© 505-Star. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05; one-way\nANOVA followed by post hoc Dunnett’s test.', 'hash': '63d007976df663fb2259a0b75041165f49d56b2e02fd66fd63c4d6e53a8f5267'}, {'image_id': 'oc-2018-00446g_0001', 'image_file_name': 'oc-2018-00446g_0001.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0001.jpg', 'caption': '(A) Workflow\nto determine the overall uptake and cytosolic concentration\nof a self-labeled CPMP, cCPP, or uCPP SNAP-tag conjugate using flow\ncytometry and FCS. The sequence of each vehicle studied in this report\nis listed above. For ZF5.3 and aPP5.3, the residues comprising the\n5.3 motif are shown in red. For CPP9 and CPP12, lowercase letters\nrepresent d-amino acids, Φ represents l-naphthylalanine,\nand PEG2 represents a 2-unit ethylene glycol spacer. (B) Mechanism\nof SNAP-tag self-labeling with a fluorophore-containing benzylguanine\nderivative.', 'hash': 'ce721269cd2797451dcc0bea108d92feb45f2e3c0dcd2ffd7d44f829e3f5a67e'}, {'image_id': 'oc-2018-00446g_m003', 'image_file_name': 'oc-2018-00446g_m003.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_m003.jpg', 'caption': 'No caption found', 'hash': 'a01976d9ed641844040a29396926afcaa2563f010e2feeee31901ea63de6c84f'}, {'image_id': 'oc-2018-00446g_m004', 'image_file_name': 'oc-2018-00446g_m004.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_m004.jpg', 'caption': 'No caption found', 'hash': '71a646548f518c4e87c27f7ee337f34e18ce0cf4b02946b978d92f5d866435e1'}, {'image_id': 'oc-2018-00446g_0002', 'image_file_name': 'oc-2018-00446g_0002.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0002.jpg', 'caption': 'Sortase-based ligation strategy for generating\nCPP9- and CPP12-SNAP.\nTo prepare SNAP-His6 bearing an N-terminal\ntriglycine sequence for sortase labeling, we expressed and purified\na variant containing an N-terminal Factor Xa recognition\nsite (MIEGR) followed by a triglycine motif to yield MIEGR-G3-SNAP-His6. MIEGR-G3-SNAP-His6 was\ncleaved with Factor Xa to yield G3-SNAP-His6. G3-SNAP-His6 was then incubated with excess\nCPP9-LPETG3 or CPP12-LPETG3 in the presence\nof the engineered sortase variant SrtA7M75 to generate the desired conjugate in good (>50%) yields. To isolate\nCPP9- and CPP12-SNAP-His6 from the reaction mixture, we\nemployed a two-step chromatographic strategy to isolate the desired\nconjugates (see Methods for additional details).', 'hash': 'bdee61a68dbedf66b2b2a7a3d86dc8d8ad13292d82912aee6157206b96a402a1'}, {'image_id': 'oc-2018-00446g_0005', 'image_file_name': 'oc-2018-00446g_0005.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0005.jpg', 'caption': '(A) Evaluation\nof the overall uptake and cytosolic delivery of\nZF5.3-SNAP-Rho and CPP12-SNAP-Rho with increasing treatment concentration\nor time using flow cytometry and FCS. (B) Flow cytometry bar plots\n(left) indicating total levels of cellular uptake and scatter plot\nrepresentation of cytosolic concentrations (right) determined by FCS\nfor varying treatment concentrations (1, 2, or 3 μM) of either\nCPP12-SNAP-Rho or ZF5.3-SNAP-Rho. (C) Flow cytometry bar plots (left)\nindicating total levels of cellular uptake and scatter plot representation\nof cytosolic concentrations (right) determined by FCS with varying\nincubation times (30 or 120 min) of either CPP12-SNAP-Rho or ZF5.3-SNAP-Rho\n(treatment concentration 1 μM). MFI values represent the average\nmedian fluorescence intensity of cells determined from 3–12\nindividual replicates (10\u202f000 cells each).', 'hash': '1c4734874e09c801fc1ebaf03ceb14158071b4a9834a8e734662d4e61718923e'}, {'image_id': 'oc-2018-00446g_m001', 'image_file_name': 'oc-2018-00446g_m001.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_m001.jpg', 'caption': 'No caption found', 'hash': '1722f1e0c68f8cdb48821eb18c7779101696a8aa36c662adcba67b226b89e219'}, {'image_id': 'oc-2018-00446g_m006', 'image_file_name': 'oc-2018-00446g_m006.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_m006.jpg', 'caption': 'No caption found', 'hash': 'a26ded886d484fe8793adec32173a1df9c9023f7acc7e1e4c96d3fc40880cea6'}, {'image_id': 'oc-2018-00446g_0004', 'image_file_name': 'oc-2018-00446g_0004.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0004.jpg', 'caption': 'Quantification of cytosolic delivery of Rho-tagged SNAP-tag\nconjugates\nusing FCS. Saos-2 cells were treated with 1 μM of each SNAP-tag\nconjugate for 30 min and replated for FCS in the same manner as described\nfor confocal microscopy in Figure 2. (A) Representative in cellulo FCS\ntraces corresponding to each indicated Rho-tagged SNAP-tag conjugate\ndisplaying the measured diffusion time (τD) as well\nas the anomalous coefficient (a) associated with each representative\ntrace. (B) Scatter plot representation of intracellular concentrations\nof Rho-tagged SNAP-tag conjugates determined from respective autocorrelation\nfits. The average cytosolic concentrations corresponding to each Rho-tagged\nSNAP-tag conjugate were statistically compared to the intracellular\nconcentration of Rho-tagged SNAP-tag lacking an appended vehicle.\n****p ≤ 0.0001, ***p ≤\n0.001, **p ≤ 0.01, *p ≤\n0.05 ; one-way ANOVA followed by post hoc Dunnett’s test.', 'hash': '7b9f1f661befacf65625af8bb1605a9258b73c2d1e505ee53154f1fb259b9d12'}, {'image_id': 'oc-2018-00446g_0003', 'image_file_name': 'oc-2018-00446g_0003.jpg', 'image_path': '../data/media_files/PMC6202653/oc-2018-00446g_0003.jpg', 'caption': 'Total cell uptake of\nRho-tagged SNAP-tag conjugates assessed by\nconfocal microscopy (A) and flow cytometry (B and C). (A) Images of\nlive Saos-2 cells treated with 1 μM of each SNAP-tag conjugate\nfor 30 min. Scale bar = 20 μm. (B) Histograms and (C) bar plots\nillustrating the relative uptake of each Rho-tagged SNAP-tag conjugate\nafter 30 min incubation and trypsin treatment to remove surface bound\nprotein. MFI values represent the average median fluorescence intensity\nof cells determined from 4–12 individual replicates (10\u202f000\ncells each). Error bars represent the standard error of the mean.\nMFI values corresponding to each SNAP-tag conjugate were statistically\ncompared to nontreated cells. ****p ≤ 0.0001,\n***p ≤ 0.001, **p ≤\n0.01, *p ≤ 0.05; one-way ANOVA followed by\npost hoc Dunnett’s test.', 'hash': '2c3e3a077f53907a6c30235dcc1960911724e451657f6eaa93ab009ca5334ba5'}]
|
{'oc-2018-00446g_0001': ['Intrigued by the efficiencies with which the isolated CPMPs aPP5.3\nand ZF5.3 traffic into the cytosol, we sought to evaluate their ability\nto deliver large proteins into cells in a direct, head-to-head comparison\nwith several other peptide-based delivery vehicles. Specifically,\nwe made use of a commercial, easy-to-use FCS system to directly quantify\nthe relative efficiencies with which two traditional uCPPs, three\nCPMPs, and two recently reported cell-penetrating cyclic peptides\n(cCPPs) deliver a full-length protein cargo—the self-labeling\nenzyme SNAP-tag67—into the cytosol\nof mammalian cells. Using FCS and this set of fluorescent SNAP-tag\nconjugates (<xref rid="oc-2018-00446g_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>A), we discovered that the CPMP ZF5.3 remains a superior cytosolic\ndelivery vehicle even when appended to an enzyme cargo of significant\nmass. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration\nas high as 250 nM, generally at least 2-fold and as much as 6-fold\nhigher than any other CPP evaluated. As we have reported previously,\nthe extent of cytosolic localization does not always mirror the extent\nof overall uptake ascertained by flow cytometry, demonstrating the\nvalue of a technique that measures concentration directly, such as\nFCS. To demonstrate that ZF5.3-mediated delivery is not restricted\nto a single protein, we conjugated it to the enzyme APEX2,<xref rid="oc-2018-00446g_0001" ref-type="fig">1</xref>A), we discovered that the CPMP ZF5.3 remains a superior cytosolic\ndelivery vehicle even when appended to an enzyme cargo of significant\nmass. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration\nas high as 250 nM, generally at least 2-fold and as much as 6-fold\nhigher than any other CPP evaluated. As we have reported previously,\nthe extent of cytosolic localization does not always mirror the extent\nof overall uptake ascertained by flow cytometry, demonstrating the\nvalue of a technique that measures concentration directly, such as\nFCS. To demonstrate that ZF5.3-mediated delivery is not restricted\nto a single protein, we conjugated it to the enzyme APEX2,68 assessed cytosolic trafficking using FCS, and\nevaluated peroxidase activity in cellulo. Taken together,\nwe have shown that FCS can realistically assess the relative merits\nof protein transduction domains for shuttling protein cargo beyond\nmembrane barriers, and that the CPMP ZF5.3 holds particular promise\nas a robust tool for delivering active enzymes into cells.', 'In order to provide a direct and quantitative comparison\nof protein delivery by previously reported uCPPs, CPMPs, and cCPPs,\nwe sought a well-characterized protein that could be labeled in a\nhomogeneous and stoichiometric manner with a bright photostable dye.\nWe envisioned that this goal would be achieved most easily using a\nwell-studied, self-labeling protein tag such as SNAP,67 CLIP,69 or Halo.70 While we considered using FCS-compatible red\nfluorescent proteins as model cargo, we were concerned by their complex\nphotophysical properties and known tendency to form multimers in vitro and in live cells.71,72 By contrast,\nSNAP-tag, an engineered human O6-alkylguanine-DNA-alkyltransferase\nvariant, reacts efficiently, in vitro and in vivo, with fluorophore-substituted O6-benzylguanine (BG) or chloropyrimidine (CP) substrates\nto form covalent SNAP-tag-fluorophore conjugates (<xref rid="oc-2018-00446g_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>B). Virtually all SNAP-tag-protein\nfusions retain <xref rid="oc-2018-00446g_0001" ref-type="fig">1</xref>B). Virtually all SNAP-tag-protein\nfusions retain O6-alkylguanine-DNA-alkyltransferase\nactivity and the ability to self-label.73 Moreover, the SNAP-tag enzyme is monomeric, highly thermostable\n(TM = ∼70 °C), and resists\ndegradation by intracellular proteases (t1/2 = ∼42 h in HEK293T cells).74 These\nfeatures render SNAP-tag an attractive model enzyme for the comparative\nevaluation of uCPP, CPMP, and cCPP-mediated cytosolic delivery.'], 'oc-2018-00446g_0002': ['We\nalso sought to compare aPP5.3 and ZF5.3 to a set of cyclic peptides\nthat have been reported by others to enter the cell cytosol, most\nnotably CPP9 and CPP12, sequence variants of the cyclic peptide known\nas cFΦR4.35,76 In previous work, cytosolic localization\nof the isolated CPP9 and CPP12 peptides (no cargo) was estimated from\nflow cytometry experiments using variants linked covalently to a dye\nthat is ∼10-fold more fluorescent at neutral pH than at pH\n≤ 6.0; like other assays based on fluorescence intensity, this\nmethod provides only an estimate of cytosolic concentration.57,76 Using this assay, the cytosolic trafficking of CPP9 and CPP12 was\nestimated to approach that of aPP5.3, which encouraged us to evaluate\ntheir relative merits using the more quantitative method of FCS and\nin a more relevant context in which both are linked to a model cargo\nprotein.76 However, unlike the uCPPs and\nCPMPs described above, CPP9 and CPP12 contain nonproteinogenic amino\nacids and cannot be easily genetically encoded to produce the requisite\nfusion protein. In an earlier study, cFΦR4 was conjugated to\nthe N-terminus of a protein using the peptide carrier\nprotein Sfp phosphopantetheinyl transferase (Sfp).35 While this method allows for site-specific labeling, it\nrequires modification of the protein of interest with an 11-residue\nSfp recognition sequence.77 Moreover, Sfp\nlabeling requires CoA-derivatized peptides, which require multiple\nsteps after solid phase synthesis to prepare and are costly to scale.35 Rabideau et al. have shown that the enzyme sortase\ncan ligate unnatural synthetic peptides, including cyclic peptides,\nonto the C-terminus of full-length proteins.78 Inspired by this report, we designed a straightforward strategy\nto ligate CPP9 and CPP12 to the N-terminus of SNAP-tag\nthrough a covalent linkage (<xref rid="oc-2018-00446g_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>). The identity and purity of each cCPP-SNAP-tag conjugate\nwere ultimately assessed by MS and SDS-PAGE, respectively (<xref rid="oc-2018-00446g_0002" ref-type="fig">2</xref>). The identity and purity of each cCPP-SNAP-tag conjugate\nwere ultimately assessed by MS and SDS-PAGE, respectively (Figure S1). To prepare for FCS, each SNAP-tag\nconjugate was incubated with BG-Lissamine rhodamine B (Rho) for 2\nh at 37 °C. Rho was selected as the dye of choice because it\nis compatibile with FCS and cannot penetrate cells on its own.47 All of the SNAP-tag conjugates that we examined\nretained robust self-labeling activity in vitro;\nin each case, we observed quantitative and homogeneous labeling. SDS-PAGE\nand MS analyses confirmed the identity, homogeneity, and purity of\neach Rho-labeled SNAP-tag conjugate used in cellular assays (Figure S2).', 'Quantification of cytosolic delivery of Rho-tagged SNAP-tag\nconjugates\nusing FCS. Saos-2 cells were treated with 1 μM of each SNAP-tag\nconjugate for 30 min and replated for FCS in the same manner as described\nfor confocal microscopy in <xref rid="oc-2018-00446g_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>. (A) Representative <xref rid="oc-2018-00446g_0002" ref-type="fig">2</xref>. (A) Representative in cellulo FCS\ntraces corresponding to each indicated Rho-tagged SNAP-tag conjugate\ndisplaying the measured diffusion time (τD) as well\nas the anomalous coefficient (a) associated with each representative\ntrace. (B) Scatter plot representation of intracellular concentrations\nof Rho-tagged SNAP-tag conjugates determined from respective autocorrelation\nfits. The average cytosolic concentrations corresponding to each Rho-tagged\nSNAP-tag conjugate were statistically compared to the intracellular\nconcentration of Rho-tagged SNAP-tag lacking an appended vehicle.\n****p ≤ 0.0001, ***p ≤\n0.001, **p ≤ 0.01, *p ≤\n0.05 ; one-way ANOVA followed by post hoc Dunnett’s test.'], 'oc-2018-00446g_0003': ['With\na set of fluorescently tagged SNAP-tag conjugates in hand, we first\nsought to evaluate their overall uptake into cells using confocal\nmicroscopy (<xref rid="oc-2018-00446g_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>). Saos-2 cells were incubated for 30 min with 1 μM of each\nRho-labeled SNAP-tag conjugate and then with 300 nM Hoechst 33342\nto visualize the cell nucleus. The cells were washed, treated with\ntrypsin to remove surface-bound protein and lift the cells, replated\nonto fibronectin-coated glass microscopy slides, and imaged. These\nimages (<xref rid="oc-2018-00446g_0003" ref-type="fig">3</xref>). Saos-2 cells were incubated for 30 min with 1 μM of each\nRho-labeled SNAP-tag conjugate and then with 300 nM Hoechst 33342\nto visualize the cell nucleus. The cells were washed, treated with\ntrypsin to remove surface-bound protein and lift the cells, replated\nonto fibronectin-coated glass microscopy slides, and imaged. These\nimages (<xref rid="oc-2018-00446g_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>A)\nrevealed low levels of intracellular fluorescence when cells were\ntreated with SNAP-Rho and CPP9-SNAP-Rho, suggesting that under these\nconditions, the presence of CPP9 does not significantly enhance the\nuptake of an appended SNAP-tag cargo by Saos-2 cells. Cells treated\nwith CPP12-SNAP-Rho, aPP5.3-SNAP-Rho, R8-SNAP-Rho, ZiF-SNAP-Rho, and\nPen-SNAP-Rho exhibited bright punctate fluorescence, suggesting significant\nlevels of endocytic uptake, but no observable cytosolic fluorescence.\nSaos-2 cells treated with ZF5.3-SNAP-Rho displayed bright punctate\nfluorescence as well as diffuse cytosolic fluorescence, suggesting\nsignificant levels of both endocytic uptake and cytosolic release.\nOverall, these qualitative confocal microscopy results suggest that\nZF5.3-SNAP-Rho reaches the cytosol of Saos-2 cells, whereas the other\nconjugates do so to a lesser extent or not at all.<xref rid="oc-2018-00446g_0003" ref-type="fig">3</xref>A)\nrevealed low levels of intracellular fluorescence when cells were\ntreated with SNAP-Rho and CPP9-SNAP-Rho, suggesting that under these\nconditions, the presence of CPP9 does not significantly enhance the\nuptake of an appended SNAP-tag cargo by Saos-2 cells. Cells treated\nwith CPP12-SNAP-Rho, aPP5.3-SNAP-Rho, R8-SNAP-Rho, ZiF-SNAP-Rho, and\nPen-SNAP-Rho exhibited bright punctate fluorescence, suggesting significant\nlevels of endocytic uptake, but no observable cytosolic fluorescence.\nSaos-2 cells treated with ZF5.3-SNAP-Rho displayed bright punctate\nfluorescence as well as diffuse cytosolic fluorescence, suggesting\nsignificant levels of both endocytic uptake and cytosolic release.\nOverall, these qualitative confocal microscopy results suggest that\nZF5.3-SNAP-Rho reaches the cytosol of Saos-2 cells, whereas the other\nconjugates do so to a lesser extent or not at all.', 'The differences\nin the overall uptake of each SNAP-tag conjugate suggested by confocal\nmicroscopy were studied further using flow cytometry. Treatment of\nSaos-2 cells with 1 μM of each Rho-labeled SNAP-tag conjugate\nas described above led to evenly distributed populations of fluorescent\ncells (<xref rid="oc-2018-00446g_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>B);\nthe median fluorescence intensity of this distribution over 3–5\nindependent replicates is also shown (<xref rid="oc-2018-00446g_0003" ref-type="fig">3</xref>B);\nthe median fluorescence intensity of this distribution over 3–5\nindependent replicates is also shown (<xref rid="oc-2018-00446g_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>C). Overall, the CPPs and CPMPs studied fall\ninto four categories: cells treated with CPP9-SNAP-Rho were not measurably\nmore fluorescent than cells treated with SNAP-Rho, as expected from\nthe microscopy experiments described above. Cells treated with CPP12-SNAP-Rho\nand aPP5.3-SNAP-Rho showed comparable levels of fluorescence throughout\nthe cell interior, reaching values approximately 2-fold higher than\nthat observed when cells were treated with SNAP-Rho. Cells treated\nwith R8-SNAP-Rho and ZiF-SNAP-Rho exhibited higher levels of total\ncellular fluorescence that were nearly 5-fold higher than that of\ncells treated with SNAP-Rho. The highest levels of total intracellular\nfluorescence were observed when cells were treated with Pen-SNAP-Rho\n(an 11-fold increase relative to cells treated with SNAP-Rho) and\nZF5.3-SNAP-Rho (a 17-fold increase relative to cells treated with\nSNAP-Rho). Overall, we observed good agreement between data from confocal\nmicroscopy and flow cytometry experiments performed using Saos-2 cells.<xref rid="oc-2018-00446g_0003" ref-type="fig">3</xref>C). Overall, the CPPs and CPMPs studied fall\ninto four categories: cells treated with CPP9-SNAP-Rho were not measurably\nmore fluorescent than cells treated with SNAP-Rho, as expected from\nthe microscopy experiments described above. Cells treated with CPP12-SNAP-Rho\nand aPP5.3-SNAP-Rho showed comparable levels of fluorescence throughout\nthe cell interior, reaching values approximately 2-fold higher than\nthat observed when cells were treated with SNAP-Rho. Cells treated\nwith R8-SNAP-Rho and ZiF-SNAP-Rho exhibited higher levels of total\ncellular fluorescence that were nearly 5-fold higher than that of\ncells treated with SNAP-Rho. The highest levels of total intracellular\nfluorescence were observed when cells were treated with Pen-SNAP-Rho\n(an 11-fold increase relative to cells treated with SNAP-Rho) and\nZF5.3-SNAP-Rho (a 17-fold increase relative to cells treated with\nSNAP-Rho). Overall, we observed good agreement between data from confocal\nmicroscopy and flow cytometry experiments performed using Saos-2 cells.'], 'oc-2018-00446g_0004': ['After assessing cellular uptake by confocal\nmicroscopy and flow cytometry, we used FCS to determine the amount\nof labeled SNAP-tag protein that reaches the cytosol of live cells.\nSaos-2 cells were prepared for FCS experiments in the same manner\nas described for confocal microscopy. After confocal images were acquired,\ncells were scanned visually to identify locations for cytosolic focal\nvolume placement; nuclear regions were avoided, as were regions with\nhigh punctate signal representative of endosomes. The acquired correlation\ndata were fit to a three-dimensional (3D) diffusion model containing\na parameter for anomalous subdiffusion using a custom MATLAB script\nas previously described.47 The average\ndiffusion times (τD) of the SNAP-tag conjugates (<xref rid="oc-2018-00446g_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>A) measured in the\ncytosol ranged between 1.20 ± 0.66 and 2.67 ± 0.37 ms, approximately\n6–14-fold higher than values acquired <xref rid="oc-2018-00446g_0004" ref-type="fig">4</xref>A) measured in the\ncytosol ranged between 1.20 ± 0.66 and 2.67 ± 0.37 ms, approximately\n6–14-fold higher than values acquired in vitro (Figure S4), in good agreement with the\nobserved increase in cytoplasmic diffusion times observed for intact\npeptides/proteins in living cells.47,81 Notably, the\nintracellular diffusion time of each SNAP-tag conjugate is at least\n5-fold longer than the average intracellular diffusion time measured\nfor the 3.4 kDa Rho-tagged stable peptide ZF5.3R (0.21\n± 0.025 ms).47 Since diffusion time\nis proportional to molecular mass, this difference provides strong\nevidence that the detected fluorescent signals represent dye-protein\nconjugates and not released dye or small protein fragments. To confirm\nthat SNAP-tag remains intact once delivered to the cytosol, cytosolic\nfractions obtained from nontreated cells and from cells treated with\nZF5.3-SNAP-Rho were loaded onto an SDS-PAGE gel and analyzed by in-gel\nfluorescence scanning followed by Western blotting with an anti-SNAP-tag\nantibody. The cytosolic fraction obtained from cells treated with\nZF5.3-SNAP-Rho contained a single fluorescent band that was positively\nidentified as SNAP-tag by Western blot analysis (Figure S5).', 'When examined by FCS, the SNAP-tag\nconjugates studied fall into two categories with respect to whether\nthey reach the cell cytosol after a 30 min incubation: those that\naccumulate to a detectable level compared to SNAP-Rho and those that\ndo not (<xref rid="oc-2018-00446g_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>B).\nSNAP-tag conjugates in the latter category include CPP9-SNAP-Rho,\nCPP12-SNAP-Rho, aPP5.3-SNAP-Rho, R8-SNAP-Rho, and ZiF-SNAP-Rho. Cells\ntreated with 1 μM of these conjugates displayed a small (3–5-fold)\nbut statistically insignificant increase in cytosolic localization\nrelative to cells treated with SNAP-Rho, which accumulated in the\ncytosol to reach a concentration of 1.8 ± 0.20 nM. Cells treated\nwith Pen-SNAP-Rho yielded a statistically significant increase in\ncytosolic fluorescence that correlated with intracellular concentrations\nbetween 5 and 65 nM, with an average of 23 ± 2.9 nM. FCS measurements\nrevealed that the cytosolic concentration achieved by ZF5.3-SNAP-Rho\nwas at least 2-fold higher than that measured for Pen-SNAP-Rho, and\nmore than 6-fold higher than the concentration measured for any other\nSNAP-tag conjugate tested. For ZF5.3-SNAP-Rho, the calculated cytosolic\nconcentration after a 30 min incubation ranged from 14 to 144 nM,\nwith an average of 58 ± 6.1 nM. Overall, our results demonstrate\nthat both ZF5.3 and Pen are effective vehicles for delivering SNAP-tag\ninto the cytosol of Saos-2 cells.<xref rid="oc-2018-00446g_0004" ref-type="fig">4</xref>B).\nSNAP-tag conjugates in the latter category include CPP9-SNAP-Rho,\nCPP12-SNAP-Rho, aPP5.3-SNAP-Rho, R8-SNAP-Rho, and ZiF-SNAP-Rho. Cells\ntreated with 1 μM of these conjugates displayed a small (3–5-fold)\nbut statistically insignificant increase in cytosolic localization\nrelative to cells treated with SNAP-Rho, which accumulated in the\ncytosol to reach a concentration of 1.8 ± 0.20 nM. Cells treated\nwith Pen-SNAP-Rho yielded a statistically significant increase in\ncytosolic fluorescence that correlated with intracellular concentrations\nbetween 5 and 65 nM, with an average of 23 ± 2.9 nM. FCS measurements\nrevealed that the cytosolic concentration achieved by ZF5.3-SNAP-Rho\nwas at least 2-fold higher than that measured for Pen-SNAP-Rho, and\nmore than 6-fold higher than the concentration measured for any other\nSNAP-tag conjugate tested. For ZF5.3-SNAP-Rho, the calculated cytosolic\nconcentration after a 30 min incubation ranged from 14 to 144 nM,\nwith an average of 58 ± 6.1 nM. Overall, our results demonstrate\nthat both ZF5.3 and Pen are effective vehicles for delivering SNAP-tag\ninto the cytosol of Saos-2 cells.', 'Many of the differences between FCS and other, perhaps\nless technical,\nassays stem from the fact that CPP-mediated protein delivery into\ncell cytosol, in most cases, requires at least two distinct steps:\nuptake by endocytosis and then endosomal release. While many CPP–protein\nconjugates are readily endocytosed, most fail to reach the cytosol\ndue to endosomal entrapment. In our study, we found that the cytosolic\nconcentrations measured by FCS correlated poorly with overall uptake,\nsuggesting that there are fundamental differences in the ability of\neach SNAP-tag conjugate to escape the endocytic pathway. To more carefully\nassess endosomal escape efficiency, we calculated an “endosomal\nescape ratio” (EER) for each conjugate, which corresponds to\nthe concentration of a CPP–Snap-tag conjugate that reaches\nthe cytosol divided by the overall uptake as determined by flow cytometry,\nand compared these values to SNAP-tag lacking an appended vehicle\n(Figure S10). This analysis revealed that\nonly two molecules, the miniature protein ZF5.3 and the cyclic peptide\nCPP9 promoted the endosomal release of SNAP-tag conjugate significantly\nover background. Although the EER value calculated for CPP9-SNAP-Rho\nis 1.3-fold higher than that of ZF5.3-SNAP-Rho, the cytosolic concentration\nit achieves is 9.6-fold lower (<xref rid="oc-2018-00446g_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>B). While we appreciate that this finding is likely\ncargo-dependent, it does emphasize that the combination of flow cytometry\nand FCS can be used to evaluate the endosomal escape efficiency of\nany conjugate of interest, provided that the molecule under study\ncan be site-specifically labeled with an appropriate fluorophore.\nWe anticipate that FCS techniques will dramatically accelerate our\nability to identify, evaluate, and optimize current strategies for\ndelivering large, intact proteins to intracellular locales.<xref rid="oc-2018-00446g_0004" ref-type="fig">4</xref>B). While we appreciate that this finding is likely\ncargo-dependent, it does emphasize that the combination of flow cytometry\nand FCS can be used to evaluate the endosomal escape efficiency of\nany conjugate of interest, provided that the molecule under study\ncan be site-specifically labeled with an appropriate fluorophore.\nWe anticipate that FCS techniques will dramatically accelerate our\nability to identify, evaluate, and optimize current strategies for\ndelivering large, intact proteins to intracellular locales.'], 'oc-2018-00446g_0005': ['Next, we made use\nof FCS to quantify the effects of concentration and incubation time\non the cytosolic localization of two SNAP-tag conjugates: one that\naccesses the cytosol well (ZF5.3-SNAP-Rho) and one that does not (CPP12-SNAP-Rho)\n(<xref rid="oc-2018-00446g_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>A). First,\nSaos-2 cells were treated with increasing concentrations of ZF5.3-SNAP-Rho\nor CPP12-SNAP-Rho (1–3 μM) for 30 min and analyzed by\nflow cytometry and FCS as previously described (<xref rid="oc-2018-00446g_0005" ref-type="fig">5</xref>A). First,\nSaos-2 cells were treated with increasing concentrations of ZF5.3-SNAP-Rho\nor CPP12-SNAP-Rho (1–3 μM) for 30 min and analyzed by\nflow cytometry and FCS as previously described (<xref rid="oc-2018-00446g_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>B). While we observed a dose-dependent increase\nin the total cellular uptake by flow cytometry when Saos-2 cells were\ntreated with ZF5.3-SNAP-Rho (up to 4-fold), only a modest increase\nwas observed when cells were treated with CPP12-SNAP-Rho (up to 2-fold).\nThese trends are mirrored by the FCS data: cells treated with increasing\nconcentrations of ZF5.3-SNAP-Rho showed a dose-dependent increase\nin cytosolic concentration as determined by FCS. Treatment concentrations\nof 1 and 2 μM ZF5.3-SNAP-Rho resulted in average cytosolic concentrations\nof 58 ± 6.1 nM and 110 ± 13 nM, respectively, corresponding\nto an average delivery efficiency of 6%. At the highest treatment\nconcentration of 3 μM, we observed an average cytosolic concentration\nof 251 ± 34 nM, a delivery efficiency of 8%. In contrast, the\ncytosolic concentration of CPP12-SNAP-Rho did not significantly increase\nas a function of treatment concentration. At the highest concentration\ntested, the delivery efficiency of CPP12-SNAP-Rho was less than 1%.<xref rid="oc-2018-00446g_0005" ref-type="fig">5</xref>B). While we observed a dose-dependent increase\nin the total cellular uptake by flow cytometry when Saos-2 cells were\ntreated with ZF5.3-SNAP-Rho (up to 4-fold), only a modest increase\nwas observed when cells were treated with CPP12-SNAP-Rho (up to 2-fold).\nThese trends are mirrored by the FCS data: cells treated with increasing\nconcentrations of ZF5.3-SNAP-Rho showed a dose-dependent increase\nin cytosolic concentration as determined by FCS. Treatment concentrations\nof 1 and 2 μM ZF5.3-SNAP-Rho resulted in average cytosolic concentrations\nof 58 ± 6.1 nM and 110 ± 13 nM, respectively, corresponding\nto an average delivery efficiency of 6%. At the highest treatment\nconcentration of 3 μM, we observed an average cytosolic concentration\nof 251 ± 34 nM, a delivery efficiency of 8%. In contrast, the\ncytosolic concentration of CPP12-SNAP-Rho did not significantly increase\nas a function of treatment concentration. At the highest concentration\ntested, the delivery efficiency of CPP12-SNAP-Rho was less than 1%.', 'In order to evaluate the effect of incubation time on cytosolic\ndelivery, we treated Saos-2 cells with 1 μM solutions of ZF5.3-SNAP-Rho\nand CPP12-SNAP-Rho for 2 h (as opposed to 30 min) prior to analysis\nby flow cytometry and FCS (<xref rid="oc-2018-00446g_0005" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>C). Cells treated with ZF5.3-SNAP-Rho for 2 h and evaluated\nusing flow cytometry exhibited a 6.5-fold increase in total intracellular\nfluorescence relative to cells treated with the same concentration\nfor 30 min; under these conditions the cytosolic levels of ZF5.3-SNAP-Rho\ndetermined using FCS increased by a factor of 2 (117 ± 19 nM).\nCells treated with CPP12-SNAP-Rho for 2 h and evaluated using flow\ncytometry exhibited a 2-fold increase relative to cells treated with\nCPP12-SNAP-Rho for 30 min. However, in contrast to results obtained\nusing ZF5.3-SNAP-Rho, we were unable to detect a significant increase\nin the cytosolic concentration of CPP12-SNAP-Rho after prolonged incubation\ntimes. Taken as a whole, these studies demonstrate that FCS can be\nused to precisely examine the effects of treatment concentration and\nincubation time on the delivery of protein cargo into the cytosol,\nand that the extent of cytosolic localization cannot be predicted\nby flow cytometry alone.<xref rid="oc-2018-00446g_0005" ref-type="fig">5</xref>C). Cells treated with ZF5.3-SNAP-Rho for 2 h and evaluated\nusing flow cytometry exhibited a 6.5-fold increase in total intracellular\nfluorescence relative to cells treated with the same concentration\nfor 30 min; under these conditions the cytosolic levels of ZF5.3-SNAP-Rho\ndetermined using FCS increased by a factor of 2 (117 ± 19 nM).\nCells treated with CPP12-SNAP-Rho for 2 h and evaluated using flow\ncytometry exhibited a 2-fold increase relative to cells treated with\nCPP12-SNAP-Rho for 30 min. However, in contrast to results obtained\nusing ZF5.3-SNAP-Rho, we were unable to detect a significant increase\nin the cytosolic concentration of CPP12-SNAP-Rho after prolonged incubation\ntimes. Taken as a whole, these studies demonstrate that FCS can be\nused to precisely examine the effects of treatment concentration and\nincubation time on the delivery of protein cargo into the cytosol,\nand that the extent of cytosolic localization cannot be predicted\nby flow cytometry alone.'], 'oc-2018-00446g_0006': ['Next, we devised\nan in cellulo assay to evaluate whether an unlabeled\nSNAP-tag conjugate would retain self-labeling activity following intracellular\ndelivery (<xref rid="oc-2018-00446g_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>A).\nWe treated cells with 3 μM of each unlabeled uCPP, CPMP, or\ncCPP SNAP-tag conjugate for 30 min, washed the cells with DPBS (Dulbecco’s\nphosphate buffered saline), and performed the labeling reaction <xref rid="oc-2018-00446g_0006" ref-type="fig">6</xref>A).\nWe treated cells with 3 μM of each unlabeled uCPP, CPMP, or\ncCPP SNAP-tag conjugate for 30 min, washed the cells with DPBS (Dulbecco’s\nphosphate buffered saline), and performed the labeling reaction in cellulo by incubating the cells in media containing 1\nμM SNAP-Cell© 505-Star for an additional 45 min. After\nthe labeling period, the cells were incubated with fresh media for\n30 min to remove unreacted SNAP-Cell© 505-Star, washed with DPBS,\nlifted with trypsin, and imaged using confocal microscopy (<xref rid="oc-2018-00446g_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>B). Cells treated\nwith SNAP, CPP9-SNAP, and ZiF-SNAP displayed no significant intracellular\nfluorescence after treatment with SNAP-Cell© 505-Star. Cells\ntreated with CPP12-SNAP displayed low levels of punctate fluorescence,\nwhereas cells treated with aPP5.3-SNAP and R8-SNAP displayed bright\npunctate fluorescence. Cells treated with Pen-SNAP and ZF5.3-SNAP\nexhibited exceptionally bright punctate fluorescence and low levels\nof diffuse cytosolic staining. We then quantitatively compared the\nlevels of intracellular fluorescence (endosomes plus cytosol) after\ntreatment with SNAP-Cell© 505-Star using flow cytometry (<xref rid="oc-2018-00446g_0006" ref-type="fig">6</xref>B). Cells treated\nwith SNAP, CPP9-SNAP, and ZiF-SNAP displayed no significant intracellular\nfluorescence after treatment with SNAP-Cell© 505-Star. Cells\ntreated with CPP12-SNAP displayed low levels of punctate fluorescence,\nwhereas cells treated with aPP5.3-SNAP and R8-SNAP displayed bright\npunctate fluorescence. Cells treated with Pen-SNAP and ZF5.3-SNAP\nexhibited exceptionally bright punctate fluorescence and low levels\nof diffuse cytosolic staining. We then quantitatively compared the\nlevels of intracellular fluorescence (endosomes plus cytosol) after\ntreatment with SNAP-Cell© 505-Star using flow cytometry (<xref rid="oc-2018-00446g_0006" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>C). Overall, there\nis good agreement between the results of flow cytometry and confocal\nmicroscopy. Cells treated with Pen-SNAP and ZF5.3-SNAP and subsequently\nincubated with SNAP-Cell© 505-Star were approximately 3.6- and\n4.7-fold brighter, respectively, than cells treated with SNAP-Cell©\n505-Star alone. While we intended to measure the precise concentration\nof each SNAP-tag conjugate after labeling <xref rid="oc-2018-00446g_0006" ref-type="fig">6</xref>C). Overall, there\nis good agreement between the results of flow cytometry and confocal\nmicroscopy. Cells treated with Pen-SNAP and ZF5.3-SNAP and subsequently\nincubated with SNAP-Cell© 505-Star were approximately 3.6- and\n4.7-fold brighter, respectively, than cells treated with SNAP-Cell©\n505-Star alone. While we intended to measure the precise concentration\nof each SNAP-tag conjugate after labeling in cellulo using FCS, we were unable to completely remove unreacted SNAP-Cell©\n505-Star (or three other fluorescent SNAP-tag substrates) from the\ncytosol in untreated control samples and were concerned by the heterogeneous\nnature of the acquired traces. Notwithstanding this limitation, the\nconfocal microscopy and flow cytometry measurements confirm that delivered\nSNAP-tag retains its activity in cells, despite its travels through\nthe endosomal pathway.82 Overall, the level\nof in cellulo SNAP-tag activity exhibited by a given\nSNAP-conjugate correlates well with cytosolic delivery (determined\nby FCS). The minor discrepancies between these experiments could result,\nfor example, from subtle differences in SNAP-tag activity that depend\non CPP identity, or from a dye-dependent conformational change that\nalters CPP efficacy, or a combination of these (or other) effects.'], 'oc-2018-00446g_0007': ['With a set of Rho-tagged APEX2 enzymes in hand,\nwe first evaluated their total uptake into cells using confocal microscopy\n(<xref rid="oc-2018-00446g_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>A) and flow\ncytometry (<xref rid="oc-2018-00446g_0007" ref-type="fig">7</xref>A) and flow\ncytometry (<xref rid="oc-2018-00446g_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>B). Saos-2 cells were treated with 1 μM of each labeled enzyme\nfor 30 min and prepared for confocal microscopy and flow cytometry\nmeasurements in the same manner as described for SNAP-tag. Cells treated\nwith 1 μM APEX2-Rho displayed minimal levels of punctate fluorescence,\nwhereas cells treated with 1 μM ZF5.3-APEX2-Rho displayed much\nbrighter, though predominantly punctate, intracellular fluorescence.\nFurther analysis by flow cytometry revealed that cells treated with\nZF5.3-APEX2-Rho were 2.4-fold brighter than cells treated with APEX2-Rho,\nconfirming that the appended miniature protein enhances the total\nuptake of APEX2 into cells. We then quantified the amount of APEX2-Rho\nand ZF5.3-Rho that reaches the cytosol using FCS (<xref rid="oc-2018-00446g_0007" ref-type="fig">7</xref>B). Saos-2 cells were treated with 1 μM of each labeled enzyme\nfor 30 min and prepared for confocal microscopy and flow cytometry\nmeasurements in the same manner as described for SNAP-tag. Cells treated\nwith 1 μM APEX2-Rho displayed minimal levels of punctate fluorescence,\nwhereas cells treated with 1 μM ZF5.3-APEX2-Rho displayed much\nbrighter, though predominantly punctate, intracellular fluorescence.\nFurther analysis by flow cytometry revealed that cells treated with\nZF5.3-APEX2-Rho were 2.4-fold brighter than cells treated with APEX2-Rho,\nconfirming that the appended miniature protein enhances the total\nuptake of APEX2 into cells. We then quantified the amount of APEX2-Rho\nand ZF5.3-Rho that reaches the cytosol using FCS (<xref rid="oc-2018-00446g_0007" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>C). The average diffusion times\n(τ<xref rid="oc-2018-00446g_0007" ref-type="fig">7</xref>C). The average diffusion times\n(τD) of ZF5.3-APEX2-Rho and APEX2-Rho measured in\nthe cytosol were 0.65 ± 0.057 and 0.95 ± 0.12 ms, respectively,\napproximately 4- and 6-fold longer than values acquired in\nvitro and at least 3-fold longer than the intracellular diffusion\ntime measured for ZF5.3R (Figure S9). FCS measurements revealed that the intracellular concentrations\nachieved by ZF5.3-APEX2-Rho ranged from 9 to 30 nM, with an average\nintracellular concentration of 17 ± 1.0 nM, whereas the intracellular\nconcentration of APEX2-Rho ranged from 2 to 9 nM, with an average\nof 4.7 ± 0.41 nM. The overall uptake and cytosolic concentration\nof ZF5.3-APEX2-Rho were lower compared to ZF5.3-SNAP-Rho, demonstrating\nthe impact of cargo on the potency of the appended CPMP. Certainly,\nmore work is needed to correlate cargo and CPP identity with the efficiency\nof cytosolic access and characterize in more detail the precise mechanism(s)\nby which endosomal escape occurs.'], 'oc-2018-00446g_0008': ['To evaluate whether APEX2 retained activity upon delivery to the\ncytosol by ZF5.3, we made use of an assay developed by Ting et al.68 that monitors the APEX2-dependent oxidation\nand fluorescence turn-on of the cell-permeant dye Amplex UltraRed\n(<xref rid="oc-2018-00446g_0008" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>A). Saos-2\ncells were incubated with 500 nM of heme-bound APEX2 or ZF5.3-APEX2\nfor 30 min, treated with trypsin to remove surface-bound enzyme, transferred\nto microcentrifuge tubes, and resuspended in 100 μL of DPBS\ncontaining 10 μM of Amplex UltraRed and 1 mM H<xref rid="oc-2018-00446g_0008" ref-type="fig">8</xref>A). Saos-2\ncells were incubated with 500 nM of heme-bound APEX2 or ZF5.3-APEX2\nfor 30 min, treated with trypsin to remove surface-bound enzyme, transferred\nto microcentrifuge tubes, and resuspended in 100 μL of DPBS\ncontaining 10 μM of Amplex UltraRed and 1 mM H2O2. After 1 min, intracellular fluorescence was analyzed using\nflow cytometry (<xref rid="oc-2018-00446g_0008" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>B). Cells treated with ZF5.3-APEX2 exhibited a 13-fold turn-on of\nfluorescence relative to cells treated with Amplex UltraRed alone,\nwhereas cells treated with APEX2 exhibited only a 1.2-fold increase\nin fluorescence. To visualize the APEX2-dependent turn-on of Amplex\nUltraRed fluorescence in live cells, we performed a similar experiment\nbut visualized cells individually using confocal microscopy (<xref rid="oc-2018-00446g_0008" ref-type="fig">8</xref>B). Cells treated with ZF5.3-APEX2 exhibited a 13-fold turn-on of\nfluorescence relative to cells treated with Amplex UltraRed alone,\nwhereas cells treated with APEX2 exhibited only a 1.2-fold increase\nin fluorescence. To visualize the APEX2-dependent turn-on of Amplex\nUltraRed fluorescence in live cells, we performed a similar experiment\nbut visualized cells individually using confocal microscopy (<xref rid="oc-2018-00446g_0008" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>C). Cells treated\nwith ZF5.3-APEX2 displayed exceptionally bright punctate and cytosolic\nfluorescence, whereas cells treated with APEX2 did not exhibit any\nsignificant turn-on of Amplex UltraRed within the same imaging time\nframe. Although these assays demonstrate that ZF5.3-APEX2-His<xref rid="oc-2018-00446g_0008" ref-type="fig">8</xref>C). Cells treated\nwith ZF5.3-APEX2 displayed exceptionally bright punctate and cytosolic\nfluorescence, whereas cells treated with APEX2 did not exhibit any\nsignificant turn-on of Amplex UltraRed within the same imaging time\nframe. Although these assays demonstrate that ZF5.3-APEX2-His6 is active in cells, we acknowledge that the pattern of Amplex\nUltraRed fluorescence may not accurately depict the intracellular\nlocation of ZF5.3-APEX2-His6; Amplex UltraRed that is oxidized\nin endosomal compartments by entrapped ZF5.3-APEX2-His6 may also diffuse into the cytosol. Regardless, these results demonstrate\nthat the CPMP ZF5.3 can deliver active APEX2 into cells. Moreover,\nthese results emphasize the limited extent to which APEX2 activity—which,\nby nature, is amplified—correlates with the relative levels\nof APEX2 enzymes in the cell cytosol determined by FCS.']}
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Fluorescence Correlation Spectroscopy Reveals Efficient
Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins
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ACS Cent Sci
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1540364400
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None
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other
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PMC6202653
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[
""
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ACS Cent Sci. 2018 Oct 24; 4(10):1379-1393
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NO-CC CODE
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Performance for spatially disparate conditions. a Modality index as a function of SRT for spatially aligned A1275V stimuli. MI gradually runs from values near +1 (A-response) to −1 (V-response) in this single-clustered dataset. b Disparate condition (∆Φ = ±90°). K-means cluster analysis yielded two distinct clusters: blue squares, cluster 1 data; red circles, cluster 2 data. Cluster 1 represent aurally driven saccades, cluster 2 are visually triggered responses. c Error versus SRT for all 24 AV conditions of the cluster 2 data, normalized by mean V-saccade error and SRT (red circles) d Error versus SRT for all 24 AV conditions of the cluster 1 data, normalized by mean A-saccade error and SRT (blue squares). Gray squares in (c, d) represent conditions that yielded single-clustered data (because of low silhouette-values [<0.6] or a small number of responses in one of the two K-means clusters [<5]). e MI as a function of the perceived disparity (in deg) and SRT for A1275V stimuli. f Same for V75A12 stimuli
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221_2009_1815_Fig8_HTML
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2
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d7759d168be4bbb0594252f2fd0f297f27e6d1137b34742abc7789fbedc4fce9
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221_2009_1815_Fig8_HTML.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
466,
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[{'image_id': '221_2009_1815_Fig7_HTML', 'image_file_name': '221_2009_1815_Fig7_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig7_HTML.jpg', 'caption': 'Absolute localization error as a function of SRT. The 2D response distributions for unisensory visual (orange dots, red ellipse) and auditory targets (blue dots and ellipse) are shown in (a–d) for comparison with the four spatially aligned bisensory response distributions: a V75A12 (gray diamonds), b V75A18 (gray diamonds), c A1275V (blue squares and red circles), and d A1875V (blue squares and red circles). Either the unisensory visual or auditory distribution is shifted by ±75\xa0ms, to align SRTs with the first stimulus of the AV-trials. Ellipses circumscribe 1 SD around the mean. Only unisensory data within 2 SD of the mean are shown. The blue squares and red dots in (c) and (d) were obtained through K-means cluster analysis on the bisensory data (mean silhouette-values: 0.76 and 0.73, respectively). Black ellipses indicate means and SD of the clusters. Note that bisensory distributions have on average reduced SRTs and smaller localization errors than the unisensory distributions', 'hash': '846d768c6a99e1a697a59cebd1cfae589bf2acd4b9e7483316758458f9b2515f'}, {'image_id': '221_2009_1815_Fig8_HTML', 'image_file_name': '221_2009_1815_Fig8_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig8_HTML.jpg', 'caption': 'Performance for spatially disparate conditions. a Modality index as a function of SRT for spatially aligned A1275V stimuli. MI gradually runs from values near +1 (A-response) to −1 (V-response) in this single-clustered dataset. b Disparate condition (∆Φ\xa0=\xa0±90°). K-means cluster analysis yielded two distinct clusters: blue squares, cluster 1 data; red circles, cluster 2 data. Cluster 1 represent aurally driven saccades, cluster 2 are visually triggered responses. c Error versus SRT for all 24 AV conditions of the cluster 2 data, normalized by mean V-saccade error and SRT (red circles) d Error versus SRT for all 24 AV conditions of the cluster 1 data, normalized by mean A-saccade error and SRT (blue squares). Gray squares in (c, d) represent conditions that yielded single-clustered data (because of low silhouette-values [<0.6] or a small number of responses in one of the two K-means clusters [<5]). e MI as a function of the perceived disparity (in deg) and SRT for A1275V stimuli. f Same for V75A12 stimuli', 'hash': 'd7759d168be4bbb0594252f2fd0f297f27e6d1137b34742abc7789fbedc4fce9'}, {'image_id': '221_2009_1815_Fig6_HTML', 'image_file_name': '221_2009_1815_Fig6_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig6_HTML.jpg', 'caption': 'Comparison of localization error distributions in elevation. a–d The bisensory (gray patch) and unisensory (V, red; A, blue) error distributions for a V75A12, b V75A18, c A1275V, and d A1875V. Note that, for each condition, the AV distribution is shifted toward smaller errors than the best unisensory (visual) distribution', 'hash': 'bfcc4468e2590377ad9845a0efe84dcff6cf8c4a0cf64c5e9f9a422598edc3d0'}, {'image_id': '221_2009_1815_Fig1_HTML', 'image_file_name': '221_2009_1815_Fig1_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig1_HTML.jpg', 'caption': 'Audiovisual paradigm. a Spatial representation of stimulus events. Subjects had to make a saccade to a red visual (red star) stimulus, ignoring an auditory distractor (highlighted speaker). The background consisted of diffuse noise from 9 small speakers and 85 green LEDs (green dots). Visual targets were randomly chosen from 12 locations (red diamonds). b The position of the distractor could coincide with the visual target (red star), or deviate in either direction (±45, ±90, 180°), or eccentricity (by a factor of 0.67 or 1.4–1.6). c Temporal events in a trial. The distractor could be presented at one of two temporal asynchronies (±75\xa0ms), and one of two SNRs (−12 and −18\xa0dB for background)', 'hash': 'd876047821900edcb783d67ccf2d5ea2b39a870bb862265c786f0dbf6d103bc8'}, {'image_id': '221_2009_1815_Fig2_HTML', 'image_file_name': '221_2009_1815_Fig2_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig2_HTML.jpg', 'caption': 'Predictions of models. a Hypothetical distributions of SRT-localization errors for unisensory responses (blue: A-only, orange: V-only). Blue and red ellipses denote 1 SD around the mean. A—responses are fast but inaccurate; V—saccades are accurate but slow. b Distribution of SRTs according to two conceptual models: gray shading: bistable model (with α\xa0=\xa01/3, Eq.\xa07), black curve: race model (Eq.\xa06). The blue–red curve indicates a higher probability of eliciting an auditory response in blue and a visual response in red. c Distribution of AV saccade errors according to the bistable model (gray shading). Blue and red curves indicate the unisensory error distributions (auditory and visual, respectively). Note that the race model would predict an error distribution close to the auditory response distributions', 'hash': '18d82d7a158250c85d1d06075b442e568a2e61f885cdf142aeb069a06c36769e'}, {'image_id': '221_2009_1815_Fig5_HTML', 'image_file_name': '221_2009_1815_Fig5_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig5_HTML.jpg', 'caption': 'Comparison of SRT distributions for AV-aligned trials. a–d The bisensory SRT distributions (gray patch) for a V75A12, b V75A18, c A1275V, and d A1875V, are shown as probability distributions. a–d Predicted bistable distributions (Eq.\xa07) are shown as red–blue curves, with blue indicating a larger probability of responding to A at a given SRT, and red indicating a larger probability for V. Parameter α is probability of responding to an auditory stimulus in a given trial (irrespective of SRT, Eq.\xa07). e Comparison of bisensory SRTs to the predictions of the stochastic-independent race model (Eq.\xa06)', 'hash': 'efbadf9146442e0dc6d0f0727be3f7579097ca3736a42e083792375756516544'}, {'image_id': '221_2009_1815_Fig3_HTML', 'image_file_name': '221_2009_1815_Fig3_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig3_HTML.jpg', 'caption': 'Effect of AV-background on V-saccades. Red/black symbols: V/VNOBG.a, b Stimulus–response plots of endpoints of primary V-saccades against stimulus a azimuth and b elevation. c Cumulative SRT probability functions. d Absolute localization error as a function of SRT. Ellipses circumscribe 2 SD around the mean. Data pooled across subjects', 'hash': '62b20941cee7e96546ba26c2d7e7aedcb500e3658d94309806b2bdf9fbc12d30'}, {'image_id': '221_2009_1815_Fig4_HTML', 'image_file_name': '221_2009_1815_Fig4_HTML.jpg', 'image_path': '../data/media_files/PMC2733184/221_2009_1815_Fig4_HTML.jpg', 'caption': 'Effect of AV-background of A-saccades. a, b Stimulus–response plots of A-saccade endpoints against a stimulus azimuth and b elevation. Blue/black symbols: A/ANOBG. c Cumulative SRT probability functions for the ANOBG- (black line), A6-, A12-, A18-, and A21 saccades. d Absolute localization error plotted as a function of SRT. Data pooled across subjects. Ellipses circumscribe 2 SD around the mean. e Response gains for azimuth (gray circles; black: mean across subjects) and elevation (cyan squares; blue: mean across subjects) of primary A-saccades as a function of SNR. f Final saccade response gains for V- (red) and A-saccades', 'hash': '7502e87c7e2cbfc8d89e8c3df1818f21c9548273df4b86c14c7e1f0342e9eabd'}]
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{'221_2009_1815_Fig1_HTML': ['The stimulus array consisted of 85 light emitting diodes (LED) mounted onto a thin wire frame at 85\xa0cm in front of the subject (Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>). The LEDs were arranged in 7 concentric circles at eccentricities R ∈ [2; 5; 9; 14; 20; 27; 35]°, and placed at 12 different directions (Φ ∈ [0; 30; 60; …; 330]°, where Φ\xa0=\xa00° is rightward, Φ\xa0=\xa090° is upward, etc. (Fig.\xa0). The LEDs were arranged in 7 concentric circles at eccentricities R ∈ [2; 5; 9; 14; 20; 27; 35]°, and placed at 12 different directions (Φ ∈ [0; 30; 60; …; 330]°, where Φ\xa0=\xa00° is rightward, Φ\xa0=\xa090° is upward, etc. (Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>). All LEDs could illuminate either red (0.18\xa0cd/m). All LEDs could illuminate either red (0.18\xa0cd/m2) or green (0.24\xa0cd/m2). To produce the visual background all 85 LEDs were turned green. The visual fixation point at [R,Φ]\xa0=\xa0[0,0]° and the target was subsequently specified by turning the appropriate LED from green to red.Fig.\xa01Audiovisual paradigm. a Spatial representation of stimulus events. Subjects had to make a saccade to a red visual (red star) stimulus, ignoring an auditory distractor (highlighted speaker). The background consisted of diffuse noise from 9 small speakers and 85 green LEDs (green dots). Visual targets were randomly chosen from 12 locations (red diamonds). b The position of the distractor could coincide with the visual target (red star), or deviate in either direction (±45, ±90, 180°), or eccentricity (by a factor of 0.67 or 1.4–1.6). c Temporal events in a trial. The distractor could be presented at one of two temporal asynchronies (±75\xa0ms), and one of two SNRs (−12 and −18\xa0dB for background)', 'The auditory background was generated by a circular array of nine speakers (Nellcor), mounted onto the wire frame at about 45° eccentricity (Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>a). Sound intensities were measured at the position of the subject’s head with a calibrated sound amplifier and microphone (Brüel & Kjaer BK2610/BK4144, Norcross, GA), and are expressed in dBA. The auditory background consisted of broadband Gaussian white noise (0.2–20 kHz) at a fixed intensity of 60\xa0dBA. The auditory distractor stimulus was produced by a broadband lightweight speaker (Philips AD-44725, Eindhoven, the Netherlands) mounted on a two-link robot, which allowed the speaker to be positioned in any direction at a distance of 90\xa0cm (Hofman and Van Opstal a). Sound intensities were measured at the position of the subject’s head with a calibrated sound amplifier and microphone (Brüel & Kjaer BK2610/BK4144, Norcross, GA), and are expressed in dBA. The auditory background consisted of broadband Gaussian white noise (0.2–20 kHz) at a fixed intensity of 60\xa0dBA. The auditory distractor stimulus was produced by a broadband lightweight speaker (Philips AD-44725, Eindhoven, the Netherlands) mounted on a two-link robot, which allowed the speaker to be positioned in any direction at a distance of 90\xa0cm (Hofman and Van Opstal 1998). The auditory distractor stimulus consisted of a periodic broad-band noise (period 20\xa0ms, sounding like a 50\xa0Hz buzzer) that had a flat broad-band characteristic between 0.2 and 20\xa0kHz, presented at a variable intensity (see below).', 'Subjects generated saccades amidst an AV-background to V- and AV-targets. Each trial began with the appearance of the AV-background (Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>a). After a randomly selected delay of either 150, 275, or 400\xa0ms, the central LED turned red, which the subject had to fixate for 600–850\xa0ms. The fixation LED was then turned green, and after a 200\xa0ms gap a peripheral red target LED was illuminated. Subjects had to generate a saccade quickly and accurately to the peripheral target LED. The location of the target was selected pseudo-randomly from 1 out of 12 possible locations (12 directions, a). After a randomly selected delay of either 150, 275, or 400\xa0ms, the central LED turned red, which the subject had to fixate for 600–850\xa0ms. The fixation LED was then turned green, and after a 200\xa0ms gap a peripheral red target LED was illuminated. Subjects had to generate a saccade quickly and accurately to the peripheral target LED. The location of the target was selected pseudo-randomly from 1 out of 12 possible locations (12 directions, R\xa0=\xa020, 27°; Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>a).a).', 'An auditory distractor was chosen from 8 possible locations for the visual target (Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>b): aligned (distractor presented behind the target LED); same direction but either 0.67 or 1.4–1.6\xa0times the radial eccentricity; and same eccentricity but rotated at ±45, ±90, or 180° away from the target location. The visual target either preceded the distractor by 75\xa0ms (V75A), or followed by 75\xa0ms (A75V) at equal probability. The auditory distractor was presented at one of two possible SNRs: −12\xa0dB (Ab): aligned (distractor presented behind the target LED); same direction but either 0.67 or 1.4–1.6\xa0times the radial eccentricity; and same eccentricity but rotated at ±45, ±90, or 180° away from the target location. The visual target either preceded the distractor by 75\xa0ms (V75A), or followed by 75\xa0ms (A75V) at equal probability. The auditory distractor was presented at one of two possible SNRs: −12\xa0dB (A12) or −18\xa0dB (A18) relative to the background (Fig.\xa0<xref rid="221_2009_1815_Fig1_HTML" ref-type="fig">1</xref>c). We also interspersed 36\xa0V-only trials (selected from 12 possible directions and three eccentricities: 14, 20, and 27°). Thus, the total number of different trials in the AV paradigm was 420 ([12 target locations\xa0×\xa08 spatial disparities\xa0×\xa02 asynchronies\xa0×\xa02 SNRs]\xa0+\xa036\xa0V conditions). These were presented as four blocks of 105 randomly selected trials, with a short rest in between. The order of the blocks was randomized from session to session; each subject completed multiple blocks yielding 1004–2391 saccades per subject (Table\xa0c). We also interspersed 36\xa0V-only trials (selected from 12 possible directions and three eccentricities: 14, 20, and 27°). Thus, the total number of different trials in the AV paradigm was 420 ([12 target locations\xa0×\xa08 spatial disparities\xa0×\xa02 asynchronies\xa0×\xa02 SNRs]\xa0+\xa036\xa0V conditions). These were presented as four blocks of 105 randomly selected trials, with a short rest in between. The order of the blocks was randomized from session to session; each subject completed multiple blocks yielding 1004–2391 saccades per subject (Table\xa01). Note that in each block only 15–18% of all trials contained spatially aligned AV stimuli.Table\xa01Stimulus types used in the experimentsStimulus typesNumber of responsesAbbrTargetDistractorSNRSOABCKGRMWJGJOABJVTotalVNOBGVisual––––7863106526843592791VVisual–––+1357613912543518A18,NOBGAuditory–−18––750506150236A21,NOBGAuditory–−21––750506650241A18Auditory–−18–+7418755050267A21Auditory–−21–+7315755047260V75A18VisualAuditory−18+75+3061812962591081,150V75A21VisualAuditory−21+75+288180290253991,110A1875VVisualAuditory−18−75+287171289251971,095A2175VVisualAuditory−21−75+2921702952501011,108Total2,3911,1212,2112,0491,0048,776Abbr: Abbreviations for the various stimulus types. Target: Target modality (either auditory of visual). Distractor: present or absent. SNR: The signal-to-noise ratio of the auditory distractor. SOA: stimulus onset asynchrony, either −75\xa0ms (auditory leading) or +75\xa0ms (visual leading). BCKGR: presence of the AV-background'], '221_2009_1815_Fig2_HTML': ['With P(τB) the predicted bistable distribution, α the probability of responding to an auditory stimulus, and 1-α the probability of responding to a visual stimulus, the bistable SRT distributions will resemble the weighted summed distributions of the A and V- saccades in which the probability α acts as weighting parameter. Once again, as with the race model, deviations from this independent model may indicate multisensory integration. Figure\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>b and c illustrates the predictions of the stochastically independent (Eq.\xa0b and c illustrates the predictions of the stochastically independent (Eq.\xa06) race and bistable models for simulated data (Fig.\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>a).a).Fig.\xa02Predictions of models. a Hypothetical distributions of SRT-localization errors for unisensory responses (blue: A-only, orange: V-only). Blue and red ellipses denote 1 SD around the mean. A—responses are fast but inaccurate; V—saccades are accurate but slow. b Distribution of SRTs according to two conceptual models: gray shading: bistable model (with α\xa0=\xa01/3, Eq.\xa07), black curve: race model (Eq.\xa06). The blue–red curve indicates a higher probability of eliciting an auditory response in blue and a visual response in red. c Distribution of AV saccade errors according to the bistable model (gray shading). Blue and red curves indicate the unisensory error distributions (auditory and visual, respectively). Note that the race model would predict an error distribution close to the auditory response distributions', 'The separated clusters (black ellipses) can be readily compared to the straightforward bistable model, which would yield two AV-clusters coinciding with either unisensory V- and A-distribution (Fig.\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>). For the V75A stimuli and also for larger numbers of clusters on the A75V stimuli, the silhouette-values quickly dropped to values <0.5, indicating that a larger number of clusters is not readily observed in the data.). For the V75A stimuli and also for larger numbers of clusters on the A75V stimuli, the silhouette-values quickly dropped to values <0.5, indicating that a larger number of clusters is not readily observed in the data.'], '221_2009_1815_Fig8_HTML': ['These measures quantify the distance (in deg) between an AV response and the perceived unisensory locations of V and A, respectively. Finally, from the perceptual localization errors we defined a dimensionless modality index, MI:12\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{mathrsfs}\n\\usepackage{upgreek}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}$$ {\\text{MI}} = \\frac{{\\text{PE}}_{\\text{V}} - {\\text{PE}}_{\\text{A}}}{{\\text{PE}}_{\\text{V}} + {\\text{PE}}_{\\text{A}}} $$\\end{document}\nwhich indicates, for each AV response, whether it lies closer to the A percept (MI\xa0=\xa0+1, as PEV\xa0≫\xa0PEA) or V percept (MI\xa0=\xa0−1; PEA\xa0≫\xa0PEV). A value of MI\xa0≈\xa00 suggests an integrated AV percept (see Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>).).', 'Figure\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>a, b shows the distributions of the AV modality index (“a, b shows the distributions of the AV modality index (“Methods”, Eq.\xa011) versus SRT for two AV conditions. The MI is a measure for the resemblance of a particular AV response to either a unisensory V or A-saccade. Note that it is expressed in terms of the perceived, rather than the physical disparity, so that even the spatially aligned stimuli (e.g., Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>a) can be shown to have evoked both aurally and visually driven saccades (MI close to +1 and −1, respectively). Interestingly, the spatially aligned Aa) can be shown to have evoked both aurally and visually driven saccades (MI close to +1 and −1, respectively). Interestingly, the spatially aligned A1275V (Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>a) data seemed to consist mostly of intermediate AV-responses, and MI gradually shifted from +1 to −1 as time progressed. K-means clustering of these data on two or more clusters yielded low silhouette-values (<0.6) and few responses (<6) were assigned to one of the clusters.a) data seemed to consist mostly of intermediate AV-responses, and MI gradually shifted from +1 to −1 as time progressed. K-means clustering of these data on two or more clusters yielded low silhouette-values (<0.6) and few responses (<6) were assigned to one of the clusters.Fig.\xa08Performance for spatially disparate conditions. a Modality index as a function of SRT for spatially aligned A1275V stimuli. MI gradually runs from values near +1 (A-response) to −1 (V-response) in this single-clustered dataset. b Disparate condition (∆Φ\xa0=\xa0±90°). K-means cluster analysis yielded two distinct clusters: blue squares, cluster 1 data; red circles, cluster 2 data. Cluster 1 represent aurally driven saccades, cluster 2 are visually triggered responses. c Error versus SRT for all 24 AV conditions of the cluster 2 data, normalized by mean V-saccade error and SRT (red circles) d Error versus SRT for all 24 AV conditions of the cluster 1 data, normalized by mean A-saccade error and SRT (blue squares). Gray squares in (c, d) represent conditions that yielded single-clustered data (because of low silhouette-values [<0.6] or a small number of responses in one of the two K-means clusters [<5]). e MI as a function of the perceived disparity (in deg) and SRT for A1275V stimuli. f Same for V75A12 stimuli', 'For A1275V stimuli with a considerable angular disparity (here ΔΦ\xa0=\xa0±90°; Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>b), however, K-means cluster analysis produced two clear distributions that appeared to obey the principles of a bistable mechanism: either auditory (blue), or visual (red) responses.b), however, K-means cluster analysis produced two clear distributions that appeared to obey the principles of a bistable mechanism: either auditory (blue), or visual (red) responses.', 'Figure\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>c, d summarizes our findings for all 24 AV stimulus conditions employed in this study. In 17/24 conditions the response data could be separated into two clusters (single-cluster conditions: V75Ac, d summarizes our findings for all 24 AV stimulus conditions employed in this study. In 17/24 conditions the response data could be separated into two clusters (single-cluster conditions: V75A12, Δφ\xa0=\xa090 and ΔR\xa0=\xa01.5; V75A18, Δφ\xa0=\xa00 and Δφ\xa0=\xa0180; A1275V, Δφ\xa0=\xa00; A1875V Δφ\xa0=\xa00 and Δφ\xa0=\xa090). Figure\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>c normalizes the cluster with the longest SRT against the V-responses, whereas in Fig.\xa0c normalizes the cluster with the longest SRT against the V-responses, whereas in Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>d the cluster with the shortest SRT was normalized against A-saccades (−12 and −18\xa0dB). If these responses would follow the simple bistable model of Fig.\xa0d the cluster with the shortest SRT was normalized against A-saccades (−12 and −18\xa0dB). If these responses would follow the simple bistable model of Fig.\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>, all points would scatter around the center of these plots. As data points lie predominantly in the lower-left quadrant, the interesting point of this analysis is, that for all stimulus conditions responses were actually , all points would scatter around the center of these plots. As data points lie predominantly in the lower-left quadrant, the interesting point of this analysis is, that for all stimulus conditions responses were actually better (i.e., faster and more accurate) than pure V- and A-saccades. Hence, even for spatially unaligned stimuli, AV enhancement occurs and the simple bistable model should be rejected.', 'Figure\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>e, f summarizes our analysis for all perceived disparities of the Ae, f summarizes our analysis for all perceived disparities of the A1275V and V75A12 stimuli. A clear pattern emerges in this plot: only when perceived disparity is very small, MI is close to zero (green-colored bins), indicative for multisensory integration. It rapidly splits into two clusters for larger perceived disparities, with invariably aurally guided responses (blue) for the short SRTs (<250\xa0ms), and visually guided saccades for longer SRTs (red). Hence, these plots delineate a sharply-defined spatial–temporal window of AV integration. Similar results were obtained for the A18 distractor (not shown).', 'In line with our observations on bistability, Corneil et\xa0al. (2002) found no bimodal response distributions. Note that in their study the perceived stimulus disparity was small compared to the current study (data not shown, but mean\xa0±\xa0SD: 3.3\xa0±\xa01.4 vs. 19.8\xa0±\xa015.3°, respectively). The present study indicates that a small perceived disparity (<10°) does not elicit bistable responses (e.g., Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>e, f).e, f).', 'Also in spatially unaligned conditions early responses were acoustically triggered and, therefore, typically ended near the location of the distractor (Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>). Later responses were guided toward the visual target (Fig.\xa0). Later responses were guided toward the visual target (Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>c–f). The data from those AV stimuli thus seem to follow the predictions of the bistable model (cf. Fig.\xa0c–f). The data from those AV stimuli thus seem to follow the predictions of the bistable model (cf. Fig.\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>) much better. However, the quantitative analysis of Fig.\xa0) much better. However, the quantitative analysis of Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>c, d indicates that even in the situation of large spatial disparities the system is not driven exclusively by one stimulus modality, as responses are clearly influenced by the other modality too. Hence, a weaker form of multisensory enhancement persists that allows these responses to still outperform the unisensory-evoked saccades.c, d indicates that even in the situation of large spatial disparities the system is not driven exclusively by one stimulus modality, as responses are clearly influenced by the other modality too. Hence, a weaker form of multisensory enhancement persists that allows these responses to still outperform the unisensory-evoked saccades.', 'Taken together, our data show that the saccadic system rapidly accounts for the spatial–temporal relations between an auditory and visual event, and uses this information efficiently to allow multisensory integration to occur, provided the perceived spatial disparity is small. For disparities exceeding approximately 10–15°, the stimuli are treated as arising from different objects in space (Kording et\xa0al. 2007; Sato et\xa0al. 2007), which results in a bistable response mode (Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>e, f). Thus, when forced to respond rapidly to a specified target, the system is prone to frequent localization errors. However, even in that case multisensory integration occurs, as the putative stimuli evoked faster and more accurate responses than their unisensory counterparts.e, f). Thus, when forced to respond rapidly to a specified target, the system is prone to frequent localization errors. However, even in that case multisensory integration occurs, as the putative stimuli evoked faster and more accurate responses than their unisensory counterparts.'], '221_2009_1815_Fig3_HTML': ['The AV-background hampered localization accuracy of unisensory visual targets (Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>a, b). V-trials displayed a larger amount of scatter in primary saccade responses than visual trials in the no-background condition (Va, b). V-trials displayed a larger amount of scatter in primary saccade responses than visual trials in the no-background condition (VNOBG, Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>a, b: red squares and black circles, respectively), both in azimuth (Fig.\xa0a, b: red squares and black circles, respectively), both in azimuth (Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>a) and in elevation (Fig.\xa0a) and in elevation (Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>b). This resulted in lower correlations between stimulus and response (b). This resulted in lower correlations between stimulus and response (r2\xa0=\xa00.98 for VNOBG and ~0.89 for V, P\xa0≪\xa00.001). The subject’s SRT increased by about 100\xa0ms in the presence of the AV-background (Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>c). The 2D distributions of absolute localization error versus SRT for both conditions (Fig.\xa0c). The 2D distributions of absolute localization error versus SRT for both conditions (Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>d; Vd; VNOBG and V: black circles and red squares, respectively) are clearly distinguishable from one another (2D KS-test, P\xa0≪\xa00.001).Fig.\xa03Effect of AV-background on V-saccades. Red/black symbols: V/VNOBG.a, b Stimulus–response plots of endpoints of primary V-saccades against stimulus a azimuth and b elevation. c Cumulative SRT probability functions. d Absolute localization error as a function of SRT. Ellipses circumscribe 2 SD around the mean. Data pooled across subjects'], '221_2009_1815_Fig4_HTML': ['Localization performance of A-saccades was compromised even more by the AV-background albeit in different ways than V-saccades. First, the azimuth and elevation components of A-responses were affected differently (Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>a, b). For example, the Aa, b). For example, the A12 responses (Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>a, b, black circles) were more accurate in azimuth than in elevation (i.e., less scatter and a higher response gain). This property results from the different neural processing pathways of sound–location coordinates (binaural difference cues, for azimuth, versus pinna-related spectral shape cues, for elevation; e.g., Oldfield and Parker a, b, black circles) were more accurate in azimuth than in elevation (i.e., less scatter and a higher response gain). This property results from the different neural processing pathways of sound–location coordinates (binaural difference cues, for azimuth, versus pinna-related spectral shape cues, for elevation; e.g., Oldfield and Parker 1984; Blauert 1997; Hofman and Van Opstal 1998). Second, localization performance depended strongly on the SNR too. The A18 responses (Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>a, b, purple squares) had a lower gain and more scatter in elevation than the Aa, b, purple squares) had a lower gain and more scatter in elevation than the A12 responses. Furthermore, the SRTs were prolonged for decreasing SNRs (CDFs in Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>c). Also the distributions of absolute localization error versus SRT for the various SNRs clearly differed from one another (Fig.\xa0c). Also the distributions of absolute localization error versus SRT for the various SNRs clearly differed from one another (Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>d). These features are summarized in Fig.\xa0d). These features are summarized in Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>e, which shows that azimuth gain (black circles) dropped for decreasing SNRs, but the elevation gain (blue squares) dropped even faster. These results are in accordance with earlier studies that reported a degrading effect of background noise on sound-localization performance (Good and Gilkey e, which shows that azimuth gain (black circles) dropped for decreasing SNRs, but the elevation gain (blue squares) dropped even faster. These results are in accordance with earlier studies that reported a degrading effect of background noise on sound-localization performance (Good and Gilkey 1996; Zwiers et\xa0al. 2001; Corneil et\xa0al. 2002).Fig.\xa04Effect of AV-background of A-saccades. a, b Stimulus–response plots of A-saccade endpoints against a stimulus azimuth and b elevation. Blue/black symbols: A/ANOBG. c Cumulative SRT probability functions for the ANOBG- (black line), A6-, A12-, A18-, and A21 saccades. d Absolute localization error plotted as a function of SRT. Data pooled across subjects. Ellipses circumscribe 2 SD around the mean. e Response gains for azimuth (gray circles; black: mean across subjects) and elevation (cyan squares; blue: mean across subjects) of primary A-saccades as a function of SNR. f Final saccade response gains for V- (red) and A-saccades', 'An important difference between V- and A-saccades, which cannot be readily observed from the primary saccade responses, is the difference in localization percepts induced by the AV-background (Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>f). Although it could take a few attempts/saccades, subjects eventually localized the V-target (red line). In contrast, the background noise introduced a large undershoot in azimuth and elevation also for the final A-saccades. This aspect is important for the AV-disparity experiment, since the f). Although it could take a few attempts/saccades, subjects eventually localized the V-target (red line). In contrast, the background noise introduced a large undershoot in azimuth and elevation also for the final A-saccades. This aspect is important for the AV-disparity experiment, since the stimulus disparity between A- and V-targets deviated from the perceptual disparity. We will return to this difference in a later section.', 'We studied the responses of the human saccadic system when faced with a visual orienting task in a rich AV environment and a competing auditory distractor. Our experiments extend the findings from Corneil et\xa0al. (2002) who assessed AV integration when visual and auditory stimuli both served as a target, and were always spatially aligned. Under such conditions the system responded according to a “best of both worlds” principle: as A-only saccades are typically fast but inaccurate (Fig.\xa0<xref rid="221_2009_1815_Fig4_HTML" ref-type="fig">4</xref>), and V-saccades are accurate but slow (Fig.\xa0), and V-saccades are accurate but slow (Fig.\xa0<xref rid="221_2009_1815_Fig3_HTML" ref-type="fig">3</xref>), the AV-responses were both fast ), the AV-responses were both fast and accurate. These experiments demonstrated a clear integration of AV channels, whereby the interaction strength depended on the SNR of the target sound and the temporal asynchrony of the stimuli.'], '221_2009_1815_Fig5_HTML': ['Figure\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>a–d presents the SRT distributions for the unisensory (A, blue; V, red) and AV (gray patch) stimuli. The V75A stimuli (auditory lagging; Fig.\xa0a–d presents the SRT distributions for the unisensory (A, blue; V, red) and AV (gray patch) stimuli. The V75A stimuli (auditory lagging; Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>a, b) both exhibit a single-peaked distribution with shorter SRTs than either unisensory distribution. This multisensory enhancement exceeds the prediction of statistical facilitation by the stochastically independent race model (Fig.\xa0a, b) both exhibit a single-peaked distribution with shorter SRTs than either unisensory distribution. This multisensory enhancement exceeds the prediction of statistical facilitation by the stochastically independent race model (Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>e, Eq.\xa0e, Eq.\xa06). This held in particular for the V75A18 stimulus, which also exceeded the negative-dependency race model (Eq.\xa04). This phenomenon of largest enhancement for weakest stimuli has been termed inverse effectiveness in the neurophysiological literature (Stein and Meredith 1993).Fig.\xa05Comparison of SRT distributions for AV-aligned trials. a–d The bisensory SRT distributions (gray patch) for a V75A12, b V75A18, c A1275V, and d A1875V, are shown as probability distributions. a–d Predicted bistable distributions (Eq.\xa07) are shown as red–blue curves, with blue indicating a larger probability of responding to A at a given SRT, and red indicating a larger probability for V. Parameter α is probability of responding to an auditory stimulus in a given trial (irrespective of SRT, Eq.\xa07). e Comparison of bisensory SRTs to the predictions of the stochastic-independent race model (Eq.\xa06)', 'In contrast, the A75V stimuli (auditory leading; Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>c, d) both produced bimodal SRT distributions, with longer SRTs than the fastest A-distribution. Interestingly, bimodal response distributions were not obtained in the Corneil et\xa0al. (c, d) both produced bimodal SRT distributions, with longer SRTs than the fastest A-distribution. Interestingly, bimodal response distributions were not obtained in the Corneil et\xa0al. (2002) study (see also “Discussion”). Note that the stochastically independent race model (Eq.\xa06) is also violated for these stimuli (Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>e), as it predicts a single-peaked, faster (or equally fast) SRT distribution for all AV stimuli (the response SRTs even fail to reach the lower bound of the race model of Eq.\xa0e), as it predicts a single-peaked, faster (or equally fast) SRT distribution for all AV stimuli (the response SRTs even fail to reach the lower bound of the race model of Eq.\xa05, not shown). Yet, the measured distribution does not coincide with the predicted bistable response distribution of Eq.\xa07 (e.g., Fig.\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>b) either. Thus, we conclude that both AV stimulus types underwent multisensory integration.b) either. Thus, we conclude that both AV stimulus types underwent multisensory integration.', 'In contrast, two response clusters might be expected for A75V stimuli, corresponding to bistable responses (Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>c, d). We therefore performed a K-means clustering analysis (K\xa0=\xa02, based on SRT, response azimuth, elevation, eccentricity, and direction), which indeed divided the data into distinct distributions (labeled by blue squares and red circles; Fig.\xa0c, d). We therefore performed a K-means clustering analysis (K\xa0=\xa02, based on SRT, response azimuth, elevation, eccentricity, and direction), which indeed divided the data into distinct distributions (labeled by blue squares and red circles; Fig.\xa0<xref rid="221_2009_1815_Fig7_HTML" ref-type="fig">7</xref>c, d) with relatively high silhouette-values (0.76 for Ac, d) with relatively high silhouette-values (0.76 for A1275V and 0.73 for A1875V).', 'Our data indicate that the orienting task belied a dichotomy, which was quite hard for our subjects. This was especially clear for stimuli in which the distractor preceded the visual stimulus by 75\xa0ms (A75V condition; Figs.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>c, d and c, d and <xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>). In this case, the auditory input arrives substantially earlier in the CNS (by about 130\xa0ms) and as a consequence subjects were unable to ignore the auditory distractor at short SRTs (<250\xa0ms), as responses then appeared to be triggered by the sound. This was true for both spatially aligned (Figs.\xa0). In this case, the auditory input arrives substantially earlier in the CNS (by about 130\xa0ms) and as a consequence subjects were unable to ignore the auditory distractor at short SRTs (<250\xa0ms), as responses then appeared to be triggered by the sound. This was true for both spatially aligned (Figs.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>, , <xref rid="221_2009_1815_Fig7_HTML" ref-type="fig">7</xref>, and , and <xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>a) and -disparate stimuli (Fig.\xa0a) and -disparate stimuli (Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>e) and led to bimodal SRT distributions. A similar result for large horizontal eye-head gaze shifts was reported by Corneil and Munoz (e) and led to bimodal SRT distributions. A similar result for large horizontal eye-head gaze shifts was reported by Corneil and Munoz (1996) when salient AV stimuli were presented at opposite locations (ΔΦ\xa0=\xa0180°, ΔR\xa0=\xa080°) without an AV-background. However, the stimulus uncertainty in that study was limited, as target and distractor could occupy only two possible locations.', 'Note that the height of the first SRT peak reflected the SNR of the acoustic distractor (Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>c, d), which underlines our conclusion that these responses were indeed aurally guided (Fig.\xa0c, d), which underlines our conclusion that these responses were indeed aurally guided (Fig.\xa0<xref rid="221_2009_1815_Fig8_HTML" ref-type="fig">8</xref>a). Interestingly, however, for the relatively rare spatially aligned condition the SRT distributions for A75V stimuli differed from the predictions of both the race model (Fig.\xa0a). Interestingly, however, for the relatively rare spatially aligned condition the SRT distributions for A75V stimuli differed from the predictions of both the race model (Fig.\xa0<xref rid="221_2009_1815_Fig5_HTML" ref-type="fig">5</xref>e) and the bistable model (Fig.\xa0e) and the bistable model (Fig.\xa0<xref rid="221_2009_1815_Fig2_HTML" ref-type="fig">2</xref>b) in that later responses, triggered by the visual stimulus, still had faster than visual latencies. Moreover, even though early responses were acoustically triggered, their accuracy was better than for A-only saccades (Fig.\xa0b) in that later responses, triggered by the visual stimulus, still had faster than visual latencies. Moreover, even though early responses were acoustically triggered, their accuracy was better than for A-only saccades (Fig.\xa0<xref rid="221_2009_1815_Fig7_HTML" ref-type="fig">7</xref>). Thus, similar multisensory integration mechanisms as described by Corneil et\xa0al. (). Thus, similar multisensory integration mechanisms as described by Corneil et\xa0al. (2002) also appear to operate efficiently in a rich environment that contains much more uncertainty.'], '221_2009_1815_Fig6_HTML': ['Corneil et\xa0al. (2002) also showed that localization errors for aligned AV stimuli were smaller than for either unisensory stimulus. Figure\xa0<xref rid="221_2009_1815_Fig6_HTML" ref-type="fig">6</xref> demonstrates that this was also true in the distractor paradigm. For all four aligned AV conditions (Fig.\xa0 demonstrates that this was also true in the distractor paradigm. For all four aligned AV conditions (Fig.\xa0<xref rid="221_2009_1815_Fig6_HTML" ref-type="fig">6</xref>, gray patch), subjects localized more accurately than the V condition (Fig.\xa0, gray patch), subjects localized more accurately than the V condition (Fig.\xa0<xref rid="221_2009_1815_Fig6_HTML" ref-type="fig">6</xref>: V red; A blue).: V red; A blue).Fig.\xa06Comparison of localization error distributions in elevation. a–d The bisensory (gray patch) and unisensory (V, red; A, blue) error distributions for a V75A12, b V75A18, c A1275V, and d A1875V. Note that, for each condition, the AV distribution is shifted toward smaller errors than the best unisensory (visual) distribution'], '221_2009_1815_Fig7_HTML': ['To obtain an integrated overview of these data, Fig.\xa0<xref rid="221_2009_1815_Fig7_HTML" ref-type="fig">7</xref>a–d compares the response distributions of absolute localization error versus SRT. Note that the V75A stimuli (Fig.\xa0a–d compares the response distributions of absolute localization error versus SRT. Note that the V75A stimuli (Fig.\xa0<xref rid="221_2009_1815_Fig7_HTML" ref-type="fig">7</xref>a, b) yielded a single-cluster of AV-responses (gray diamonds with black ellipse at 1 SD around the mean), with an average SRT and error that was smaller than either unisensory response distribution (A-saccades: small blue dots and ellipse; V-saccades: small yellow dots and red ellipse).a, b) yielded a single-cluster of AV-responses (gray diamonds with black ellipse at 1 SD around the mean), with an average SRT and error that was smaller than either unisensory response distribution (A-saccades: small blue dots and ellipse; V-saccades: small yellow dots and red ellipse).Fig.\xa07Absolute localization error as a function of SRT. The 2D response distributions for unisensory visual (orange dots, red ellipse) and auditory targets (blue dots and ellipse) are shown in (a–d) for comparison with the four spatially aligned bisensory response distributions: a V75A12 (gray diamonds), b V75A18 (gray diamonds), c A1275V (blue squares and red circles), and d A1875V (blue squares and red circles). Either the unisensory visual or auditory distribution is shifted by ±75\xa0ms, to align SRTs with the first stimulus of the AV-trials. Ellipses circumscribe 1 SD around the mean. Only unisensory data within 2 SD of the mean are shown. The blue squares and red dots in (c) and (d) were obtained through K-means cluster analysis on the bisensory data (mean silhouette-values: 0.76 and 0.73, respectively). Black ellipses indicate means and SD of the clusters. Note that bisensory distributions have on average reduced SRTs and smaller localization errors than the unisensory distributions', 'Taking a coarser look at the A75V data (Fig.\xa0<xref rid="221_2009_1815_Fig7_HTML" ref-type="fig">7</xref>c, d), the blue cluster best resembles the A-distribution, while the red cluster resembles the V-distribution. Yet, some responses in both clusters have SRT’s and errors that could have resulted from either cluster. A better look at the data reveals a c, d), the blue cluster best resembles the A-distribution, while the red cluster resembles the V-distribution. Yet, some responses in both clusters have SRT’s and errors that could have resulted from either cluster. A better look at the data reveals a gradual improvement in localization error as reaction time progresses, rather than a sudden drop that would have resulted from a true bistable mode as subjects would have shifted from fast and inaccurate auditory, to slow but accurate visual responses. In fact, at any given SRT AV-responses were more accurate than the unisensory responses, which further underline the evidence for multisensory interaction.']}
|
The effect of spatial–temporal audiovisual disparities on saccades in a complex scene
|
[
"Multisensory integration",
"Human",
"Gaze control",
"Race model",
"Natural scene"
] |
Exp Brain Res
|
1252134000
|
Traumatic brain injury (TBI) is a major cause of morbidity and mortality worldwide. Studies of human TBI demonstrate that the cerebellum is sometimes affected even when the initial mechanical insult is directed to the cerebral cortex. Some of the components of TBI, including ataxia, postural instability, tremor, impairments in balance and fine motor skills, and even cognitive deficits, may be attributed in part to cerebellar damage. Animal models of TBI have begun to explore the vulnerability of the cerebellum. In this paper, we review the clinical presentation, pathogenesis, and putative mechanisms underlying cerebellar damage with an emphasis on experimental models that have been used to further elucidate this poorly understood but important aspect of TBI. Animal models of indirect (supratentorial) trauma to the cerebellum, including fluid percussion, controlled cortical impact, weight drop impact acceleration, and rotational acceleration injuries, are considered. In addition, we describe models that produce direct trauma to the cerebellum as well as those that reproduce specific components of TBI including axotomy, stab injury, in vitro stretch injury, and excitotoxicity. Overall, these models reveal robust characteristics of cerebellar damage including regionally specific Purkinje cell injury or loss, activation of glia in a distinct spatial pattern, and traumatic axonal injury. Further research is needed to better understand the mechanisms underlying the pathogenesis of cerebellar trauma, and the experimental models discussed here offer an important first step toward achieving that objective.
|
[
"Animals",
"Brain Injuries",
"Cerebellum",
"Disease Models, Animal",
"Humans",
"Purkinje Cells"
] |
other
|
PMC2733184
| null |
165
|
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] |
Exp Brain Res. 2009 Sep 5; 198(2-3):425-437
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NO-CC CODE
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CLR01 inhibits ZIKV infection of Vero E6 cells. (A) Light microscopy images of Vero E6 cells infected with ZIKV MR766 in the absence or presence of 150 μM CLR01 or CLR03. Images were taken 4 days post infection (dpi). (B) ZIKV MR766 was incubated with 0.2-150 μM CLR01 or CLR03 before these mixtures were used to infect Vero E6 cells. After 4 days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the MTT assay (Müller et al., 2016). Values represent mean ± SD of percentages derived from triplicate infections. IC50 was determined using GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (*** denotes p < 0.0001) (C) Confocal microscopy images of Vero E6 cells infected with CLR01- or CLR03-treated ZIKV at day 3 post infection. Cells were stained for ZIKV E protein (green) and nuclei (Hoechst, blue) and imaged using confocal microscopy. Scale bar: 50 μm. (D) Flow cytometry of infected Vero E6 cells. Virus was pretreated with PBS, CLR01 or CLR03 and added to cells. 48 h later, cells were fixed, permeabilized, and stained with an anti E protein antibody, and quantified using an Alexa 488-coupled secondary antibody. Percentages indicate the fraction of protein E positive cells. IC, isotype control.
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gr2_lrg
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2
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8f67bd14c109d40ea0dd9e1a5b6d8715110a8ec634f496bf920aab0d32bf0c13
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gr2_lrg.jpg
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multiple
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multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
789,
1075
] |
[{'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr2_lrg.jpg', 'caption': "CLR01 inhibits ZIKV infection of Vero E6 cells. (A) Light microscopy images of Vero E6 cells infected with ZIKV MR766 in the absence or presence of 150\u202fμM CLR01 or CLR03. Images were taken 4 days post infection (dpi). (B) ZIKV MR766 was incubated with 0.2-150\u202fμM CLR01 or CLR03 before these mixtures were used to infect Vero E6 cells. After 4 days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the MTT assay (Müller et al., 2016). Values represent mean\u202f±\u202fSD of percentages derived from triplicate infections. IC50 was determined using GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (*** denotes p\xa0<\xa00.0001) (C) Confocal microscopy images of Vero E6 cells infected with CLR01- or CLR03-treated ZIKV at day 3 post infection. Cells were stained for ZIKV E protein (green) and nuclei (Hoechst, blue) and imaged using confocal microscopy. Scale bar: 50\u202fμm. (D) Flow cytometry of infected Vero E6 cells. Virus was pretreated with PBS, CLR01 or CLR03 and added to cells. 48 h later, cells were fixed, permeabilized, and stained with an anti E protein antibody, and quantified using an Alexa 488-coupled secondary antibody. Percentages indicate the fraction of protein E positive cells. IC, isotype control.", 'hash': '8f67bd14c109d40ea0dd9e1a5b6d8715110a8ec634f496bf920aab0d32bf0c13'}, {'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr6_lrg.jpg', 'caption': 'CLR01 inhibits infection of human and murine brain cells. (A) Human glioblastoma (A172) or neuroglioma (H4) cells were infected with ZIKV in the presence or absence of 1.5-150\u202fμM CLR01. Two days later, cells were stained for ZIKV E protein (green) and nuclei (with Hoechst; blue) and imaged by confocal microscopy. Scale bar: 50\u202fμm. (B) Primary murine cerebellum cultures were infected with ZIKV MR766 that had been incubated with 1.5–150\u202fμM CLR01 for 10\u202fmin\u202fat 37\u202f°C. 3 dpi, cultures were fixed, permeabilized and stained for the neuronal protein βIII tubulin (red), ZIKV E protein (green) and nuclei (with Hoechst; blue). Scale bar: 20\u202fμm.', 'hash': 'fb0f9cc8a26814f5448abbc643b1bbafe3eda64cd8213297513c814db6e2471b'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr3_lrg.jpg', 'caption': "Mechanism of CLR01 inhibition of ZIKV infectivity. (A) For virus treatment, ZIKV was incubated with CLR01 for 10\xa0min\xa0at room temperature before the mixtures were added to Vero E6 cells. For cell treatment, CLR01 was added directly to the cells; after 2\xa0h, the medium was replaced and the cells were infected with ZIKV MR766. 2 dpi, cell-based ZIKV immunodetection assay was performed. Values represent mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (* denotes p\xa0<\xa00.01; *** denotes p\xa0<\xa00.0001) (B) ZIKV was incubated for the indicated times with PBS or 10\xa0μM CLR01 before the mixture was added to Vero E6 cells. 2 dpi, cell-based ZIKV immunodetection assay was performed. Values represent mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare cells infected with CLR01-treated ZIKV to cells infected with ZIKV that had been incubated with PBS for the same time period (*** denotes p\xa0<\xa00.0001). (C) Indicated concentrations of ZIKV E protein were titrated to 100\u202fμM CLR01 or PBS before ZIKV was added. Mixtures were used to inoculate Vero E6 cells. 3 dpi, the number of adherent, viable cells were determined using the MTT assay (Müller et al., 2016). The values shown are mean\xa0±\xa0SD from triplicate infections. One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare cells treated with different concentrations of ZIKV E protein and CLR01 to cells that had been treated with the same concentrations of E protein and PBS (*** denotes p\xa0<\xa00.0001). (D) ZIKV MR766 was incubated with PBS, 150\u202fμM Triton X-100, 1.5-150\u202fμM CLR01 or 150\u202fμM CLR03 for 30\u202fmin\u202fat 37\u202f°C. Samples were inactivated by UV light of a laminar flow for 60\u202fmin. Then, 10\u202fμl of the samples were used to determine RNA concentrations using the QuantiFluor® RNA System and a Quantus Fluorometer (Promega). Background values obtained from control samples using cell supernatant of uninfected cells were subtracted from the respective signals. RNA levels of virus stock incubated with PBS were subtracted from these values. Data points represent mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare samples treated with different CLR01 concentrations, CLR03 or Triton X-100 to PBS-treated virus (*** denotes p\xa0<\xa00.0001).", 'hash': '9526678003c4e8b74744a6e15ba97cec4199382c3ef72dab40b22c9518792952'}, {'image_id': 'gr7_lrg', 'image_file_name': 'gr7_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr7_lrg.jpg', 'caption': "CLR01 loses antiviral activity in the presence of serum but is active in other body fluids. Indicated concentrations of CLR01 were incubated with ZIKV MR766 in the presence of increasing concentrations of human (A) serum, (B) urine, (C) saliva, (D) semen and (E) cerebrospinal fluid (CSF). After 10\xa0min of incubation, the mixtures were used to infect Vero E6 cells. 2 days later, infection rates were determined via a cell-based ZIKV immunodetection assay. Data points represent mean\xa0±\xa0SEM (n\xa0=\xa06), except for CSF and serum: mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01 concentrations to cells infected in the absence of CLR01 but in presence of the same respective body fluid concentration (* denotes p\xa0<\xa00.01; *** denotes p\xa0<\xa00.0001).", 'hash': '535a76de7c99657eada6d4787f6445c46f588c84d6fb211d77f7a8cb76fe0b33'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr1_lrg.jpg', 'caption': "CLR01 inhibits lentiviral infection independently of the viral glycoprotein and prevents EBOV plaque formation. (A) Luciferase-encoding lentiviral particles pseudotyped with glycoproteins of viruses of the Filoviridae (EBOV, Marburg virus),Rhabdoviridae (Rabies virus) or the Coronaviridae family (SARS-CoV), were exposed to indicated concentrations of CLR01 or CLR03 and then used to infect Huh-7\u202fcells. After three days, infection rates were determined by quantifying firefly luciferase activity and subtracting background activity derived from uninfected cells. Values represent % reporter gene activities\u202f±\u202fSD derived from triplicate infections, normalized to values obtained for cells infected in the absence of tweezers. (B) Analysis of replication competent EBOV was performed using confluent Vero E6 cells in 24-well plates. rgEBOV-eGFP was preincubated with CLR01 or CLR03 and mixtures were added to the cells. After 7 days, samples were analyzed by counting the number of plaques per well and calculating the corresponding plaque forming units per milliliter (PFU/ml) for each inhibitor and dilution. Displayed values represent the mean of three independent experiments\u202f±\u202fSD. IC50 values were calculated by GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (* denotes p\xa0<\xa00.01; ** denotes p\xa0<\xa00.001; *** denotes p\xa0<\xa00.0001).", 'hash': '09c6f42c1f5df645ab67d650960863b26045271196c38f0860d6c81df0045d18'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr5_lrg.jpg', 'caption': "CLR01 abrogates ZIKV infection of cells derived from the anogenital region. ZIKV was incubated for 10\u202fmin\u202fat 37\u202f°C with 0.2-150\u202fμM CLR01 or CLR03. Next, mixtures were used to inoculate HeLa (A), SW480 (B), or HFF (C) cells. 3 days later, infection rates were determined via quantification of the viral E protein using a cell-based ZIKV immunodetection assay. Data points represent mean\u202f±\u202fSEM (n\u202f=\u202f6). One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01 concentrations to cells infected in the absence of compound (* denotes p < 0.01; ** denotes p < 0.001; *** denotes p < 0.0001).", 'hash': 'b7ac29c5f5e72c0ba6d45fdd7f242446677eb67bf997b3da796dc46cdb0f1fcc'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC7113745/gr4_lrg.jpg', 'caption': "CLR01 inactivates epidemic ZIKV isolates. (A) ZIKV FB-GWUH-2016 or (B) PRVABC-59 was incubated for 10\u202fmin\u202fat 37\u202f°C with 0.2-150\u202fμM CLR01 or CLR03. Thereafter, Vero E6 cells were infected and 2 days later, infection rates were determined via a cell-based ZIKV immunodetection assay. Data points represent mean\u202f±\u202fSD (n\u202f=\u202f3). IC50 values were determined with GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni's multiple comparison tests were applied to compare the different CLR01 concentrations to cells infected in the absence of compound (** denotes p\xa0<\xa00.001; *** denotes p\xa0<\xa00.0001).", 'hash': 'b3d49f124b755b984bf0d700f29afa0d4c53e2ed25cb9ddd83f8f07a69b5be1d'}]
|
{'gr1_lrg': ['We have shown previously that CLR01 inhibits HIV-1 infection by disrupting the viral membrane (Lump et al., 2015), suggesting that the antiviral activity of the tweezer is independent of the presence of the viral glycoproteins. To test this hypothesis, we generated luciferase-encoding retroviral particles harboring glycoproteins derived from EBOV, the closely related Filovirus Marburg virus, Rabies virus (Rhabdoviridae) or SARS-CoV (Coronaviridae). These pseudoparticles were exposed to CLR01 and then added to Huh-7\u202fcells. CLR03, which lacks hydrophobic side arms and has no anti-HIV-1 activity (Lump et al., 2015), served as a negative control. Infection rates were determined three days later by quantifying luciferase activity and showed that CLR01, in contrast to CLR03, efficiently blocked infection by all tested pseudoparticles (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>\nA). The half-maximal inhibitory concentrations (IC\nA). The half-maximal inhibitory concentrations (IC50) of CLR01 against the four pseudoparticles were similar and ranged between 27.6\u202fμM for Rabies and 45.6\u202fμM for Marburg virus (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>A). These data corroborate our previous findings that CLR01 targets the viral membrane rather than a viral glycoprotein (A). These data corroborate our previous findings that CLR01 targets the viral membrane rather than a viral glycoprotein (Lump et al., 2015). We tested next whether CLR01 blocked infection by the replication-competent BSL4 pathogen EBOV. Vero E6 cells were inoculated with GFP-encoding EBOV (Ebihara et al., 2007, Hoenen et al., 2013) that had been exposed to CLR01 or CLR03. After 7 days, the number of virus-induced plaques revealed that CLR01, but not CLR03, reduced the infectious titer in a dose-dependent manner with an IC50 of 25.8\u202fμM (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B).B).Fig. 1CLR01 inhibits lentiviral infection independently of the viral glycoprotein and prevents EBOV plaque formation. (A) Luciferase-encoding lentiviral particles pseudotyped with glycoproteins of viruses of the Filoviridae (EBOV, Marburg virus),Rhabdoviridae (Rabies virus) or the Coronaviridae family (SARS-CoV), were exposed to indicated concentrations of CLR01 or CLR03 and then used to infect Huh-7\u202fcells. After three days, infection rates were determined by quantifying firefly luciferase activity and subtracting background activity derived from uninfected cells. Values represent % reporter gene activities\u202f±\u202fSD derived from triplicate infections, normalized to values obtained for cells infected in the absence of tweezers. (B) Analysis of replication competent EBOV was performed using confluent Vero E6 cells in 24-well plates. rgEBOV-eGFP was preincubated with CLR01 or CLR03 and mixtures were added to the cells. After 7 days, samples were analyzed by counting the number of plaques per well and calculating the corresponding plaque forming units per milliliter (PFU/ml) for each inhibitor and dilution. Displayed values represent the mean of three independent experiments\u202f±\u202fSD. IC50 values were calculated by GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (* denotes p\xa0<\xa00.01; ** denotes p\xa0<\xa00.001; *** denotes p\xa0<\xa00.0001).Fig. 1'], 'gr2_lrg': ['Next, we determined the effect of CLR01 on ZIKV infection. Vero E6 cells were inoculated with the African ZIKV MR766 strain that was isolated in 1947 from a sentinel rhesus macaque (Dick et al., 1952) in the absence or presence of CLR01 or CLR03 and viral replication was monitored by light microscopy. In the absence of CLR01, ZIKV caused a profound cytopathic effect (CPE) as evidenced by large plaques caused by detachment of cells. CLR03 had no effect on this phenotype (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\nA). However, exposure to 150\u202fμM CLR01 prevented formation of the virus-induced CPE entirely. To quantitatively assess the antiviral activity, we used a colorimetric MTT assay measuring the virally induced CPE (\nA). However, exposure to 150\u202fμM CLR01 prevented formation of the virus-induced CPE entirely. To quantitatively assess the antiviral activity, we used a colorimetric MTT assay measuring the virally induced CPE (Müller et al., 2016). In the absence of compounds or in the presence of CLR03, ZIKV resulted in more than 70% dead (detached) cells. In contrast, CLR01 suppressed CPE formation with an IC50 of 8.2\u202fμM (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>B).B).Fig. 2CLR01 inhibits ZIKV infection of Vero E6 cells. (A) Light microscopy images of Vero E6 cells infected with ZIKV MR766 in the absence or presence of 150\u202fμM CLR01 or CLR03. Images were taken 4 days post infection (dpi). (B) ZIKV MR766 was incubated with 0.2-150\u202fμM CLR01 or CLR03 before these mixtures were used to infect Vero E6 cells. After 4 days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the MTT assay (Müller et al., 2016). Values represent mean\u202f±\u202fSD of percentages derived from triplicate infections. IC50 was determined using GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (*** denotes p\xa0<\xa00.0001) (C) Confocal microscopy images of Vero E6 cells infected with CLR01- or CLR03-treated ZIKV at day 3 post infection. Cells were stained for ZIKV E protein (green) and nuclei (Hoechst, blue) and imaged using confocal microscopy. Scale bar: 50\u202fμm. (D) Flow cytometry of infected Vero E6 cells. Virus was pretreated with PBS, CLR01 or CLR03 and added to cells. 48 h later, cells were fixed, permeabilized, and stained with an anti E protein antibody, and quantified using an Alexa 488-coupled secondary antibody. Percentages indicate the fraction of protein E positive cells. IC, isotype control.Fig. 2', 'The MTT assay allows an indirect measurement of ZIKV infection as the tetrazolium dye is metabolized only in living, non-infected cells. To evaluate the effect of CLR01 on ZIKV more directly, infected cells were stained for the viral E protein and analyzed by confocal microscopy (Schandock et al., 2017). Upon treatment with 15 or 150\u202fμM of CLR01, E protein-specific fluorescence could not be detected, whereas CLR03 had no effect, as expected (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>C). Lack of E protein expression in cells infected with CLR01-treated virus was confirmed by flow cytometry (C). Lack of E protein expression in cells infected with CLR01-treated virus was confirmed by flow cytometry (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>D, D, Supplementary Fig. S1A). Of note, CLR01 was not cytotoxic at concentrations active against ZIKV (Supplementary Fig. S1B), and did not cause alterations in cell morphology (compare confocal images and dot plots of uninfected cells vs. 150\u202fμM CLR01 exposed cells in <xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>C and D). Thus, CLR01 prevents ZIKV infection of Vero E6 cells.C and D). Thus, CLR01 prevents ZIKV infection of Vero E6 cells.'], 'gr3_lrg': ['We hypothesized that if CLR01 inhibited ZIKV infection by a similar mechanism to other enveloped viruses, its activity would be directed against the viral membrane (Lump et al., 2015). Indeed, when CLR01 was added to cells, rather than to the virus, and washed out prior to infection, no antiviral effect was observed in a cell-based ZIKV immunodetection assay (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\nA). We performed next a time-course analysis in which ZIKV was exposed to 10\u202fμM CLR01 for 10 to 3600\u202fs prior to infection. Already after exposure for only 10\u202fs, CLR01 reduced infection by ∼43%. Infection declined further with increasing incubation time and was nearly at the level of uninfected cells after 1800\u202fs incubation (\nA). We performed next a time-course analysis in which ZIKV was exposed to 10\u202fμM CLR01 for 10 to 3600\u202fs prior to infection. Already after exposure for only 10\u202fs, CLR01 reduced infection by ∼43%. Infection declined further with increasing incubation time and was nearly at the level of uninfected cells after 1800\u202fs incubation (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>B). In agreement with a direct activity against the virion, the ICB). In agreement with a direct activity against the virion, the IC50 of CLR01 increased from 4.3\u202fμM to 32.3\u202fμM when a 10-fold higher multiplicity of infection was applied (Supplementary Fig. S2). CLR01 was shown to destroy HIV-1 membrane integrity through interaction with raft-rich regions in the retroviral envelope (Lump et al., 2015). In contrast to HIV-1, the ZIKV particle is relatively densely packed with glycoproteins (Sirohi et al., 2016), which might hamper the interaction of the tweezer with the ZIKV membrane. To determine whether CLR01 might inhibit ZIKV infection through direct interaction with glycoproteins, increasing amounts of recombinant viral E protein were titrated to CLR01, and then these solutions were assayed for anti-ZIKV activity. As shown in <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C, even E-antigen concentrations of 1000\u202fμg/ml did not affect the antiviral activity of CLR01, suggesting that the tweezer did not reduce ZIKV infectivity through binding to the viral E protein. Interestingly, we also observed that elevated E-antigen concentrations of 333 and 1000\u202fμg/ml reduced infection in the absence of CLR01 (C, even E-antigen concentrations of 1000\u202fμg/ml did not affect the antiviral activity of CLR01, suggesting that the tweezer did not reduce ZIKV infectivity through binding to the viral E protein. Interestingly, we also observed that elevated E-antigen concentrations of 333 and 1000\u202fμg/ml reduced infection in the absence of CLR01 (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>C), which is likely due to the competition between the virion-associated E protein and the recombinant E protein for cellular receptors.C), which is likely due to the competition between the virion-associated E protein and the recombinant E protein for cellular receptors.Fig. 3Mechanism of CLR01 inhibition of ZIKV infectivity. (A) For virus treatment, ZIKV was incubated with CLR01 for 10\xa0min\xa0at room temperature before the mixtures were added to Vero E6 cells. For cell treatment, CLR01 was added directly to the cells; after 2\xa0h, the medium was replaced and the cells were infected with ZIKV MR766. 2 dpi, cell-based ZIKV immunodetection assay was performed. Values represent mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare the different CLR01/CLR03 concentrations to cells infected in the absence of compound (* denotes p\xa0<\xa00.01; *** denotes p\xa0<\xa00.0001) (B) ZIKV was incubated for the indicated times with PBS or 10\xa0μM CLR01 before the mixture was added to Vero E6 cells. 2 dpi, cell-based ZIKV immunodetection assay was performed. Values represent mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare cells infected with CLR01-treated ZIKV to cells infected with ZIKV that had been incubated with PBS for the same time period (*** denotes p\xa0<\xa00.0001). (C) Indicated concentrations of ZIKV E protein were titrated to 100\u202fμM CLR01 or PBS before ZIKV was added. Mixtures were used to inoculate Vero E6 cells. 3 dpi, the number of adherent, viable cells were determined using the MTT assay (Müller et al., 2016). The values shown are mean\xa0±\xa0SD from triplicate infections. One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare cells treated with different concentrations of ZIKV E protein and CLR01 to cells that had been treated with the same concentrations of E protein and PBS (*** denotes p\xa0<\xa00.0001). (D) ZIKV MR766 was incubated with PBS, 150\u202fμM Triton X-100, 1.5-150\u202fμM CLR01 or 150\u202fμM CLR03 for 30\u202fmin\u202fat 37\u202f°C. Samples were inactivated by UV light of a laminar flow for 60\u202fmin. Then, 10\u202fμl of the samples were used to determine RNA concentrations using the QuantiFluor® RNA System and a Quantus Fluorometer (Promega). Background values obtained from control samples using cell supernatant of uninfected cells were subtracted from the respective signals. RNA levels of virus stock incubated with PBS were subtracted from these values. Data points represent mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare samples treated with different CLR01 concentrations, CLR03 or Triton X-100 to PBS-treated virus (*** denotes p\xa0<\xa00.0001).Fig. 3', 'To test whether CLR01 interaction with the viral particle results in loss of membrane integrity, as was shown in the case of HIV-1, (Lump et al., 2015), we measured viral RNA genome release. Sucrose cushion purified ZIKV was incubated with CLR01, CLR03, PBS or Triton X-100, as a positive control, for 30\u202fmin\u202fat 37\u202f°C and the amount of total RNA in the solution was determined fluorometrically. As expected, no viral RNA was detectable when ZIKV was incubated with buffer or CLR03 (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>D). In contrast, incubation with Triton X-100 resulted in readily detectable viral RNA suggesting that the detergent lysed the ZIKV particle. Similarly, elevated amounts of RNA were detected upon treatment of ZIKV with CLR01, demonstrating the physical destruction of the viral particle by the tweezer. Of note, CLR01 itself did not act as detergent but targets the viral membrane (D). In contrast, incubation with Triton X-100 resulted in readily detectable viral RNA suggesting that the detergent lysed the ZIKV particle. Similarly, elevated amounts of RNA were detected upon treatment of ZIKV with CLR01, demonstrating the physical destruction of the viral particle by the tweezer. Of note, CLR01 itself did not act as detergent but targets the viral membrane (Lump et al., 2015).', 'Several lines of evidence demonstrate that the antiviral activity of CLR01 is directed against the ZIKV particle itself. First, treatment of cells with CLR01 prior to infection had no antiviral effect (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>A). Second, the ICA). Second, the IC50 of CLR01 increased with increasing viral concentrations (Supplementary Fig. S2). Third, the integrity of the ZIKV particle was lost upon CLR01 treatment, as shown by the release of viral RNA from CLR01-treated virions (<xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>D). These findings were in agreement with our previous observations that CLR01 selectively disrupted viral membranes (D). These findings were in agreement with our previous observations that CLR01 selectively disrupted viral membranes (Lump et al., 2015). Interestingly, the selectivity stems from CLR01\'s preferential interaction with membranes containing high levels of sphingomyelin and cholesterol, two lipids that are enriched in membranes of enveloped viruses, such as HIV-1 or EBOV (Bavari et al., 2002, Brügger et al., 2006, Chazal and Gerlier, 2003, Lorizate et al., 2013). The selective interaction of CLR01 with lipids that are enriched in the viral but not the cellular membrane may also explain its minimal effects on cell viability (Attar et al., 2012, Attar et al., 2014, Ferreira et al., 2014, Lopes et al., 2015, Prabhudesai et al., 2012, Sinha et al., 2011) (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>C, D, C, D, Supplementary Fig. S1B). CLR01 is slightly cytotoxic at concentrations of ∼1\u202fmM, corresponding to selectivity indices of 50–100, which is in a reasonable range for drug development (Buschmann and Mannhold, 2017, Food and Drug Administration, 2006).'], 'gr4_lrg': ['Next, we analyzed whether CLR01 was active against epidemic ZIKV strains. The FB-GWUH-2016 isolate was derived in 2016 from the fetus of a pregnant Finnish tourist returning from South America (Driggers et al., 2016), and the PRVABC-59 isolate represents the current American epidemic strain, isolated in 2015 from a Puerto Rican patient (Lanciotti et al., 2016). Both ZIKV strains were exposed to CLR01 upon infection of Vero E6 cells. Cell-based immunodetection assay and flow cytometry experiments demonstrated that the tweezer suppressed infection of both strains with IC50 values of 6.7\u202fμM for FB-GWUH-2016 (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\nA and \nA and Supplementary Fig. S3), and 4.2\u202fμM against PRVABC-59 (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>B), respectively.B), respectively.Fig. 4CLR01 inactivates epidemic ZIKV isolates. (A) ZIKV FB-GWUH-2016 or (B) PRVABC-59 was incubated for 10\u202fmin\u202fat 37\u202f°C with 0.2-150\u202fμM CLR01 or CLR03. Thereafter, Vero E6 cells were infected and 2 days later, infection rates were determined via a cell-based ZIKV immunodetection assay. Data points represent mean\u202f±\u202fSD (n\u202f=\u202f3). IC50 values were determined with GraphPad Prism. One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare the different CLR01 concentrations to cells infected in the absence of compound (** denotes p\xa0<\xa00.001; *** denotes p\xa0<\xa00.0001).Fig. 4'], 'gr5_lrg': ['As ZIKV can be transmitted via sexual intercourse, we studied whether ZIKV infects cells derived from the anogenital region and if so, whether infection can be blocked by CLR01. ZIKV effectively infected cell lines derived from cervix (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>\nA, \nA, Supplementary Fig. S4), colon (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>B, B, Supplementary Fig. S4), and primary foreskin cells (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>C, C, Supplementary Fig. S4). Viral infectivity was suppressed by CLR01 but not CLR03, with IC50 values in the μM range (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref> and and Supplementary Fig. S4). Because ZIKV is neurotropic (Cao-Lormeau et al., 2016, Mlakar et al., 2016) and CLR01 has been shown previously to penetrate through the blood–brain barrier (BBB) when administered systemically (Attar et al., 2012, Attar et al., 2014, Richter et al., 2017), we also tested whether CLR01 blocked ZIKV infection of brain-derived cells. Both A172 glioblastoma and H4 neuroglioma cells were susceptible to ZIKV infection and CLR01 entirely abrogated infection at 150\u202fμM (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>\nA). Finally, we confirmed these findings obtained in human cell lines using primary murine cerebellar neurons. ZIKV infected neuronal bystander cells, and this was blocked by CLR01 (\nA). Finally, we confirmed these findings obtained in human cell lines using primary murine cerebellar neurons. ZIKV infected neuronal bystander cells, and this was blocked by CLR01 (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>B). There was no apparent toxicity of CLR01 in the primary neurons at this concentration.B). There was no apparent toxicity of CLR01 in the primary neurons at this concentration.Fig. 5CLR01 abrogates ZIKV infection of cells derived from the anogenital region. ZIKV was incubated for 10\u202fmin\u202fat 37\u202f°C with 0.2-150\u202fμM CLR01 or CLR03. Next, mixtures were used to inoculate HeLa (A), SW480 (B), or HFF (C) cells. 3 days later, infection rates were determined via quantification of the viral E protein using a cell-based ZIKV immunodetection assay. Data points represent mean\u202f±\u202fSEM (n\u202f=\u202f6). One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare the different CLR01 concentrations to cells infected in the absence of compound (* denotes p < 0.01; ** denotes p < 0.001; *** denotes p < 0.0001).Fig. 5Fig. 6CLR01 inhibits infection of human and murine brain cells. (A) Human glioblastoma (A172) or neuroglioma (H4) cells were infected with ZIKV in the presence or absence of 1.5-150\u202fμM CLR01. Two days later, cells were stained for ZIKV E protein (green) and nuclei (with Hoechst; blue) and imaged by confocal microscopy. Scale bar: 50\u202fμm. (B) Primary murine cerebellum cultures were infected with ZIKV MR766 that had been incubated with 1.5–150\u202fμM CLR01 for 10\u202fmin\u202fat 37\u202f°C. 3 dpi, cultures were fixed, permeabilized and stained for the neuronal protein βIII tubulin (red), ZIKV E protein (green) and nuclei (with Hoechst; blue). Scale bar: 20\u202fμm.Fig. 6'], 'gr7_lrg': ['Its broad antiviral activity makes CLR01 an interesting lead compound for antiviral prevention or treatment. For a systemic application, CLR01 must retain antiviral activity in blood. To test whether this was the case, CLR01 was diluted in human serum, and then exposed to ZIKV. As shown in <xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>\nA, serum concentrations of 2.5% and higher abrogated the anti-ZIKV activity of the tweezer. Similarly, serum also abrogated the anti-HIV-1 activity of CLR01 (\nA, serum concentrations of 2.5% and higher abrogated the anti-ZIKV activity of the tweezer. Similarly, serum also abrogated the anti-HIV-1 activity of CLR01 (Supplementary Fig. S5), precluding its use as a systemically applied antiviral drug. In contrast, CLR01 remained active and inhibited ZIKV infection in the presence of up to 45% of urine (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>B), saliva (B), saliva (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>C), cerebrospinal fluid (C), cerebrospinal fluid (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>E) and 5% human semen (E) and 5% human semen (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>D). Thus, CLR01 inactivates ZIKV in the presence of body fluids associated with virus transmission, and could be administered locally to halt virus replication, or applied topically as a microbicide to prevent e.g. sexual virus transmission.D). Thus, CLR01 inactivates ZIKV in the presence of body fluids associated with virus transmission, and could be administered locally to halt virus replication, or applied topically as a microbicide to prevent e.g. sexual virus transmission.Fig. 7CLR01 loses antiviral activity in the presence of serum but is active in other body fluids. Indicated concentrations of CLR01 were incubated with ZIKV MR766 in the presence of increasing concentrations of human (A) serum, (B) urine, (C) saliva, (D) semen and (E) cerebrospinal fluid (CSF). After 10\xa0min of incubation, the mixtures were used to infect Vero E6 cells. 2 days later, infection rates were determined via a cell-based ZIKV immunodetection assay. Data points represent mean\xa0±\xa0SEM (n\xa0=\xa06), except for CSF and serum: mean\xa0±\xa0SD (n\xa0=\xa03). One-way ANOVA (non-parametric, grouped), followed by Bonferroni\'s multiple comparison tests were applied to compare the different CLR01 concentrations to cells infected in the absence of CLR01 but in presence of the same respective body fluid concentration (* denotes p\xa0<\xa00.01; *** denotes p\xa0<\xa00.0001).Fig. 7'], 'gr6_lrg': ['Our results establish CLR01 as a broad-spectrum antiviral agent, which not only inhibits infection of established viral pathogens (Lump et al., 2015), but also of emerging viruses, such as EBOV and ZIKV. We characterized here mainly the effect of CLR01 on ZIKV, as there is currently no specific antiviral therapy nor a preventive vaccine available. CLR01 blocked ZIKV infection of primary brain-derived murine cells (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>B) as well as human cell lines derived from the anogenital region (B) as well as human cell lines derived from the anogenital region (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>, , Supplementary Fig. S4) and the brain (<xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>A) with ICA) with IC50 values between 5 and 50\u202fμM. These values are in the same range as IC50 values obtained against EBOV (26\u202fμM, <xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B) and HIV-1 (14-20\u202fμM) (B) and HIV-1 (14-20\u202fμM) (Lump et al., 2015).']}
|
The molecular tweezer CLR01 inhibits Ebola and Zika virus infection
|
[
"Zika virus",
"Ebola virus",
"Broadly active antiviral agents",
"Virus inactivation",
"Lipid rafts"
] |
Antiviral Res
|
1523170800
|
Ebola (EBOV) and Zika viruses (ZIKV) are responsible for recent global health threats. As no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. This small molecule has previously been shown to inactivate HIV-1 and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. We found that CLR01 indeed blocked infection of EBOV and ZIKV in a dose-dependent manner. The tweezer inhibited infection of epidemic ZIKV strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. Our findings show that CLR01 is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent.
|
[
"Animals",
"Antiviral Agents",
"Bridged-Ring Compounds",
"Cell Line",
"Chlorocebus aethiops",
"Ebolavirus",
"Hemorrhagic Fever, Ebola",
"Humans",
"Organophosphates",
"Vero Cells",
"Virus Replication",
"Zika Virus",
"Zika Virus Infection"
] |
other
|
PMC7113745
| null |
55
|
[
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"{'Citation': 'Zhu J.-D., Meng W., Wang X.-J., Wang H.-C.R. Broad-spectrum antiviral agents. Front. Microbiol. 2015;6', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4440912'}, {'@IdType': 'pubmed', '#text': '26052325'}]}}"
] |
Antiviral Res. 2018 Apr 8; 152:26-35
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NO-CC CODE
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(a) Schematic illustration
indicating the FRET change of UCNP@p-QD
in the presence of MMP2. (b) In vivo MMP2 sensitivity
of UCNP@p-QD was performed in Cal27/VC and Cal27/MMP2. The FRET change
showed FRET-induced fluorescence (600 nm) and upconversion fluorescence
(475 nm) were discriminately expressed by MMP2 alternation. Dynamic
tracking of UCNP@p-QD performed in the mouse tumor model. The scale
bar represents 50 μm. (c) By intratumoral injection, UCNP@p-QD
was administrated into Cal27/VC (left side of mice) and Cal27/MMP2
(right side of mice) induced tumors. FRET-induced images were detected
at 1 h, 8 h, 16 h, and 48 h under 808 nm irradiation. (d) FRET change,
focusing on 475 and 600 nm, was observed from a tumor section that
was stained with propidium iodide (PI). These tumor sections were
prepared from NO.1 mouse. The scale bar represents 50 μm.
|
ao-2017-014948_0004
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2
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04138e4d284da6e9b696724add23d2dd2cc64ac2f30ea239f7705ff4600b73bf
|
ao-2017-014948_0004.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
730,
772
] |
[{'image_id': 'ao-2017-014948_0005', 'image_file_name': 'ao-2017-014948_0005.jpg', 'image_path': '../data/media_files/PMC6045330/ao-2017-014948_0005.jpg', 'caption': 'No caption found', 'hash': '6e8412f5f9171dcfa6a7989790b5e7d42dc06c5b47b8aa39cf018505567f19b5'}, {'image_id': 'ao-2017-014948_0002', 'image_file_name': 'ao-2017-014948_0002.jpg', 'image_path': '../data/media_files/PMC6045330/ao-2017-014948_0002.jpg', 'caption': 'Optical\nproperties of UCNP, CIS/ZnS QD, and UCNP@p-QD. (a) Photoluminescence\nanalysis (PL) of UCNP. Inset showed upconversion fluorescence under\n808 nm excitation. (b) Ultraviolet–visible spectroscopy of\nCIS/ZnS QD. Blue fluctuation showed QD absorption, and red fluctuation\nwas the emission region of the QD. Inset represented fluorescence\nfrom the QD. (c) PL spectra of conjugating with different QD concentration.\nUCNP@p-QD showed the FRET fluorescence under 808 nm irradiation (inset).\n(d) The normalized PL spectra of QD, UCNP@p-QD, UCNP@p-QD*, UCNP,\nand UCNP*. The attached symbol (*) indicated UCNP@p-QD decorated with\nPEG polymers. PL showed the emissions under 808 nm excitation except\nfor unconjugated QD (360 nm excitation).', 'hash': 'b1b2d07a0cac5f7919fa9c05295cd1756e9978d4162c54e68d2f5ffb80e4910f'}, {'image_id': 'ao-2017-014948_0003', 'image_file_name': 'ao-2017-014948_0003.jpg', 'image_path': '../data/media_files/PMC6045330/ao-2017-014948_0003.jpg', 'caption': 'Photoluminescence\nanalysis showed the FRET change of UCNP@p-QD\nin the presence of rhMMP2 under 808 nm irradiation. The blue line\nindicated the visible fluorescence of UCNP at 475 nm, and the red\nline indicated the FRET-induced florescence at 600 nm. (a) UCNP@p-QD\nwas incubated with different concentrations of rhMMP2. The concentration\nof rhMMP2 was 0, 10–6, 10–5, 10–4, 10–3, 10–2,\n10–1, and 1 pg/mL, respectively. (b) The time-lapse\nPL analysis showed that UCNP@p-QD was incubated with 1 pg/mL of rhMMP2.\nThe checkpoint was 20, 40, 60, 80, 100, and 120 min.', 'hash': 'd87ad3f187e310430ad31ed80e6b1b0b11556beecb028fe27cd6360d8e89c6f1'}, {'image_id': 'ao-2017-014948_0004', 'image_file_name': 'ao-2017-014948_0004.jpg', 'image_path': '../data/media_files/PMC6045330/ao-2017-014948_0004.jpg', 'caption': '(a) Schematic illustration\nindicating the FRET change of UCNP@p-QD\nin the presence of MMP2. (b) In vivo MMP2 sensitivity\nof UCNP@p-QD was performed in Cal27/VC and Cal27/MMP2. The FRET change\nshowed FRET-induced fluorescence (600 nm) and upconversion fluorescence\n(475 nm) were discriminately expressed by MMP2 alternation. Dynamic\ntracking of UCNP@p-QD performed in the mouse tumor model. The scale\nbar represents 50 μm. (c) By intratumoral injection, UCNP@p-QD\nwas administrated into Cal27/VC (left side of mice) and Cal27/MMP2\n(right side of mice) induced tumors. FRET-induced images were detected\nat 1 h, 8 h, 16 h, and 48 h under 808 nm irradiation. (d) FRET change,\nfocusing on 475 and 600 nm, was observed from a tumor section that\nwas stained with propidium iodide (PI). These tumor sections were\nprepared from NO.1 mouse. The scale bar represents 50 μm.', 'hash': '04138e4d284da6e9b696724add23d2dd2cc64ac2f30ea239f7705ff4600b73bf'}, {'image_id': 'ao-2017-014948_0001', 'image_file_name': 'ao-2017-014948_0001.jpg', 'image_path': '../data/media_files/PMC6045330/ao-2017-014948_0001.jpg', 'caption': 'Preparation and characterization of UCNP@p-QD. (a) Schematic showing\nthe fabrication strategy of UCNP@p-QD. UCNP was coated with SiO2 and decorated with MMP2-sensitive peptide. CIS/ZnS QD conjugated\nonto the peptide on the UCNP. (b) The X-ray diffractions of NaYF4:Yb/Tm@NaYF4:Yb/Nd\nand NaYF4:Yb/Tm were compared with the β-NaYF4 database (JCPDS\n16-0334). (c) The uniform UCNP was observed by TEM. The scale bar\nrepresents 50 nm. (d) The UCNP was coated with SiO2. The\nscale bar represents 50 nm. (e) High-resolution TEM showed the QD\ncould be conjugated onto UCNP. The arrow indicated the QD (inset).\nThe scale bar represents 10 nm. (f) High-resolution TEM image and\nthe elemental mapping (g) of UCNP (F, Y, Nd) and CIS/ZnS QD (In).\nThe scale bar represents 20 nm. The yellow rectangular frame indicated\nthe same proportions in EDS mapping analysis.', 'hash': 'b460690d929ef05dbd55043458c9ea7baeeae95d1665e72fbc21179ada008531'}]
|
{'ao-2017-014948_0001': ['In this study, we developed a FRET-based nanocomposite\nthat detected\nMMP2 expression in vivo. This nanocomposite consists\nof UCNP and CIS/ZnS QDs, which were linked by an MMP2-sensitive peptide\n(<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>a). UCNP@p-QD\nhas FRET-induced red fluorescence by 808 nm irradiation. This nanocomposite\nrevealed the blue fluorescence in the presence of MMP2. At first,\nwe fabricated a core/shell type NaYF<xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>a). UCNP@p-QD\nhas FRET-induced red fluorescence by 808 nm irradiation. This nanocomposite\nrevealed the blue fluorescence in the presence of MMP2. At first,\nwe fabricated a core/shell type NaYF4:Yb/Tm@NaYF4:Yb/Nd UCNP. The core was NaYF4:Yb3+/Tm3+ that emitted multifluorescence under NIR irradiation. The\ndata of the hexagonal β-NaYF4 database (JCPDS 16-0334) were\ncompared with NaYF4:Yb,Tm and NaYF4:Yb,Tm. The\ndiffraction peaks were similar to those of the standard peaks (<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>b). UCNPs were uniform\nin aqueous solution of which the diameter was approximately 20 nm\n(<xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>b). UCNPs were uniform\nin aqueous solution of which the diameter was approximately 20 nm\n(<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>c). Doped\nwith Yb<xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>c). Doped\nwith Yb3+ and Nd3+, the core/shell type, which\nformed a hexagonal structure, was in response to 808 nm excitation.\nIn order to graft the MMP2-sensitive peptide, UCNP was modified with\nprimary amine on the SiO2 layer.29 The SiO2 absorption was revealed at 1100 cm–1 compared to ligand-free UCNP (Figure S1a, Supporting Information). The absorption of UCNP@p at the Si–O–Si\n(1100 cm–1), N–C=O (1660 cm–1), and C–O (1100–1350 cm–1) corresponded\nwith the standard curve of UCNP and peptide. The SiO2-coated\nUCNP showed the size of 25 nm ±1 nm (<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>d). The N-terminal part from UCNP embedded\na cysteine residue, to which the QD conjugated through disulfide linkage.<xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>d). The N-terminal part from UCNP embedded\na cysteine residue, to which the QD conjugated through disulfide linkage.30 X-ray diffraction (XRD) of the CIS/ZnS showed\nthe major peaks corresponding to the standard diffraction patterns\nof CIS crystal (JCPDS No. 85-1175) and ZnS crystal (JCPDS No. 77-2100)\nat 27°, 47°, and 55° (Figure S1b, Supporting Information). The CIS/ZnS QD is a stable and biocompatible\nnanoparticle that is appropriate to bioapplication.23 The distance between QD and UCNP was close to 10 nm that\nwas FRET allowed between donor and acceptor. UCNP@p-QD was approximate\nto 30 nm in size (<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>e). CIS/ZnS QD with size of 3.5 nm ± 0.2 nm was surrounded\naround the UCNP@p (inset, <xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>e). CIS/ZnS QD with size of 3.5 nm ± 0.2 nm was surrounded\naround the UCNP@p (inset, <xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>e). The EDS analysis indicated that QDs were bound\ntightly with UCNP (Figure S1c, <xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>e). The EDS analysis indicated that QDs were bound\ntightly with UCNP (Figure S1c, Supporting Information), in which the elements of QDs, such as In, S, and Cu, were located\nnext to the elemental cluster within Nd and Yb. Moreover, the elements\nof UCNP and CIS/ZnS QD were identified from the conjugated structure\nusing elemental mapping (<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>f). According to the elemental distribution, the fluorine\n(F), yttrium (Y), and neodymium (Nd) elements of UCNP appeared in\nthe core, while the indium (In) of CIS/ZnS QD was distributed in the\nperiphery (<xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>f). According to the elemental distribution, the fluorine\n(F), yttrium (Y), and neodymium (Nd) elements of UCNP appeared in\nthe core, while the indium (In) of CIS/ZnS QD was distributed in the\nperiphery (<xref rid="ao-2017-014948_0001" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>g).<xref rid="ao-2017-014948_0001" ref-type="fig">1</xref>g).'], 'ao-2017-014948_0002': ['UCNP showed antistoke shift fluorescence under 808 nm laser\nirradiation\n(<xref rid="ao-2017-014948_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>a). The Nd<xref rid="ao-2017-014948_0002" ref-type="fig">2</xref>a). The Nd3+ ion was a sensitizer, in which the electron was excited\nfrom the ground state to the excited state.31 Meanwhile, a nonradiative energy transfers to the excited level 2F5/2 of Yb3+. The excited state of Tm3+ has four energy relaxations at wavelengths of 340, 360,\n450, and 475 nm. 340 and 360 nm were due to the electronic transition 1I6 → 3F4 and 1D2 → 3H6. The blue-visible\nlight was contributed by transitions of 1D2 → 3F4 and 1G4 → 3H6. QD has a broad absorption below 500 nm and\nhas red fluorescence emission at 625 nm (<xref rid="ao-2017-014948_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>b). QD showed an orange emission under UV\nirradiation (inset, <xref rid="ao-2017-014948_0002" ref-type="fig">2</xref>b). QD showed an orange emission under UV\nirradiation (inset, <xref rid="ao-2017-014948_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>b). To develop not only high penetration of excitation source\nbut also a red shift, the fluorophore is desired for clinical use.\nUCNP acted as a donor that was excited by 808 nm irradiation. The\nacceptor CIS/ZnS QD that was excited by the upconversion fluorescence\nwas a red fluorophore. The FRET process was manifested by the fact\nthat the fluorescence intensity of the UV and visible-light spectrum\nshowed a gradual decrease depending on increasing QD concentration\n(<xref rid="ao-2017-014948_0002" ref-type="fig">2</xref>b). To develop not only high penetration of excitation source\nbut also a red shift, the fluorophore is desired for clinical use.\nUCNP acted as a donor that was excited by 808 nm irradiation. The\nacceptor CIS/ZnS QD that was excited by the upconversion fluorescence\nwas a red fluorophore. The FRET process was manifested by the fact\nthat the fluorescence intensity of the UV and visible-light spectrum\nshowed a gradual decrease depending on increasing QD concentration\n(<xref rid="ao-2017-014948_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>c). To delineate\nthe FRET dynamic change, we focused on the wavelengths at 475 and\n600 nm, to which UCNP and QD contributed, respectively (Figure S2a, <xref rid="ao-2017-014948_0002" ref-type="fig">2</xref>c). To delineate\nthe FRET dynamic change, we focused on the wavelengths at 475 and\n600 nm, to which UCNP and QD contributed, respectively (Figure S2a, Supporting Information). The red fluorescence\nrevealed at 25 mg/mL of QD conjugation implied a threshold of FRET-induced\nred fluorescence. Furthermore, the zeta-potential analysis indicated\nthat the positive charge of UCNP@p decreased by the increasing QDs\n(Figure S2b, Supporting Information). Since\nthe CIS/ZnS QD was hydrophobic, it dissolved in organic solvent rather\nthan in H2O. We then modified the UCNP@p-QD synthesis process\nby PEGlyation onto the surface of the QD.32 PEGlyation was carried out by methyl-PEG4-thiol and carboxy-PEG12-thiol, which increased the hydrophilicity and minimized\nnonspecific binding (Figure S2c, Supporting Information). The 2853 and 2925 cm–1 were from the methylene-stretching\ngroup of PEG. The emission of UCNP@p-QD with 808 nm irradiation is\nsimilar to QD under UV excitation. The PEGlyated UCNP@p-QD has orange\nfluorescence at wavelength of 550 nm (<xref rid="ao-2017-014948_0002" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>d). Obviously, there is about a 100 nm blue\nshift after PEGlyation. Park et al. suggested that cation exchange\ncould promote a blue shift in emission.<xref rid="ao-2017-014948_0002" ref-type="fig">2</xref>d). Obviously, there is about a 100 nm blue\nshift after PEGlyation. Park et al. suggested that cation exchange\ncould promote a blue shift in emission.33 Ryu et al. suggested that the addition of surfactant could contribute\nto the blue shift.34 In our experimental\nresult, the surface reconstruction of UCNP@p-QD led to an apparent\nblue-shift emission. The blue-shift mechanism should be further demonstrated.'], 'ao-2017-014948_0003': ['We soaked 10 μg of UCNP@QD into 1 mL of PBS at different\npH values of 2, 4, 7, 9, and 12, respectively (Figure S3a, Supporting Information). Dynamic light scattering\n(DLS) showed that the hydration radii between pH 4 and pH 9 were not\nobviously changed, implying that UCNP@p-QD was stable in biological\nsolution. UCNP@p-QD (0.2 mg/mL) was incubated with different concentration\nof MMP2 (Figure S3b, Supporting Information). The 600 and 475 nm emissions delineated a FRET change that originated\nfrom QD and UCNP, respectively. The fluctuations showed that the FRET-induced\nfluorescence (600 nm) gradually decreased; meanwhile, upconversion\nfluorescence (475 nm) increased depending on increasing rhMMP2 (<xref rid="ao-2017-014948_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>a). The recovery\nof upconversion fluorescence indicated that MMP2-sensitive peptides\nwere digested, and then QDs were separated from UCNP. The FRET-induced\nand upconversion fluorescence were simultaneously detected at the\ninterval of 10<xref rid="ao-2017-014948_0003" ref-type="fig">3</xref>a). The recovery\nof upconversion fluorescence indicated that MMP2-sensitive peptides\nwere digested, and then QDs were separated from UCNP. The FRET-induced\nand upconversion fluorescence were simultaneously detected at the\ninterval of 10–5 to 10–2 pg/mL.\nIn the concentration of 10–1 mg/mL, the intensity\nof upconversion fluorescence reached a plateau, and the FRET-induced\nred fluorescence declined to the zero, indicating that the 0.2 mg/mL\nof UCNP@p-QD could be the effective dose. In time-dependent rhMMP2\ndigestion, the FRET was consistently expressed until the cleavage\ntime reached 60 min (Figure S3c, Supporting Information). Not only the increase of upconversion fluorescence but also the\ndecrease of FRET-induced fluorescence showed exponential fluctuations.\nThe FRET-induced fluorescence severely decreased between 0 and 20\nmin. The upconversion fluorescence intensity was retrieved serially\nalong with reaction time until a plateau at 100 min (<xref rid="ao-2017-014948_0003" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>b). The FRET elimination of\nUCNP@p-QD showed the presence of rhMMP2. This implied the UCNP@p-QD\ncould be employed in clinical MMP2 detection such as a blood specimen.<xref rid="ao-2017-014948_0003" ref-type="fig">3</xref>b). The FRET elimination of\nUCNP@p-QD showed the presence of rhMMP2. This implied the UCNP@p-QD\ncould be employed in clinical MMP2 detection such as a blood specimen.'], 'ao-2017-014948_0004': ['The extracellular UCNP@p-QD showed red\nemission under 808 nm irradiation.\nAfter MMP2 digestion, the UCNP@p showed blue upconversion fluorescence\n(<xref rid="ao-2017-014948_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>a). To detect\nthe MMP2 in the cell model, constituted MMP2-overexpressing cells\n(Cal27/MMP2) and their cognate cells (Cal27/VC) were cultured for\nUCNP@p-QD detection. Cal27/MMP2 overexpressed the MMP2 protein not\nonly in cytoplasm but also in the culture medium.<xref rid="ao-2017-014948_0004" ref-type="fig">4</xref>a). To detect\nthe MMP2 in the cell model, constituted MMP2-overexpressing cells\n(Cal27/MMP2) and their cognate cells (Cal27/VC) were cultured for\nUCNP@p-QD detection. Cal27/MMP2 overexpressed the MMP2 protein not\nonly in cytoplasm but also in the culture medium.35 The FRET-induced red fluorescence showed in Cal27/VC but\nalso in Cal27/MMP2 (<xref rid="ao-2017-014948_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>b). The UCNP@p-QD accumulated around Cal27/VC cells, indicating\nthere was no MMP2 expression; oppositely, the FRET-induced fluorescence\nwas significantly reduced in Cal27/MMP2. Moreover, UCNP@p-QD showed\nthe same consequence in OEC-M1 and FADU cells (Figure S5, <xref rid="ao-2017-014948_0004" ref-type="fig">4</xref>b). The UCNP@p-QD accumulated around Cal27/VC cells, indicating\nthere was no MMP2 expression; oppositely, the FRET-induced fluorescence\nwas significantly reduced in Cal27/MMP2. Moreover, UCNP@p-QD showed\nthe same consequence in OEC-M1 and FADU cells (Figure S5, Supporting Information), which were MMP2-null\nand MMP2-overexpressing cells, respectively.35 This result indicated that UCNP@p-QD could be detected in extracellular\nMMP2. The effective penetration is usually an obstacle for scientists\nto develop the photodependent biosensors, so the high penetrating\ninfrared has attracted the attention of investigators.', 'In order to take an in\nvivo image, we refitted\nan\xa0 optical imaging system to employ the upconversion luminescence\ndetection (Figure S6a, Supporting Information). Under 808 nm irradiation, the FRET-induced fluorescence was detected\non the separated dorsal side within 1 h (<xref rid="ao-2017-014948_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>c; column 1H). Distribution of UCNP@p-QD\nwas visualized uniformly in tumor and its adjacent part. Additionally,\nthe RFP-harbor Cal27/VC cell could be specifically examined on the\nleft dorsal side (Figure S6b, <xref rid="ao-2017-014948_0004" ref-type="fig">4</xref>c; column 1H). Distribution of UCNP@p-QD\nwas visualized uniformly in tumor and its adjacent part. Additionally,\nthe RFP-harbor Cal27/VC cell could be specifically examined on the\nleft dorsal side (Figure S6b, Supporting Information). The FRET-induced fluorescence significantly decreased over time\nin Cal27/MMP2-induced tumors (1H to 48H). Finally, the FRET-induced\nfluorescence almost disappeared from the Cal27/MMP2-induced tumor\npart (<xref rid="ao-2017-014948_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>c; column\n48H). Although there is a little FRET-induced fluorescence from Cal27/MMP2-induced\ntumor (<xref rid="ao-2017-014948_0004" ref-type="fig">4</xref>c; column\n48H). Although there is a little FRET-induced fluorescence from Cal27/MMP2-induced\ntumor (<xref rid="ao-2017-014948_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>c; NO.3),\nmost UCNP@p-QDs were significantly degraded by MMP2. The harvested\ntumors showed the similar size and pathological feature between Cal27/VC-\nand Cal27/MMP2-induced tumors (Figures S6c and S6d, <xref rid="ao-2017-014948_0004" ref-type="fig">4</xref>c; NO.3),\nmost UCNP@p-QDs were significantly degraded by MMP2. The harvested\ntumors showed the similar size and pathological feature between Cal27/VC-\nand Cal27/MMP2-induced tumors (Figures S6c and S6d, Supporting Information). Because the blue-visible filter is\nnot equipped in an in vivo imaging system, the tumor\nsections were examined using a multiphoton microscope (<xref rid="ao-2017-014948_0004" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>d). The UCNP@p-QDs were shown\nubiquitously in the Cal27/VC tumor tissue. Oppositely, the upconversion\nfluorescence was detected in the Cal27/MMP2 tumor. Hence, UCNP@p-QD\ndetected not only rhMMP2 protein <xref rid="ao-2017-014948_0004" ref-type="fig">4</xref>d). The UCNP@p-QDs were shown\nubiquitously in the Cal27/VC tumor tissue. Oppositely, the upconversion\nfluorescence was detected in the Cal27/MMP2 tumor. Hence, UCNP@p-QD\ndetected not only rhMMP2 protein in vitro but also\nMMP2 expression in the cell model and animal tumor imaging.']}
|
Near-Infrared-Activated Fluorescence Resonance Energy
Transfer-Based Nanocomposite to Sense MMP2-Overexpressing Oral Cancer
Cells
| null |
ACS Omega
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1518076800
|
The matrix metalloproteinases (MMPs) are well-known mediators that are activated in tumor progression. MMP2 is a kind of gelatinase in extracellular matrix remodeling and cancer metastasis processes. MMP2 secretion increased in many types of cancer diseases, and its abnormal expression is associated with a poor prognosis. We fabricated a nanocomposite that sensed MMP2 expression by a red and blue light change. This nanocomposite consisted of an upconversion nanoparticle (UCNP), MMP2-sensitive peptide, and CuInS/ZnS quantum dot (CIS/ZnS QD). An UCNP is composed of NaYF:Tm/Yb@NaYF:Nd/Yb, which has multiple emissions at UV/blue-visible wavelengths under 808 nm laser excitation. The conjugated CIS/ZnS QD showed the red-visible fluorescence though the FRET process. The two fluorophores were connected by a MMP2-sensitive peptide to form a novel MMP2 biosensor, named UCNP@p-QD. UCNP@p-QD was highly biocompatible according to cell viability assay. The FRET-based biosensor was employed in the MMP2 determination and . Furthermore, it was administrated into the tumor-bearing mouse to check MMP2 expression. UCNP@p-QD could be a promising tool for biological study and biomedical application. In this study, we demonstrated that the CIS/ZnS QD improved the upconversion intensity through a near-infrared-induced FRET process. This nanocomposite has the advantage of light penetration, excellent biocompatibility, and high sensitivity to sense MMP2. The near-infrared-induced composites are a potential inspiration for use in biomedical applications.
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[] |
other
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PMC6045330
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38
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[
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] |
ACS Omega. 2018 Feb 8; 3(2):1627-1634
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NO-CC CODE
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Fas receptor is recruited into lipid rafts after GPR55 receptor activation. (A) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for 24 hr and detergent-resistant lipid rafts were isolated on a discontinuous 5%-40% sucrose gradient. The resulting fractions were analyzed by immunoblotting using a Fas-specific antibody to determine the subcellular location of these proteins. (B) Co-localization of Fas immunoreactivity and lipid raft staining after AEA and O-1602 treatment. Mz-ChA-1 cells were treated with AEA and O-1602 (both at 10−5 M) and the lipid rafts were stained with Alexa Fluor 488-conjugated cholera toxin B subunit (green) as well as Fas immunoreactivity (red). Co-localization is indicated by yellow areas. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (C) Mz-Neo neg and Mz-GPR55 shRNA were treated with AEA or O-1602 (both at 10−5 M) for 24 hr and detergent-resistant lipid rafts were isolated. In addition Mz-ChA-1 cells were pretreated with the JNK inhibitor, SP600125 (10−7 M) for 1 hr prior to the addition of AEA or O-1602 (both at 10−5 M) for 24 hr followed by lipid raft fractionation. The resulting fractions were analyzed by immunoblotting using a Fas-specific antibody to determine its subcellular location.
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nihms-269270-f0007
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8f550e0888161246c89aceb805f6b12a5586395b53c199444a12a0297aeb7604
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nihms-269270-f0007.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
601,
382
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[{'image_id': 'nihms-269270-f0005', 'image_file_name': 'nihms-269270-f0005.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0005.jpg', 'caption': 'GPR55 activation by AEA and O-1602 increases JNK activity. (A) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for various time points up to 6 hr. JNK activity was assessed by commercially available ELISA kits (average ± SEM, n=4; * p<0.05 compared to basal treatment. (B) Mz-ChA-1 cells were pretreated with the JNK inhibitor SP600125 (10−7 M) for 1 hr prior to the addition of AEA or O-1602 (both at 10−5 M) for 48 hr. Cell viability was assessed by MTS assays. Data are expressed as fold change in viability (average ± SEM, n=7, *p<0.05 compared to basal treatment within each cell line). (C) Mz-Neo neg, Mz-GPR55 shRNA and Mz-Gα12 shRNA cells were treated with AEA or O-1602 (both at 10−5 M) for 6 hr. JNK activity was assessed by commercially available ELISA kits. * p<0.05 compared to basal treatment within each cell line.', 'hash': '6152261e2530fe98b7d1aa6fcd092cc8bacc792e29e5c5708c2d38d7d72e75b5'}, {'image_id': 'nihms-269270-f0002', 'image_file_name': 'nihms-269270-f0002.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0002.jpg', 'caption': '(A) Cholangiocarcinoma cells (Mz-ChA-1, HuCCT-1, CCLP-1 and SG231) and non-malignant H69 and HIBEC cells were treated with various concentrations of the GPR55 agonist, O-1602 (10−9 to 10−5 M) for 48 hr. Cell viability was assessed by MTS assays. Data are expressed as fold change in viability (average ± SEM, n=7) * p<0.05 compared to basal treatment within each cell line. (B) Mz-ChA-1 cells were injected into the flank of athymic mice. After tumors were established, mice were treated with 10 mg/kg/day (ip) O-1602, three days per week and tumor volume assessed.', 'hash': '9ca36d96de3b93cc470c4de2b1910e9d5b3454037108f0fb9337ecd7b12aba53'}, {'image_id': 'nihms-269270-f0003', 'image_file_name': 'nihms-269270-f0003.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0003.jpg', 'caption': 'Specific knockdown of GPR55 receptor expression prevents the antiproliferative effects of AEA in vitro and in vivo. Mz-ChA-1 cells were stably transfected with GPR55 shRNA vectors. The effect of AEA and O-1602 on cells with low levels of GPR55 expression (Mz-GPR55 shRNA) was assessed in vitro by MTS assay (A). Mz-Neo neg and Mz-GPR55 shRNA cells were treated with various concentrations of the GPR55 agonists, AEA and O-1602 (10−5 M) for 48 hr. Cell viability was assessed by MTS assays. Data are expressed as fold change in viability (average ± SEM, n=7; * p<0.05 compared to basal treatment within each cell line). The effect of AEA and O-1602 on cells with low levels of GPR55 expression was assessed in vivo using the xenograft model of cholangiocarcinoma (B). Mz-Neo neg cells and Mz-GPR55 shRNA cells were injected into the flank of athymic mice. After tumors were established, mice were treated with 10 mg/kg/day (ip) O-1602, three days per week and tumor volume assessed.', 'hash': '1a97a0146bb545f3c4fc2ae765593afc8dff5ae50da93855c196a5caeeb9e22b'}, {'image_id': 'nihms-269270-f0004', 'image_file_name': 'nihms-269270-f0004.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0004.jpg', 'caption': 'Specific knockdown of Gα12 G protein expression prevents the antiproliferative effects of GPR55 activation in vitro. Mz-ChA-1 cells were stably transfected with Gα12 shRNA vectors (Mz-Gα12 shRNA). The effect of AEA and O-1602 on cells with low levels of Gα12 expression was assessed in vitro by MTS assay (A). Mz-Neo neg and Mz-Gα12 shRNA cells were treated with various concentrations of the GPR55 agonist, O-1602 (10−9 to 10−5 M) for 48 hr. Cell viability was assessed using an MTS cell proliferation assay. Data are expressed as fold change in viability (average ± SEM, n=7, *p<0.05 compared to basal treatment within each cell line).', 'hash': 'a796330276b019fc41fe7edb68f9421323b15799716b42a835f3c89355b9c32c'}, {'image_id': 'nihms-269270-f0007', 'image_file_name': 'nihms-269270-f0007.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0007.jpg', 'caption': 'Fas receptor is recruited into lipid rafts after GPR55 receptor activation. (A) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for 24 hr and detergent-resistant lipid rafts were isolated on a discontinuous 5%-40% sucrose gradient. The resulting fractions were analyzed by immunoblotting using a Fas-specific antibody to determine the subcellular location of these proteins. (B) Co-localization of Fas immunoreactivity and lipid raft staining after AEA and O-1602 treatment. Mz-ChA-1 cells were treated with AEA and O-1602 (both at 10−5 M) and the lipid rafts were stained with Alexa Fluor 488-conjugated cholera toxin B subunit (green) as well as Fas immunoreactivity (red). Co-localization is indicated by yellow areas. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (C) Mz-Neo neg and Mz-GPR55 shRNA were treated with AEA or O-1602 (both at 10−5 M) for 24 hr and detergent-resistant lipid rafts were isolated. In addition Mz-ChA-1 cells were pretreated with the JNK inhibitor, SP600125 (10−7 M) for 1 hr prior to the addition of AEA or O-1602 (both at 10−5 M) for 24 hr followed by lipid raft fractionation. The resulting fractions were analyzed by immunoblotting using a Fas-specific antibody to determine its subcellular location.', 'hash': '8f550e0888161246c89aceb805f6b12a5586395b53c199444a12a0297aeb7604'}, {'image_id': 'nihms-269270-f0001', 'image_file_name': 'nihms-269270-f0001.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0001.jpg', 'caption': 'GPR55 is expressed in cholangiocyte and cholangiocarcinoma cell lines. GPR55 levels were assessed in four cholangiocarcinoma cell lines as well as the non-malignant cholangiocyte cell lines H69 and HIBEC, by real time PCR (A), immunoblotting (B). Data are expressed as average relative GPR55 mRNA expression (± SEM) in each cell line compared to H69 cells after using GAPDH expression as a loading control (n=3; A).', 'hash': '2ef5956d9012343a28c21db2f27f5f7f9758ee56eb570baefa5020c5b9aed078'}, {'image_id': 'nihms-269270-f0006', 'image_file_name': 'nihms-269270-f0006.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0006.jpg', 'caption': 'Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. (A) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10−5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p<0.05) compared to basal treatment (n=7). (B) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for 4 hr and detergent-resistant lipid rafts were isolated on a discontinuous 5%-40% sucrose gradient. The resulting fractions were analyzed by immunoblotting using GPR55 and Gα12-specific antibodies. (D) Co-localization of GPR55 immunoreactivity and lipid raft staining after AEA and O-1602 treatment. Mz-ChA-1 cells were treated with AEA and O-1602 (both at 10−5 M) and the lipid rafts were stained with Alexa Fluor 488-conjugated cholera toxin B subunit (green) as well as GPR55 immunoreactivity (red). Co-localization is indicated by yellow areas. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm.', 'hash': '77be13d3d48447053c5175ec1b1a5da435d865522baef9ab086da298be41cc0b'}]
|
{'nihms-269270-f0001': ['All of the cholangiocarcinoma cell lines studied here, as well as the non-malignant cell lines (H69 and HIBEC) expressed the mRNA (<xref ref-type="fig" rid="nihms-269270-f0001">Figure 1A</xref>) and protein () and protein (<xref ref-type="fig" rid="nihms-269270-f0001">Figure 1B</xref>) for GPR55. There was no obvious or consistent difference between the GPR55 mRNA and protein expression in the malignant and non-malignant cell lines () for GPR55. There was no obvious or consistent difference between the GPR55 mRNA and protein expression in the malignant and non-malignant cell lines (<xref ref-type="fig" rid="nihms-269270-f0001">Figure 1A, 1B</xref>). By immunofluorescence, GPR55 immunoreactivity was predominantly found in the membrane and cytoplasm in all cell lines studied (). By immunofluorescence, GPR55 immunoreactivity was predominantly found in the membrane and cytoplasm in all cell lines studied (Supplemental Figure S1 A). Furthermore, immunohistochemical analysis of human liver biopsy samples indicated similar intensity and subcellular location of GPR55 immunoreactivity in malignant and non-malignant cholangiocytes (Supplemental Figure S1 B).'], 'nihms-269270-f0002': ['Activation of GPR55 with the specific agonist O-1602 in vitro significantly decreased cell viability in all cholangiocarcinoma cell lines studied, but not in the non-malignant cholangiocyte cell lines (<xref ref-type="fig" rid="nihms-269270-f0002">Figure 2A</xref>). Furthermore, there was an increase in apoptosis in all cholangiocarcinoma cell lines after O-1602 treatment as demonstrated by Annexin V staining (). Furthermore, there was an increase in apoptosis in all cholangiocarcinoma cell lines after O-1602 treatment as demonstrated by Annexin V staining (Supplemental Figure S2). Treating in vivo xenograft cholangiocarcinoma tumors with O-1602 significantly inhibited tumor growth (<xref ref-type="fig" rid="nihms-269270-f0002">Figure 2B</xref>). In addition, the latency of tumor growth (i.e., time taken for tumor volume to increase to 150% of the original size) was increased from 5.8 ± 0.76 to 11.2 ± 2.74 days after chronic O-1602 treatment. Histological analysis of the excised tumors revealed that all cancer cells within tumors from O-1602-treated and vehicle-treated animals were CK-19 positive, indicating a cholangiocyte phenotype (). In addition, the latency of tumor growth (i.e., time taken for tumor volume to increase to 150% of the original size) was increased from 5.8 ± 0.76 to 11.2 ± 2.74 days after chronic O-1602 treatment. Histological analysis of the excised tumors revealed that all cancer cells within tumors from O-1602-treated and vehicle-treated animals were CK-19 positive, indicating a cholangiocyte phenotype (Supplemental Figure S3). Furthermore, all cholangiocarcinoma cells in the tumor retained GPR55 immunoreactivity, which appeared to increase after O-1602 treatment (Supplemental Figure S3). Using TUNEL staining as a marker of apoptosis, O-1602 treatment increased the incidence of apoptosis in the cholangiocarcinoma tumors (Supplemental Figure S3).'], 'nihms-269270-f0003': ['We have previously shown that AEA exerts antiproliferative effects on cholangiocarcinoma (12) in a similar manner to that shown here for GPR55 activation. Therefore, we wanted to assess definitively if AEA is working through a GPR55-dependent mechanism. To this end, we stably knocked down the expression of GPR55 by transfecting GPR55-specific shRNA constructs into Mz-ChA-1 cells. Characterization of the resulting cell line (Mz-GPR55 shRNA) revealed a 60-70% knock down in GPR55 mRNA and protein expression when compared to the mock-transfected cell line (Mz-Neo neg; Supplemental Figure S4). Using these cell lines, we assessed the antiproliferative effects of both AEA and O-1602 in vitro and in vivo. Both AEA and O-1602 decreased cell viability in Mz-Neo neg to a similar degree, but failed to have any effect in Mz-GPR55 shRNA cells (<xref ref-type="fig" rid="nihms-269270-f0003">Figure 3A</xref>). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-GPR55 shRNA cells (). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-GPR55 shRNA cells (Supplemental Figure S5). The growth suppressive effects of AEA and O-1602 were evident in the xenograft model of cholangiocarcinoma using Mz-Neo neg cells (<xref ref-type="fig" rid="nihms-269270-f0003">Figure 3B</xref>), whereas tumors derived from the implantation of Mz-GPR55 shRNA were not sensitive to AEA or O-1602 (), whereas tumors derived from the implantation of Mz-GPR55 shRNA were not sensitive to AEA or O-1602 (<xref ref-type="fig" rid="nihms-269270-f0003">Figure 3B</xref>).).'], 'nihms-269270-f0004': ['GPR55 is a Gα12 G-protein-coupled receptor (8) and as such, we wanted to provide further evidence of the involvement of this receptor system in the antiproliferative actions of AEA. Again, we used a genetic approach and stably knocked down the expression of Gα12 by transfecting Gα12-specific shRNA constructs into Mz-ChA-1 cells. Characterization of the resulting cell line (designated Mz-Gα12 shRNA) revealed a 60% knock down in both mRNA and protein expression compared to the Mz-Neo neg cells (Supplemental Figure S6). We then assessed the effects of AEA and O-1602 on these cells in vitro and clearly demonstrated that, as seen above, both AEA and O-1602 decreased cell viability in Mz-Neo neg to a similar degree, but failed to have any effect in Mz-Gα12 shRNA cells (<xref ref-type="fig" rid="nihms-269270-f0004">Figure 4</xref>). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-Gα12 shRNA cells (). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-Gα12 shRNA cells (Supplemental Figure S7), suggesting a dependence on Gα12 for the antiproliferative effects of AEA and O-1602.'], 'nihms-269270-f0005': ['One of the downstream consequences of the activation of Gα12 G-protein-coupled receptors is the activation of JNK (11). We therefore assessed the effects of AEA and O-1602 on JNK activity. Treatment of Mz-ChA-1 cells with AEA and O-1602 increased JNK activity 1 hr after stimulation, an effect that continued up to 6 hours (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5A</xref>). Furthermore, pretreatment of Mz-ChA-1 cells with the JNK inhibitor attenuated the antiproliferative effects of both AEA and O-1602 (). Furthermore, pretreatment of Mz-ChA-1 cells with the JNK inhibitor attenuated the antiproliferative effects of both AEA and O-1602 (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5B</xref>). To demonstrate the requirement of GPR55 and Gα12 in the AEA-induced activation of JNK activity, we assessed the effects of AEA and O-1602 on JNK activity in our knockdown cell lines. Treatment of the Mz-Neo neg cells with AEA and O-1602 caused an increase in JNK activity in a similar manner to that seen in the parental cell line (). To demonstrate the requirement of GPR55 and Gα12 in the AEA-induced activation of JNK activity, we assessed the effects of AEA and O-1602 on JNK activity in our knockdown cell lines. Treatment of the Mz-Neo neg cells with AEA and O-1602 caused an increase in JNK activity in a similar manner to that seen in the parental cell line (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5C</xref>). However, treatment of cells with reduced expression of either GPR55 (Mz-GPR55 shRNA) or Gα12 (Mz-Gα12 shRNA) with AEA or O-1602 failed to induce JNK activation (). However, treatment of cells with reduced expression of either GPR55 (Mz-GPR55 shRNA) or Gα12 (Mz-Gα12 shRNA) with AEA or O-1602 failed to induce JNK activation (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5C</xref>). Taken together, these data suggest that the activation of GPR55 by AEA requires JNK activity to exert its antiproliferative effects ). Taken together, these data suggest that the activation of GPR55 by AEA requires JNK activity to exert its antiproliferative effects in vitro.'], 'nihms-269270-f0006': ['We have previously shown that AEA exerts its tumor-suppressive effects via the stabilization of lipid raft structures and that disrupting lipid rafts prevents these antiproliferative effects (12). If our hypothesis that AEA is working via the activation of GPR55 is correct, the specific GPR55 agonist should also require lipid rafts for its function. To this end, cholangiocarcinoma cell lines were pretreated with the lipid raft disruptors, β-methyl cyclodextrin and fillipin III, prior to the addition of O-1602. Disruption of lipid rafts by these two agents prevented the antiproliferative effects of GPR55 activation in Mz-ChA-1 cells (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6A</xref>) and the other cholangiocarcinoma cell lines studied () and the other cholangiocarcinoma cell lines studied (Supplemental Figure S8).', 'We then wanted to determine if GPR55 itself was recruited into lipid rafts or whether the lipid raft-mediated effects were downstream of receptor activation. Using sucrose density centrifugation, we isolated the detergent-resistant membrane fractions that contain the lipid raft structures. We have previously shown that under the conditions used in the experiments outlined here, lipid raft-enriched fractions can be found at the interface between the 5% sucrose and 30% sucrose layers (12), which corresponds to fractions 3 and 4. Using flotillin-1 as a lipid raft marker, it can be seen that the lipid rafts did indeed float to the interface between 5% and 30% sucrose layers under basal conditions and after AEA and O-1602 treatment, whereas the non-raft associated β-actin was used as a negative control to show that under our experimental conditions, non-raft-associated proteins were not found in the lipid raft fractions (Supplemental Figure S9 A). Using these characterized fractions, we showed that under basal conditions, GPR55 was exclusively found in the non-lipid raft fractions. However, after AEA and O-1602 treatment, GPR55 could be found in the lipid raft fractions as well (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6B</xref>). This suggests that upon activation, GPR55 migrates into lipid rafts in the plasma membrane. As mentioned previously, GPR55 is coupled to Gα12 and therefore if GPR55 migrates into lipid rafts, Gα12 should also be found in lipid rafts. Indeed, Gα12 was found predominantly in non-lipid raft fractions under basal conditions; however, after AEA and O-1602 treatment, Gα12 was also found in lipid raft fractions (). This suggests that upon activation, GPR55 migrates into lipid rafts in the plasma membrane. As mentioned previously, GPR55 is coupled to Gα12 and therefore if GPR55 migrates into lipid rafts, Gα12 should also be found in lipid rafts. Indeed, Gα12 was found predominantly in non-lipid raft fractions under basal conditions; however, after AEA and O-1602 treatment, Gα12 was also found in lipid raft fractions (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6B</xref>). Furthermore, Gα12 migration into lipid rafts after AEA and O-1602 treatment was observed in Mz-Neo neg (). Furthermore, Gα12 migration into lipid rafts after AEA and O-1602 treatment was observed in Mz-Neo neg (Supplemental Figure S9 B) but was absent in cells with GPR55 expression knocked down (Mz-GPR55 shRNA; Supplemental Figure S9 B), suggesting a requirement for GPR55 expression for the translocation of Gα12 into lipid rafts in response to AEA or O-1602 treatment.', 'In support of these data, double-staining experiments showed that under basal conditions there was a moderate overlap between GPR55 (red) and lipid raft-specific staining (green), indicated by the yellow color (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6C</xref>). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.36 to 0.57 in 6 random regions. However, after both AEA and O-1602 treatment, there was a greater degree of correlation and co-localization between GPR55 and lipid raft staining (). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.36 to 0.57 in 6 random regions. However, after both AEA and O-1602 treatment, there was a greater degree of correlation and co-localization between GPR55 and lipid raft staining (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6C</xref>), resulting in a Pearson\'s co-efficient in the range of 0.75 to 0.93.), resulting in a Pearson\'s co-efficient in the range of 0.75 to 0.93.'], 'nihms-269270-f0007': ['We have previously shown that AEA appears to activate pro-apoptotic events in cholangiocarcinoma that require the involvement of the TNF super family, the recruitment of Fas and Fas ligand into lipid raft structures and the downstream effector molecule, Fas-associated death domain (FADD) (12). Therefore, we wanted to assess if this phenomenon also occurred with the GPR55 agonists. Under basal conditions Fas was found exclusively in the non-lipid raft fractions, but after AEA treatment, Fas could also be found in the lipid raft fractions (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7A</xref>). Similarly, the recruitment of Fas into lipid raft fractions could also be detected after treatment with O-1602 (). Similarly, the recruitment of Fas into lipid raft fractions could also be detected after treatment with O-1602 (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7A</xref>), suggesting that this recruitment of Fas into lipid rafts may be via GPR55 activation.), suggesting that this recruitment of Fas into lipid rafts may be via GPR55 activation.', 'In support of these data, co-localization experiments showed that under basal conditions there was a moderate overlap between Fas (red) and lipid raft-specific staining (green), indicated by the yellow color (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7B</xref>). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.33 to 0.65 in 6 random regions. However, after both AEA and O-1602 treatment there was a greater degree of correlation and co-localization between Fas and lipid raft staining (). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.33 to 0.65 in 6 random regions. However, after both AEA and O-1602 treatment there was a greater degree of correlation and co-localization between Fas and lipid raft staining (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7B</xref>) resulting in a Pearson\'s co-efficient of 0.74 to 0.94.) resulting in a Pearson\'s co-efficient of 0.74 to 0.94.', 'To clearly demonstrate the requirement of GPR55 expression in the AEA- and O-1602-mediated recruitment of Fas into lipid rafts, we evaluated this phenomenon in cells with suppressed GPR55 expression. As with the parental cell line, AEA and O-1602 treatment of mock-transfected cells (Mz-Neo neg) resulted in the recruitment of Fas into lipid raft structures (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7C</xref>), whereas Fas recruitment into lipid rafts was not observed in Mz-GPR55 shRNA after AEA or O-1602 treatment (), whereas Fas recruitment into lipid rafts was not observed in Mz-GPR55 shRNA after AEA or O-1602 treatment (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7C</xref>).).', 'In addition, because we have demonstrated that GPR55-mediated effects on cholangiocarcinoma cell viability require the activation of the JNK pathway, we wished to assess if the AEA- and O-1602-mediated recruitment of Fas into lipid rafts is also dependent on JNK activation. Indeed, pretreatment of cholangiocarcinoma cells with a JNK inhibitor prevented the translocation of Fas into lipid raft fractions after GPR55 activation (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7C</xref>).).']}
|
Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor
|
[
"Cannabinoids",
"lipid rafts",
"JNK",
"Fas",
"Gα12 G protein"
] |
Lab Invest
|
1309762800
|
Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. It has previously been shown that the endocannabinoid anandamide exerts antiproliferative effects on cholangiocarcinoma independent of any known cannabinoid receptors, and by the stabilization of lipid rafts, thereby allowing the recruitment and activation of the Fas death receptor complex. Recently, GPR55 was identified as a putative cannabinoid receptor; therefore, the role of GPR55 in the antiproliferative effects of anandamide was evaluated. GPR55 is expressed in all cholangiocarcinoma cells and liver biopsy samples to a similar level as in non-malignant cholangiocytes. Treatment with either anandamide or the GPR55 agonist, O-1602, reduced cholangiocarcinoma cell proliferation in vitro and in vivo. Furthermore, knocking down the expression of GPR55 prevented the antiproliferative effects of anandamide. Coupled to these effects was an increase in JNK activity. The antiproliferative effects of anandamide could be blocked by pretreatment with a JNK inhibitor and the lipid raft disruptors β-methylcyclodextrin and fillipin III. Activation of GPR55 by anandamide or O-1602 increased the amount of Fas in the lipid raft fractions, which could be blocked by pretreatment with the JNK inhibitor. These data represent the first evidence that GPR55 activation by anandamide can lead to the recruitment and activation of the Fas death receptor complex and that targeting GPR55 activation may be a viable option for the development of therapeutic strategies to treat cholangiocarcinoma.
|
[
"Animals",
"Arachidonic Acids",
"Bile Duct Neoplasms",
"Bile Ducts, Intrahepatic",
"Cannabinoid Receptor Modulators",
"Cell Line, Tumor",
"Cell Proliferation",
"Cholangiocarcinoma",
"Endocannabinoids",
"Humans",
"Mice",
"Mice, Nude",
"Polyunsaturated Alkamides",
"Receptors, Cannabinoid",
"Receptors, G-Protein-Coupled"
] |
other
|
PMC3126905
| null |
49
|
[
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] |
Lab Invest. 2011 Jul 4; 91(7):1007-1017
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NO-CC CODE
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Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. (A) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10−5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p<0.05) compared to basal treatment (n=7). (B) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for 4 hr and detergent-resistant lipid rafts were isolated on a discontinuous 5%-40% sucrose gradient. The resulting fractions were analyzed by immunoblotting using GPR55 and Gα12-specific antibodies. (D) Co-localization of GPR55 immunoreactivity and lipid raft staining after AEA and O-1602 treatment. Mz-ChA-1 cells were treated with AEA and O-1602 (both at 10−5 M) and the lipid rafts were stained with Alexa Fluor 488-conjugated cholera toxin B subunit (green) as well as GPR55 immunoreactivity (red). Co-localization is indicated by yellow areas. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm.
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multiple panels: images & plots
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"Microscopy",
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"Immuno Assays"
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[{'image_id': 'nihms-269270-f0005', 'image_file_name': 'nihms-269270-f0005.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0005.jpg', 'caption': 'GPR55 activation by AEA and O-1602 increases JNK activity. (A) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for various time points up to 6 hr. JNK activity was assessed by commercially available ELISA kits (average ± SEM, n=4; * p<0.05 compared to basal treatment. (B) Mz-ChA-1 cells were pretreated with the JNK inhibitor SP600125 (10−7 M) for 1 hr prior to the addition of AEA or O-1602 (both at 10−5 M) for 48 hr. Cell viability was assessed by MTS assays. Data are expressed as fold change in viability (average ± SEM, n=7, *p<0.05 compared to basal treatment within each cell line). (C) Mz-Neo neg, Mz-GPR55 shRNA and Mz-Gα12 shRNA cells were treated with AEA or O-1602 (both at 10−5 M) for 6 hr. JNK activity was assessed by commercially available ELISA kits. * p<0.05 compared to basal treatment within each cell line.', 'hash': '6152261e2530fe98b7d1aa6fcd092cc8bacc792e29e5c5708c2d38d7d72e75b5'}, {'image_id': 'nihms-269270-f0002', 'image_file_name': 'nihms-269270-f0002.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0002.jpg', 'caption': '(A) Cholangiocarcinoma cells (Mz-ChA-1, HuCCT-1, CCLP-1 and SG231) and non-malignant H69 and HIBEC cells were treated with various concentrations of the GPR55 agonist, O-1602 (10−9 to 10−5 M) for 48 hr. Cell viability was assessed by MTS assays. Data are expressed as fold change in viability (average ± SEM, n=7) * p<0.05 compared to basal treatment within each cell line. (B) Mz-ChA-1 cells were injected into the flank of athymic mice. After tumors were established, mice were treated with 10 mg/kg/day (ip) O-1602, three days per week and tumor volume assessed.', 'hash': '9ca36d96de3b93cc470c4de2b1910e9d5b3454037108f0fb9337ecd7b12aba53'}, {'image_id': 'nihms-269270-f0003', 'image_file_name': 'nihms-269270-f0003.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0003.jpg', 'caption': 'Specific knockdown of GPR55 receptor expression prevents the antiproliferative effects of AEA in vitro and in vivo. Mz-ChA-1 cells were stably transfected with GPR55 shRNA vectors. The effect of AEA and O-1602 on cells with low levels of GPR55 expression (Mz-GPR55 shRNA) was assessed in vitro by MTS assay (A). Mz-Neo neg and Mz-GPR55 shRNA cells were treated with various concentrations of the GPR55 agonists, AEA and O-1602 (10−5 M) for 48 hr. Cell viability was assessed by MTS assays. Data are expressed as fold change in viability (average ± SEM, n=7; * p<0.05 compared to basal treatment within each cell line). The effect of AEA and O-1602 on cells with low levels of GPR55 expression was assessed in vivo using the xenograft model of cholangiocarcinoma (B). Mz-Neo neg cells and Mz-GPR55 shRNA cells were injected into the flank of athymic mice. After tumors were established, mice were treated with 10 mg/kg/day (ip) O-1602, three days per week and tumor volume assessed.', 'hash': '1a97a0146bb545f3c4fc2ae765593afc8dff5ae50da93855c196a5caeeb9e22b'}, {'image_id': 'nihms-269270-f0004', 'image_file_name': 'nihms-269270-f0004.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0004.jpg', 'caption': 'Specific knockdown of Gα12 G protein expression prevents the antiproliferative effects of GPR55 activation in vitro. Mz-ChA-1 cells were stably transfected with Gα12 shRNA vectors (Mz-Gα12 shRNA). The effect of AEA and O-1602 on cells with low levels of Gα12 expression was assessed in vitro by MTS assay (A). Mz-Neo neg and Mz-Gα12 shRNA cells were treated with various concentrations of the GPR55 agonist, O-1602 (10−9 to 10−5 M) for 48 hr. Cell viability was assessed using an MTS cell proliferation assay. Data are expressed as fold change in viability (average ± SEM, n=7, *p<0.05 compared to basal treatment within each cell line).', 'hash': 'a796330276b019fc41fe7edb68f9421323b15799716b42a835f3c89355b9c32c'}, {'image_id': 'nihms-269270-f0007', 'image_file_name': 'nihms-269270-f0007.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0007.jpg', 'caption': 'Fas receptor is recruited into lipid rafts after GPR55 receptor activation. (A) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for 24 hr and detergent-resistant lipid rafts were isolated on a discontinuous 5%-40% sucrose gradient. The resulting fractions were analyzed by immunoblotting using a Fas-specific antibody to determine the subcellular location of these proteins. (B) Co-localization of Fas immunoreactivity and lipid raft staining after AEA and O-1602 treatment. Mz-ChA-1 cells were treated with AEA and O-1602 (both at 10−5 M) and the lipid rafts were stained with Alexa Fluor 488-conjugated cholera toxin B subunit (green) as well as Fas immunoreactivity (red). Co-localization is indicated by yellow areas. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (C) Mz-Neo neg and Mz-GPR55 shRNA were treated with AEA or O-1602 (both at 10−5 M) for 24 hr and detergent-resistant lipid rafts were isolated. In addition Mz-ChA-1 cells were pretreated with the JNK inhibitor, SP600125 (10−7 M) for 1 hr prior to the addition of AEA or O-1602 (both at 10−5 M) for 24 hr followed by lipid raft fractionation. The resulting fractions were analyzed by immunoblotting using a Fas-specific antibody to determine its subcellular location.', 'hash': '8f550e0888161246c89aceb805f6b12a5586395b53c199444a12a0297aeb7604'}, {'image_id': 'nihms-269270-f0001', 'image_file_name': 'nihms-269270-f0001.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0001.jpg', 'caption': 'GPR55 is expressed in cholangiocyte and cholangiocarcinoma cell lines. GPR55 levels were assessed in four cholangiocarcinoma cell lines as well as the non-malignant cholangiocyte cell lines H69 and HIBEC, by real time PCR (A), immunoblotting (B). Data are expressed as average relative GPR55 mRNA expression (± SEM) in each cell line compared to H69 cells after using GAPDH expression as a loading control (n=3; A).', 'hash': '2ef5956d9012343a28c21db2f27f5f7f9758ee56eb570baefa5020c5b9aed078'}, {'image_id': 'nihms-269270-f0006', 'image_file_name': 'nihms-269270-f0006.jpg', 'image_path': '../data/media_files/PMC3126905/nihms-269270-f0006.jpg', 'caption': 'Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. (A) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10−5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p<0.05) compared to basal treatment (n=7). (B) Mz-ChA-1 cells were treated with AEA or O-1602 (both at 10−5 M) for 4 hr and detergent-resistant lipid rafts were isolated on a discontinuous 5%-40% sucrose gradient. The resulting fractions were analyzed by immunoblotting using GPR55 and Gα12-specific antibodies. (D) Co-localization of GPR55 immunoreactivity and lipid raft staining after AEA and O-1602 treatment. Mz-ChA-1 cells were treated with AEA and O-1602 (both at 10−5 M) and the lipid rafts were stained with Alexa Fluor 488-conjugated cholera toxin B subunit (green) as well as GPR55 immunoreactivity (red). Co-localization is indicated by yellow areas. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm.', 'hash': '77be13d3d48447053c5175ec1b1a5da435d865522baef9ab086da298be41cc0b'}]
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{'nihms-269270-f0001': ['All of the cholangiocarcinoma cell lines studied here, as well as the non-malignant cell lines (H69 and HIBEC) expressed the mRNA (<xref ref-type="fig" rid="nihms-269270-f0001">Figure 1A</xref>) and protein () and protein (<xref ref-type="fig" rid="nihms-269270-f0001">Figure 1B</xref>) for GPR55. There was no obvious or consistent difference between the GPR55 mRNA and protein expression in the malignant and non-malignant cell lines () for GPR55. There was no obvious or consistent difference between the GPR55 mRNA and protein expression in the malignant and non-malignant cell lines (<xref ref-type="fig" rid="nihms-269270-f0001">Figure 1A, 1B</xref>). By immunofluorescence, GPR55 immunoreactivity was predominantly found in the membrane and cytoplasm in all cell lines studied (). By immunofluorescence, GPR55 immunoreactivity was predominantly found in the membrane and cytoplasm in all cell lines studied (Supplemental Figure S1 A). Furthermore, immunohistochemical analysis of human liver biopsy samples indicated similar intensity and subcellular location of GPR55 immunoreactivity in malignant and non-malignant cholangiocytes (Supplemental Figure S1 B).'], 'nihms-269270-f0002': ['Activation of GPR55 with the specific agonist O-1602 in vitro significantly decreased cell viability in all cholangiocarcinoma cell lines studied, but not in the non-malignant cholangiocyte cell lines (<xref ref-type="fig" rid="nihms-269270-f0002">Figure 2A</xref>). Furthermore, there was an increase in apoptosis in all cholangiocarcinoma cell lines after O-1602 treatment as demonstrated by Annexin V staining (). Furthermore, there was an increase in apoptosis in all cholangiocarcinoma cell lines after O-1602 treatment as demonstrated by Annexin V staining (Supplemental Figure S2). Treating in vivo xenograft cholangiocarcinoma tumors with O-1602 significantly inhibited tumor growth (<xref ref-type="fig" rid="nihms-269270-f0002">Figure 2B</xref>). In addition, the latency of tumor growth (i.e., time taken for tumor volume to increase to 150% of the original size) was increased from 5.8 ± 0.76 to 11.2 ± 2.74 days after chronic O-1602 treatment. Histological analysis of the excised tumors revealed that all cancer cells within tumors from O-1602-treated and vehicle-treated animals were CK-19 positive, indicating a cholangiocyte phenotype (). In addition, the latency of tumor growth (i.e., time taken for tumor volume to increase to 150% of the original size) was increased from 5.8 ± 0.76 to 11.2 ± 2.74 days after chronic O-1602 treatment. Histological analysis of the excised tumors revealed that all cancer cells within tumors from O-1602-treated and vehicle-treated animals were CK-19 positive, indicating a cholangiocyte phenotype (Supplemental Figure S3). Furthermore, all cholangiocarcinoma cells in the tumor retained GPR55 immunoreactivity, which appeared to increase after O-1602 treatment (Supplemental Figure S3). Using TUNEL staining as a marker of apoptosis, O-1602 treatment increased the incidence of apoptosis in the cholangiocarcinoma tumors (Supplemental Figure S3).'], 'nihms-269270-f0003': ['We have previously shown that AEA exerts antiproliferative effects on cholangiocarcinoma (12) in a similar manner to that shown here for GPR55 activation. Therefore, we wanted to assess definitively if AEA is working through a GPR55-dependent mechanism. To this end, we stably knocked down the expression of GPR55 by transfecting GPR55-specific shRNA constructs into Mz-ChA-1 cells. Characterization of the resulting cell line (Mz-GPR55 shRNA) revealed a 60-70% knock down in GPR55 mRNA and protein expression when compared to the mock-transfected cell line (Mz-Neo neg; Supplemental Figure S4). Using these cell lines, we assessed the antiproliferative effects of both AEA and O-1602 in vitro and in vivo. Both AEA and O-1602 decreased cell viability in Mz-Neo neg to a similar degree, but failed to have any effect in Mz-GPR55 shRNA cells (<xref ref-type="fig" rid="nihms-269270-f0003">Figure 3A</xref>). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-GPR55 shRNA cells (). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-GPR55 shRNA cells (Supplemental Figure S5). The growth suppressive effects of AEA and O-1602 were evident in the xenograft model of cholangiocarcinoma using Mz-Neo neg cells (<xref ref-type="fig" rid="nihms-269270-f0003">Figure 3B</xref>), whereas tumors derived from the implantation of Mz-GPR55 shRNA were not sensitive to AEA or O-1602 (), whereas tumors derived from the implantation of Mz-GPR55 shRNA were not sensitive to AEA or O-1602 (<xref ref-type="fig" rid="nihms-269270-f0003">Figure 3B</xref>).).'], 'nihms-269270-f0004': ['GPR55 is a Gα12 G-protein-coupled receptor (8) and as such, we wanted to provide further evidence of the involvement of this receptor system in the antiproliferative actions of AEA. Again, we used a genetic approach and stably knocked down the expression of Gα12 by transfecting Gα12-specific shRNA constructs into Mz-ChA-1 cells. Characterization of the resulting cell line (designated Mz-Gα12 shRNA) revealed a 60% knock down in both mRNA and protein expression compared to the Mz-Neo neg cells (Supplemental Figure S6). We then assessed the effects of AEA and O-1602 on these cells in vitro and clearly demonstrated that, as seen above, both AEA and O-1602 decreased cell viability in Mz-Neo neg to a similar degree, but failed to have any effect in Mz-Gα12 shRNA cells (<xref ref-type="fig" rid="nihms-269270-f0004">Figure 4</xref>). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-Gα12 shRNA cells (). Similarly, AEA and O-1602 increased the amount of apoptotic cells in Mz-Neo neg cells, but had no effect in the Mz-Gα12 shRNA cells (Supplemental Figure S7), suggesting a dependence on Gα12 for the antiproliferative effects of AEA and O-1602.'], 'nihms-269270-f0005': ['One of the downstream consequences of the activation of Gα12 G-protein-coupled receptors is the activation of JNK (11). We therefore assessed the effects of AEA and O-1602 on JNK activity. Treatment of Mz-ChA-1 cells with AEA and O-1602 increased JNK activity 1 hr after stimulation, an effect that continued up to 6 hours (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5A</xref>). Furthermore, pretreatment of Mz-ChA-1 cells with the JNK inhibitor attenuated the antiproliferative effects of both AEA and O-1602 (). Furthermore, pretreatment of Mz-ChA-1 cells with the JNK inhibitor attenuated the antiproliferative effects of both AEA and O-1602 (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5B</xref>). To demonstrate the requirement of GPR55 and Gα12 in the AEA-induced activation of JNK activity, we assessed the effects of AEA and O-1602 on JNK activity in our knockdown cell lines. Treatment of the Mz-Neo neg cells with AEA and O-1602 caused an increase in JNK activity in a similar manner to that seen in the parental cell line (). To demonstrate the requirement of GPR55 and Gα12 in the AEA-induced activation of JNK activity, we assessed the effects of AEA and O-1602 on JNK activity in our knockdown cell lines. Treatment of the Mz-Neo neg cells with AEA and O-1602 caused an increase in JNK activity in a similar manner to that seen in the parental cell line (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5C</xref>). However, treatment of cells with reduced expression of either GPR55 (Mz-GPR55 shRNA) or Gα12 (Mz-Gα12 shRNA) with AEA or O-1602 failed to induce JNK activation (). However, treatment of cells with reduced expression of either GPR55 (Mz-GPR55 shRNA) or Gα12 (Mz-Gα12 shRNA) with AEA or O-1602 failed to induce JNK activation (<xref ref-type="fig" rid="nihms-269270-f0005">Figure 5C</xref>). Taken together, these data suggest that the activation of GPR55 by AEA requires JNK activity to exert its antiproliferative effects ). Taken together, these data suggest that the activation of GPR55 by AEA requires JNK activity to exert its antiproliferative effects in vitro.'], 'nihms-269270-f0006': ['We have previously shown that AEA exerts its tumor-suppressive effects via the stabilization of lipid raft structures and that disrupting lipid rafts prevents these antiproliferative effects (12). If our hypothesis that AEA is working via the activation of GPR55 is correct, the specific GPR55 agonist should also require lipid rafts for its function. To this end, cholangiocarcinoma cell lines were pretreated with the lipid raft disruptors, β-methyl cyclodextrin and fillipin III, prior to the addition of O-1602. Disruption of lipid rafts by these two agents prevented the antiproliferative effects of GPR55 activation in Mz-ChA-1 cells (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6A</xref>) and the other cholangiocarcinoma cell lines studied () and the other cholangiocarcinoma cell lines studied (Supplemental Figure S8).', 'We then wanted to determine if GPR55 itself was recruited into lipid rafts or whether the lipid raft-mediated effects were downstream of receptor activation. Using sucrose density centrifugation, we isolated the detergent-resistant membrane fractions that contain the lipid raft structures. We have previously shown that under the conditions used in the experiments outlined here, lipid raft-enriched fractions can be found at the interface between the 5% sucrose and 30% sucrose layers (12), which corresponds to fractions 3 and 4. Using flotillin-1 as a lipid raft marker, it can be seen that the lipid rafts did indeed float to the interface between 5% and 30% sucrose layers under basal conditions and after AEA and O-1602 treatment, whereas the non-raft associated β-actin was used as a negative control to show that under our experimental conditions, non-raft-associated proteins were not found in the lipid raft fractions (Supplemental Figure S9 A). Using these characterized fractions, we showed that under basal conditions, GPR55 was exclusively found in the non-lipid raft fractions. However, after AEA and O-1602 treatment, GPR55 could be found in the lipid raft fractions as well (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6B</xref>). This suggests that upon activation, GPR55 migrates into lipid rafts in the plasma membrane. As mentioned previously, GPR55 is coupled to Gα12 and therefore if GPR55 migrates into lipid rafts, Gα12 should also be found in lipid rafts. Indeed, Gα12 was found predominantly in non-lipid raft fractions under basal conditions; however, after AEA and O-1602 treatment, Gα12 was also found in lipid raft fractions (). This suggests that upon activation, GPR55 migrates into lipid rafts in the plasma membrane. As mentioned previously, GPR55 is coupled to Gα12 and therefore if GPR55 migrates into lipid rafts, Gα12 should also be found in lipid rafts. Indeed, Gα12 was found predominantly in non-lipid raft fractions under basal conditions; however, after AEA and O-1602 treatment, Gα12 was also found in lipid raft fractions (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6B</xref>). Furthermore, Gα12 migration into lipid rafts after AEA and O-1602 treatment was observed in Mz-Neo neg (). Furthermore, Gα12 migration into lipid rafts after AEA and O-1602 treatment was observed in Mz-Neo neg (Supplemental Figure S9 B) but was absent in cells with GPR55 expression knocked down (Mz-GPR55 shRNA; Supplemental Figure S9 B), suggesting a requirement for GPR55 expression for the translocation of Gα12 into lipid rafts in response to AEA or O-1602 treatment.', 'In support of these data, double-staining experiments showed that under basal conditions there was a moderate overlap between GPR55 (red) and lipid raft-specific staining (green), indicated by the yellow color (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6C</xref>). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.36 to 0.57 in 6 random regions. However, after both AEA and O-1602 treatment, there was a greater degree of correlation and co-localization between GPR55 and lipid raft staining (). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.36 to 0.57 in 6 random regions. However, after both AEA and O-1602 treatment, there was a greater degree of correlation and co-localization between GPR55 and lipid raft staining (<xref ref-type="fig" rid="nihms-269270-f0006">Figure 6C</xref>), resulting in a Pearson\'s co-efficient in the range of 0.75 to 0.93.), resulting in a Pearson\'s co-efficient in the range of 0.75 to 0.93.'], 'nihms-269270-f0007': ['We have previously shown that AEA appears to activate pro-apoptotic events in cholangiocarcinoma that require the involvement of the TNF super family, the recruitment of Fas and Fas ligand into lipid raft structures and the downstream effector molecule, Fas-associated death domain (FADD) (12). Therefore, we wanted to assess if this phenomenon also occurred with the GPR55 agonists. Under basal conditions Fas was found exclusively in the non-lipid raft fractions, but after AEA treatment, Fas could also be found in the lipid raft fractions (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7A</xref>). Similarly, the recruitment of Fas into lipid raft fractions could also be detected after treatment with O-1602 (). Similarly, the recruitment of Fas into lipid raft fractions could also be detected after treatment with O-1602 (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7A</xref>), suggesting that this recruitment of Fas into lipid rafts may be via GPR55 activation.), suggesting that this recruitment of Fas into lipid rafts may be via GPR55 activation.', 'In support of these data, co-localization experiments showed that under basal conditions there was a moderate overlap between Fas (red) and lipid raft-specific staining (green), indicated by the yellow color (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7B</xref>). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.33 to 0.65 in 6 random regions. However, after both AEA and O-1602 treatment there was a greater degree of correlation and co-localization between Fas and lipid raft staining (). Pearson\'s correlation analysis of the degree of co-localization revealed a Pearson\'s coefficient in the range of 0.33 to 0.65 in 6 random regions. However, after both AEA and O-1602 treatment there was a greater degree of correlation and co-localization between Fas and lipid raft staining (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7B</xref>) resulting in a Pearson\'s co-efficient of 0.74 to 0.94.) resulting in a Pearson\'s co-efficient of 0.74 to 0.94.', 'To clearly demonstrate the requirement of GPR55 expression in the AEA- and O-1602-mediated recruitment of Fas into lipid rafts, we evaluated this phenomenon in cells with suppressed GPR55 expression. As with the parental cell line, AEA and O-1602 treatment of mock-transfected cells (Mz-Neo neg) resulted in the recruitment of Fas into lipid raft structures (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7C</xref>), whereas Fas recruitment into lipid rafts was not observed in Mz-GPR55 shRNA after AEA or O-1602 treatment (), whereas Fas recruitment into lipid rafts was not observed in Mz-GPR55 shRNA after AEA or O-1602 treatment (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7C</xref>).).', 'In addition, because we have demonstrated that GPR55-mediated effects on cholangiocarcinoma cell viability require the activation of the JNK pathway, we wished to assess if the AEA- and O-1602-mediated recruitment of Fas into lipid rafts is also dependent on JNK activation. Indeed, pretreatment of cholangiocarcinoma cells with a JNK inhibitor prevented the translocation of Fas into lipid raft fractions after GPR55 activation (<xref ref-type="fig" rid="nihms-269270-f0007">Figure 7C</xref>).).']}
|
Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor
|
[
"Cannabinoids",
"lipid rafts",
"JNK",
"Fas",
"Gα12 G protein"
] |
Lab Invest
|
1309762800
|
Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. It has previously been shown that the endocannabinoid anandamide exerts antiproliferative effects on cholangiocarcinoma independent of any known cannabinoid receptors, and by the stabilization of lipid rafts, thereby allowing the recruitment and activation of the Fas death receptor complex. Recently, GPR55 was identified as a putative cannabinoid receptor; therefore, the role of GPR55 in the antiproliferative effects of anandamide was evaluated. GPR55 is expressed in all cholangiocarcinoma cells and liver biopsy samples to a similar level as in non-malignant cholangiocytes. Treatment with either anandamide or the GPR55 agonist, O-1602, reduced cholangiocarcinoma cell proliferation in vitro and in vivo. Furthermore, knocking down the expression of GPR55 prevented the antiproliferative effects of anandamide. Coupled to these effects was an increase in JNK activity. The antiproliferative effects of anandamide could be blocked by pretreatment with a JNK inhibitor and the lipid raft disruptors β-methylcyclodextrin and fillipin III. Activation of GPR55 by anandamide or O-1602 increased the amount of Fas in the lipid raft fractions, which could be blocked by pretreatment with the JNK inhibitor. These data represent the first evidence that GPR55 activation by anandamide can lead to the recruitment and activation of the Fas death receptor complex and that targeting GPR55 activation may be a viable option for the development of therapeutic strategies to treat cholangiocarcinoma.
|
[
"Animals",
"Arachidonic Acids",
"Bile Duct Neoplasms",
"Bile Ducts, Intrahepatic",
"Cannabinoid Receptor Modulators",
"Cell Line, Tumor",
"Cell Proliferation",
"Cholangiocarcinoma",
"Endocannabinoids",
"Humans",
"Mice",
"Mice, Nude",
"Polyunsaturated Alkamides",
"Receptors, Cannabinoid",
"Receptors, G-Protein-Coupled"
] |
other
|
PMC3126905
| null |
49
|
[
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] |
Lab Invest. 2011 Jul 4; 91(7):1007-1017
|
NO-CC CODE
|
|
Incorporating EcR and E75 in the molecular clockEcR is expressed in clock cells of larval (A) and adult brains (B). EcR is detected with two different mouse anti-EcR antibodies (EcR-A and EcR-C) and PDF is stained with a rabbit anti-PDF antibody. EcR-C antibody detects all isoforms of EcR (A, B1 and B2), whereas EcR-A detects the RA specific isoform of EcR. Scale bar = 10μm. (C) Model for the role of E75 in the Drosophila molecular clock. E75 represses Clk transcription, and this repression is inhibited by PER, which thus acts as a de-repressor of Clk. PER can also modulate Clk expression through VRI (as VRI is a transcriptional target of CLK, which is regulated by PER), but this is not shown here for the sake of simplicity. In addition, E75 also regulates VRI expression in such a way that overexpression or knockdown of E75 increases or reduces the VRI levels respectively, thus indirectly affecting the CLK expression. Under stress (nutritional and temperature) conditions, E75 is required to maintain robust rhythms.
|
nihms639102f6
|
2
|
2c67fcd4ba42b71a00a9abc06874f10a9f51346f1580bc3452838511e37817e1
|
nihms639102f6.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
667,
376
] |
[{'image_id': 'nihms639102f1', 'image_file_name': 'nihms639102f1.jpg', 'image_path': '../data/media_files/PMC4269253/nihms639102f1.jpg', 'caption': 'Effect of E75 overexpression on the expression of per and Clk in adult heads(A)\nper mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies during the indicated phases of an LD cycle. (B)\nClk mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies under LD cycle. (C) PER and CLK levels in the genotypes indicated above. A representative western blot is shown. PER and CLK levels are significantly lower in the TG27 >UAS-E75 (II) flies than in TG27 control flies particularly at peak time points. HSP70 antibodies are used to control for loading. Quantification of four independent experiments shows significantly decreased (D) PER and (E) CLK levels in TG27 >UAS-E75 (II) flies relative to the TG27 control flies. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. A molecular marker (Precision Plus Protein™ Dual Color Standards) was run to detect the exact molecular size of different proteins.', 'hash': '21cd1dadea10e0099435386942ec958437426b152b3f72da7b1267f443edf06f'}, {'image_id': 'nihms639102f6', 'image_file_name': 'nihms639102f6.jpg', 'image_path': '../data/media_files/PMC4269253/nihms639102f6.jpg', 'caption': 'Incorporating EcR and E75 in the molecular clockEcR is expressed in clock cells of larval (A) and adult brains (B). EcR is detected with two different mouse anti-EcR antibodies (EcR-A and EcR-C) and PDF is stained with a rabbit anti-PDF antibody. EcR-C antibody detects all isoforms of EcR (A, B1 and B2), whereas EcR-A detects the RA specific isoform of EcR. Scale bar = 10μm. (C) Model for the role of E75 in the Drosophila molecular clock. E75 represses Clk transcription, and this repression is inhibited by PER, which thus acts as a de-repressor of Clk. PER can also modulate Clk expression through VRI (as VRI is a transcriptional target of CLK, which is regulated by PER), but this is not shown here for the sake of simplicity. In addition, E75 also regulates VRI expression in such a way that overexpression or knockdown of E75 increases or reduces the VRI levels respectively, thus indirectly affecting the CLK expression. Under stress (nutritional and temperature) conditions, E75 is required to maintain robust rhythms.', 'hash': '2c67fcd4ba42b71a00a9abc06874f10a9f51346f1580bc3452838511e37817e1'}, {'image_id': 'nihms639102f5', 'image_file_name': 'nihms639102f5.jpg', 'image_path': '../data/media_files/PMC4269253/nihms639102f5.jpg', 'caption': 'PER interacts with E75 to de-repress expression of the Clk promoter(A) Co-immunoprecipitation assay showed that PER physically interacts with E75. 100 ng of CMV-per HA was transfected with 200 ng of CMV-E75 V5 (with two different isoforms RC and RA), CMV-cry V5, CMV-GABA-Transaminase (GABAT) V5 and empty CMV vector. Anti-V5 antibody was used to pull down protein complexes. PER specifically binds with CRY but not with GABA-T, PER also binds strongly with E75-RC. The RA isoform was poorly expressed, hence weaker interaction with PER (lane 5 in IP: αV5). (B) Knockdown of E75 increases Clk mRNA expression in wild type and per0 flies. As in Figure 2, E75 knock down by TG27 significantly increases Clk mRNA levels in wild type flies relative to TG27 controls at ZT14. This effect is more striking in per0 flies where baseline Clk mRNA levels are quite low. (C) Overexpression of E75 reduces Clk mRNA expression in wild type and per0 flies. As in Figure 1, E75 overexpression by TG27 significantly decreases Clk mRNA levels in wild type and per0 flies relative to TG27 controls at ZT02. (D) CLK levels in TG27 control, TG27 >UAS-E75 RNAi (GD) and TG27 > UAS-E75 (II) in wild type and per0 backgrounds are shown for two time points. HSP70 antibodies are used to control for loading. Molecular marker (Precision Plus Protein™ Dual Color Standards) were run to facilitate detection of different proteins. (D) Quantification of four independent western blots is shown. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM and indicate variability across flies of a specific genotype.', 'hash': '6a3fe388ad21ee2826c0a35885f96369701a8f0ae7421ae13a95751305e1574f'}, {'image_id': 'nihms639102f2', 'image_file_name': 'nihms639102f2.jpg', 'image_path': '../data/media_files/PMC4269253/nihms639102f2.jpg', 'caption': 'Effects of E75 knockdown on the expression of per and Clk in adult heads(A)\nper mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies at the indicated time points of an LD cycle. (B)\nClk mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies under LD cycle. (C) PER and CLK levels in the same genotypes as above. A representative western blot is shown. HSP70 antibodies are used to control for loading. Quantification of six independent experiments shows (D) PER and (E) CLK levels in TG27 >UAS-E75 RNAi (GD) flies and TG27 control flies. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. A molecular marker (Precision Plus Protein™ Dual Color Standards) was run to detect the exact molecular size of different proteins.', 'hash': 'ff0bb86d8bfeae6b6994b5b6b84ecb17c9a233b480ab2a672c1d275dd109db3e'}, {'image_id': 'nihms639102f3', 'image_file_name': 'nihms639102f3.jpg', 'image_path': '../data/media_files/PMC4269253/nihms639102f3.jpg', 'caption': 'Knockdown of E75 increases PER expression in brain clock cellsFlies from TG27 and TG27> E75 RNAi genotypes were tested for their circadian behavior under DD conditions on 6th day and 8–10 rhythmic TG27 controls and 8–10 arrhythmic E75 knockdown flies were used for IHC at each of the indicated time points. PER expression at different times of day in TG27>UAS-E75 RNAi (GD) flies and TG27 controls in the (A) small and large LNvs and (B) Dorsal LNs (LNds). Quantification of PER staining from (C) s-LNvs and (D) l-LNvs subset of neurons in the TG27 >UAS-E75 RNAi (GD) flies (n=10) relative to TG27 control flies (n=9). Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. Time is indicated as CT (circadian time) where CT0 is 12h after lights-off of the last LD cycle. Scale bar = 10 μm.', 'hash': 'b30b9fbb1006f7db5ac4a2c1ef39490416c3d482cfb41f08521ef4382a99f45e'}, {'image_id': 'nihms639102f4', 'image_file_name': 'nihms639102f4.jpg', 'image_path': '../data/media_files/PMC4269253/nihms639102f4.jpg', 'caption': 'E75 represses transcriptional activity of the native Clk promoter(A) Mammalian HEK-293T cells were transfected with Clk-luc (50ng) and renilla-luc (10ng) reporter (internal control) and with increasing doses of CMV-Pdp1ε (10 and 50 ng) or CMV-E75 (50, 100, and 250 ng) and in some cases with CMV-per (10, 50, 100 ng). The (+), (++) and (+++).denote 10, 50, and 100 ng of DNA respectively, except for the empty pCDNA3.1 vector. Additionally, CMV-gfp (10, 50, 100 ng) was used a control for per. The firefly luciferase activity was normalized to renilla-luc activity, and the fold induction (Y axis) was based upon comparisons with 10 ng of CMV expression vector alone transfections. Data represent an average of four experiments each performed in duplicate. Error bars depict standard error of the mean. C3m-TK luc (a mutant promoter, which cannot be activated by PDP1), was used as an additional control. PDP1 ε and E75 significantly activated and repressed Clk-luc respectively, compared to empty vector pCDNA3.1 controls. PER significantly de-repressed the E75 mediated repression of Clk promoter repression. (B) An artificial promoter, C3-TK luc, was used instead of the native Clk-luc promoter to determine if E75 acts as a co-repressor with VRI. C3-TK luc contains 3 tandem consensus binding sites for PDP1/VRI but none for E75. Other details, including the dosage used, are same to panel (A). PDP1 activates this promoter in a dose dependent manner, and this is repressed by VRI. The highest dose of E75 enhances repression by VRI. (*p < 0.05. Two-way ANOVA, Tukey’s test post-hoc comparison).', 'hash': '5b85705304b8c3fd9b752a40efc2b5a7c81c793cccebc651f48db2f753037a53'}]
|
{'nihms639102f1': ['To determine whether E75 affects molecular clock components, we first examined transcript levels of per and Clk in whole head extracts of flies overexpressing E75 (UAS-E75 II) via the TG27 driver. Oscillations of per and Clk were dampened by E75 over-expression, in particular through a reduction in peak levels (<xref rid="nihms639102f1" ref-type="fig">Figure 1A and B</xref>). We also measured PER and CLK protein levels through western blots of whole head lysates and found that these were significantly reduced in ). We also measured PER and CLK protein levels through western blots of whole head lysates and found that these were significantly reduced in E75 overexpressing flies under LD conditions (<xref rid="nihms639102f1" ref-type="fig">Figure 1C,D and E</xref>). Similar effects were observed under constant dark (DD) conditions; CLK expression was significantly reduced in flies overexpressing ). Similar effects were observed under constant dark (DD) conditions; CLK expression was significantly reduced in flies overexpressing E75 as compared to the controls (Supplementary Figure 2A and B).', 'Tissue culture experiments do not indicate a direct effect of E75 on per expression, although we cannot exclude the possibility that it does so in flies, as suggested by the robust effect of E75 overexpression on per mRNA and protein levels (<xref rid="nihms639102f1" ref-type="fig">Figure 1</xref>). However, E75 interacts with PER to regulate transcription of ). However, E75 interacts with PER to regulate transcription of Clk. Indeed, this work reveals a new role for Drosophila PER as a de-repressor. As noted above, earlier studies showed that PER promotes expression of Clk, but the underlying mechanisms were not identified. We find that it does so by reducing the inhibitory effect of E75 on CLK. It may do so by affecting DNA-binding of E75 or perhaps even by destabilizing it. Regardless, these data are reminiscent of mammalian PER, which acts as a de-repressor with some nuclear receptors and a co-activator with others32. Indeed, mammalian PER2 and REVERB-α physically interact32; 33, as we show here for PER and E75. We suggest that crosstalk between components of the two loops is a conserved mechanism that serves to maintain a robust cycle. On the one hand Drosophila PER inhibits activity of the CLK/CYC complex to generate a negative feedback loop; meanwhile, it interacts with nuclear hormone receptors like E75 to promote Clk gene expression in the positive feedback loop. Although this study only examined effects of PER on E75-mediated repression of CLK, it is likely that there are other circadian targets of E75 that are modulated by PER. Future studies should help to clarify the extent to which E75 impacts transcription within the circadian network. Importantly, the data on E75 presented here provide insight into some of the unresolved questions in the clock field— for instance, why Clk mRNA levels are low in per0 flies.'], 'nihms639102f2': ['Next we examined the effects of knocking down E75 using the same TG27 driver to express UAS-E75 RNAi (GD) constructs. As shown in Table 1B, the GD line in particular led to a strong behavioral phenotype. As predicted by the overexpression data, per transcript levels were slightly, although not significantly, increased in flies in which E75 was knocked down, although PER protein levels were significantly different at ZT08 (<xref rid="nihms639102f2" ref-type="fig">Figure 2A, C and D</xref>). ). Clk mRNA and protein levels were also significantly higher at specific times of day (<xref rid="nihms639102f2" ref-type="fig">Figure 2B, C and E</xref>). Notably under these conditions, ). Notably under these conditions, per and Clk mRNA cycling did not appear to be affected (<xref rid="nihms639102f2" ref-type="fig">Figure 2A–E</xref>). As with ). As with E75 overexpression, knockdown had robust effects on CLK expression under freerunning conditions. CLK levels were significantly higher at CT08 and CT14 on the first day of DD in E75 knockdown flies (Supplementary Figure 2C and D).', 'We also assayed Clk mRNA levels in wild type and per0 backgrounds under conditions where E75 was overexpressed with the TG27 driver. As noted in <xref rid="nihms639102f2" ref-type="fig">Figure 2</xref>, overexpression of E75 reduced , overexpression of E75 reduced Clk mRNA in wild type flies. In per0 flies also, overexpression of E75 reduced expression of Clk mRNA, although the difference was small, perhaps because Clk levels were already low (<xref rid="nihms639102f5" ref-type="fig">Figure 5C</xref>). The effect of knockdown and overexpression of E75 on ). The effect of knockdown and overexpression of E75 on Clk mRNA levels in a per0 background indicate that endogenous PER affects E75 action at the Clk promoter.', '(A) Co-immunoprecipitation assay showed that PER physically interacts with E75. 100 ng of CMV-per HA was transfected with 200 ng of CMV-E75 V5 (with two different isoforms RC and RA), CMV-cry V5, CMV-GABA-Transaminase (GABAT) V5 and empty CMV vector. Anti-V5 antibody was used to pull down protein complexes. PER specifically binds with CRY but not with GABA-T, PER also binds strongly with E75-RC. The RA isoform was poorly expressed, hence weaker interaction with PER (lane 5 in IP: αV5). (B) Knockdown of E75 increases Clk mRNA expression in wild type and per0 flies. As in <xref rid="nihms639102f2" ref-type="fig">Figure 2</xref>, , E75 knock down by TG27 significantly increases Clk mRNA levels in wild type flies relative to TG27 controls at ZT14. This effect is more striking in per0 flies where baseline Clk mRNA levels are quite low. (C) Overexpression of E75 reduces Clk mRNA expression in wild type and per0 flies. As in <xref rid="nihms639102f1" ref-type="fig">Figure 1</xref>, , E75 overexpression by TG27 significantly decreases Clk mRNA levels in wild type and per0 flies relative to TG27 controls at ZT02. (D) CLK levels in TG27 control, TG27 >UAS-E75 RNAi (GD) and TG27 > UAS-E75 (II) in wild type and per0 backgrounds are shown for two time points. HSP70 antibodies are used to control for loading. Molecular marker (Precision Plus Protein™ Dual Color Standards) were run to facilitate detection of different proteins. (D) Quantification of four independent western blots is shown. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM and indicate variability across flies of a specific genotype.'], 'nihms639102f3': ['Clock proteins in adult head extracts are derived largely from the eyes, which do not contribute to the behavioral rhythm. Therefore we also assayed PER levels through immunohistochemistry in circadian behavior-relevant brain clock neurons of flies with reduced levels of E75. As the behavioral phenotype produced by E75 knockdown was somewhat variable (perhaps due to inefficient knockdown), we first selected arrhythmic flies by assaying their behavior and then collected 8–10 flies from each experimental and control group at four different times of day. TG27 mediated knockdown of E75 resulted in dampened cycling of PER in constant darkness in different subsets of clock neurons (<xref rid="nihms639102f3" ref-type="fig">Figure 3A and, B</xref>). The dampening appeared to arise from significantly higher expression at trough time points (CT8 and CT14) (). The dampening appeared to arise from significantly higher expression at trough time points (CT8 and CT14) (<xref rid="nihms639102f3" ref-type="fig">Figure 3A and B</xref>). As the PDF cells are the ones most relevant for free-running behavior, we quantified PER expression in these cells, and found significant differences at CT8 and 14 in the small LN). As the PDF cells are the ones most relevant for free-running behavior, we quantified PER expression in these cells, and found significant differences at CT8 and 14 in the small LNvs and at CT8 in the large LNvs (<xref rid="nihms639102f3" ref-type="fig">Figure 3C, D</xref>). PER cycling was also dampened under light:dark (LD) cycles; in fact, under these conditions, PER levels appeared to be relatively higher at all times in LN). PER cycling was also dampened under light:dark (LD) cycles; in fact, under these conditions, PER levels appeared to be relatively higher at all times in LNvs and LNds of the E75 knockdown flies (Supplementary Figure 3A and B).'], 'nihms639102f4': ['The behavioral and molecular effects of E75 overexpression and knockdown strongly suggested an important role of this nuclear receptor in the molecular clock. To test for a possible function in the transcription of clock genes, we used cell culture assays. Given that the Drosophila E75 and mammalian REV-ERB proteins are so well conserved (~70%) in their DNA binding domains (Supplementary Figure 4A), we first tested E75 for effects on the native mouse Bmal1 promoter. We transfected Bmal1-luc (Bmal1 promoter fused to luciferase) constructs into HEK293T cells and activated expression using mammalian ROR-α. Upon co-transfection with CMV-E75, Bmal1-luc activity was significantly reduced ~ 4 fold (Supplementary Figure 4B). We then used a reporter construct for Clk in which the native promoter of Clk (~ 3.2 Kb) was fused to a luciferase reporter gene2. The Clk promoter contains multiple PDP1/VRI and E75 sites, the latter based on their homology to target sites of mammalian REV-ERB proteins, which bind AGGTCA sites in A/T rich regions20. We transfected the Clk-luc construct into HEK293T cells and assayed its expression in response to E75. As E75 is known to be a transcriptional repressor, but basal levels of Clk-luc are too low to detect further repression, we first activated expression of Clk-luc using Pdp1 (pCDNA3-CMV-Pdp1 ε) (<xref rid="nihms639102f4" ref-type="fig">Figure 4A</xref>). As reported previously). As reported previously2, we saw strong activation of the Clk promoter by PDP1ε in a dose dependent manner (<xref rid="nihms639102f4" ref-type="fig">Figure 4A</xref>). Next we co-transfected these cells with different doses of ). Next we co-transfected these cells with different doses of E75 (driven by the CMV promoter in pCDNA3.1) and found that E75 strongly repressed Clk-luc activity (<xref rid="nihms639102f4" ref-type="fig">Figure 4A</xref>). To verify that E75 acts on the ). To verify that E75 acts on the Clk promoter, we constructed an E75-VP16 fusion protein, which is expected to turn E75 into an activator by introducing a VP16 activation domain, and tested its efficacy in regulating Clk-luc. We observed an increase of luciferase activity (Supplementary Figure 4C), suggesting that E75 binds directly to the Clk promoter.', 'Clk transcription is also known to be repressed by a well-known bZIP transcriptional factor, VRI, which directly competes with PDP1 to bind at the V/P box2. Interestingly, a genome-wide study aimed at identifying novel molecules induced by ecdysone signaling showed that vri expression was significantly increased 22. In a separate study, vri expression was found elevated by ecdysone treatment in a tissue culture system15. As E75 expression is also induced by ecdysone signaling10, we examined whether E75 affects repression of Clk transcription by VRI. We used an artificial promoter containing 3 tandem consensus-binding sites for PDP1/VRI, but importantly lacking E75 binding sites, and co-expressed PDP1, VRI and E75. As expected, PDP1 activated Clk promoter driven luciferase activity, whereas VRI repressed this activity in a dose-dependent manner (<xref rid="nihms639102f4" ref-type="fig">Figure 4B</xref>). Interestingly, E75 further repressed expression of this promoter, suggesting that E75 modulates repression by VRI (). Interestingly, E75 further repressed expression of this promoter, suggesting that E75 modulates repression by VRI (<xref rid="nihms639102f4" ref-type="fig">Figure 4B</xref>). To further address whether the two proteins act together to regulate transcription, we used a VRI-VP16 construct to directly activate the artificial ). To further address whether the two proteins act together to regulate transcription, we used a VRI-VP16 construct to directly activate the artificial Clk promoter, and found that the activation was potentiated by E75 (Supplementary Figure 5A).', 'To address this question, we used the same luciferase based transcription assays in cell culture. As above, the native Clk-luc promoter was activated by PDP1ε and repressed by E75, and subsequently PER (driven by the CMV promoter) was added in a dose-dependent manner. Interestingly PER strongly inhibited repression of Clk by E75, suggesting that it acts as a de-repressor (<xref rid="nihms639102f4" ref-type="fig">Figure 4A</xref>). A construct expressing GFP did not affect repression by E75, demonstrating that the de-repression did not result merely from the presence of another transfected protein (). A construct expressing GFP did not affect repression by E75, demonstrating that the de-repression did not result merely from the presence of another transfected protein (<xref rid="nihms639102f4" ref-type="fig">Figure 4A</xref>). In the absence of E75, PER did not affect transcriptional activity of the ). In the absence of E75, PER did not affect transcriptional activity of the Clk promoter, supporting the idea that it acts as a de-repressor rather than a co-activator (<xref rid="nihms639102f4" ref-type="fig">Figure 4A</xref>).).'], 'nihms639102f5': ['To determine whether E75 and PER physically interact, we co-transfected them into mammalian HEK293T cells as well as into Drosophila S2 cells and conducted co-immunoprecipitation assays. E75 tagged with V5 pulled down PER in HEK 293T cells (<xref rid="nihms639102f5" ref-type="fig">Figure 5A</xref> and and Supplementary Figure 6A). CRY-V5 also pulled down PER under these conditions, whereas a non-specific GABA-T-V5 (GABA-transaminase fused to a V5 tag) protein did not, indicating specificity of the interaction between E75 and PER and also between CRY and PER (<xref rid="nihms639102f5" ref-type="fig">Figure 5A</xref> and and Supplementary Figure 6A). Similar results were obtained in Drosophila S2 cells, where PER pulled down V5-tagged E75 (Supplementary Figure 6B).', 'We next determined whether PER contributes to the regulation of Clk via E75 in vivo. Thus, we examined Clk mRNA levels in response to manipulations of E75 in the presence and absence of PER. As reported earlier, we found that per0 flies express relatively low levels of Clk mRNA4 (<xref rid="nihms639102f5" ref-type="fig">Figure 5B, C</xref>). Knockdown of ). Knockdown of E75 in a per0 background increased Clk mRNA to peak levels in wild type, supporting the idea that E75 keeps Clk mRNA low in per0 flies (<xref rid="nihms639102f5" ref-type="fig">Figure 5B</xref>). In wild type flies, the effect of ). In wild type flies, the effect of E75 knockdown on Clk mRNA was restricted to the trough time point and was less than in per0, perhaps because E75 has limited contribution to Clk expression in the presence of the de-repressor PER.', 'To further analyze the genetic interaction between PER and E75, we also compared CLK protein levels in wild type and per0 genetic backgrounds, following over-expression or knockdown of E75. Knockdown of E75 increased CLK protein in wild type and per0 flies, but in these experiments the difference was significant only in per0 (note that there is variability in the effect on CLK), again supporting the idea that E75 contributes more to CLK expression in per0 flies. However, the increase in the protein was not as great as in the mRNA, suggesting that other factors keep CLK low in per0 flies. Over-expression of E75 resulted in a greater overall reduction of CLK in a per0 background than in wild type, again most likely because CLK levels are already low in per0 (<xref rid="nihms639102f5" ref-type="fig">Figure 5D, E</xref>).).'], 'nihms639102f6': ['We first established that the ecdysone receptor (EcR) is expressed in circadian neurons. EcR has three isoforms A, B1 and B2, which differ in their N termini26. We obtained antibodies to EcR isoforms and verified that they recognize these specific proteins, based on their reduced levels in EcR RNAi lines and increased levels in flies that over-express EcR (data not shown). The EcR-A antibody is specific for EcR-A isoform, and the EcR-C antibody is known to detect all 3 isoforms of EcR27. Through immunohistochemistry experiments, we identified distinct expression of the EcR-A specific isoform and perhaps other isoforms (as detected by EcR-C) in adult LNvs as well as in the 3rd instar larval stage (<xref rid="nihms639102f6" ref-type="fig">Figure 6A, B</xref>). To alter EcR activity in clock cells, we utilized RNAi, dominant negative and overexpression approaches, as null mutations of ). To alter EcR activity in clock cells, we utilized RNAi, dominant negative and overexpression approaches, as null mutations of EcR are lethal. The dominant negative form of EcR (EcRΔ) cannot be activated by ecdysone and interferes with the activity of endogenous EcR, leading to deficiencies in EcR function26. Expression of EcR-B1Δ by Pdf-Gal4 resulted in a significant increase in period and decreased rhythm strength (Table 2A). Using TUG, UAS-EcR-B1Δ was expressed in broader sets of clock cells, and resulted in a much longer period (~26 hrs) and ~ 30% arrhythmicity, but surprisingly less of an effect on the strength of rhythms in rhythmic flies (Table 2A). We also expressed EcR-B1Δ using the even stronger clock cell Gal4 (TG27), which resulted in 100% lethality. As reported by Itoh et al15, knockdown of EcR using RNAi also yielded circadian phenotypes. EcR-A RNAi in PDF positive cells did not alter period but significantly reduced rhythm strength (Table 2A). On the other hand, EcR-A RNAi using the TUG driver led to a significantly longer period (Table 2A), although again, with less of an effect on rhythm strength.', 'In vertebrates, the closest homologs of E75 are members of the REV-ERB family. While REV-ERB is a part of the mammalian clock mechanism, the Drosophila ortholog, i.e. E75, was previously not known to have a circadian function. Here we show that E75 is an inhibitor of Clk transcription, by itself and also in conjunction with VRI. Prior to this work, it was thought that the role of nuclear hormone receptors in mammalian clocks was served by PAR domain containing proteins, PDP1 and VRI, in Drosophila2. Thus, while REV-ERB and ROR regulate expression of Bmal1 in mammals, PDP and VRI regulate expression of the other transcriptional activator, Clk, in flies. Our data indicate that E75 does indeed function in the Drosophila clock, much as its mammalian counterpart does (<xref rid="nihms639102f6" ref-type="fig">Figure 6C</xref>). One may ask why E75 is required if the second feedback loop is maintained by PDP1 and VRI. We suggest that E75 couples the clock to extracellular cues. Induction of E75 by the steroid hormone, ecdysone, likely allows the clock to respond to endocrine signals and perhaps other ligands (further discussed below). As reported here, E75 signaling may be particularly relevant under conditions of stress.). One may ask why E75 is required if the second feedback loop is maintained by PDP1 and VRI. We suggest that E75 couples the clock to extracellular cues. Induction of E75 by the steroid hormone, ecdysone, likely allows the clock to respond to endocrine signals and perhaps other ligands (further discussed below). As reported here, E75 signaling may be particularly relevant under conditions of stress.']}
|
Drosophila An ecdysone-responsive nuclear receptor regulates circadian rhythms in
|
[
"circadian clock",
"nuclear hormone receptor",
"ecdysone",
"E75",
"ecdysone induced protein",
"stress"
] |
Nat Commun
|
1418716800
|
Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. Here we report a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which we identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, CLOCK (CLK), and may also affect circadian output. PER inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of PER on Clk transcription. The ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. We find that E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila.
|
[
"Animals",
"CLOCK Proteins",
"Circadian Clocks",
"Circadian Rhythm",
"DNA-Binding Proteins",
"Drosophila Proteins",
"Drosophila melanogaster",
"Gene Expression Regulation",
"Neurons",
"Period Circadian Proteins",
"Promoter Regions, Genetic",
"RNA, Small Interfering",
"Receptors, Steroid",
"Signal Transduction",
"Stress, Physiological",
"Transcription Factors",
"Transcription, Genetic"
] |
other
|
PMC4269253
| null |
41
|
[
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Nat Commun. 2014 Dec 16; 5:5697
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α5 integrin expression after 4 h on the different substrates. (a) Representative Western blot bands for α5 integrin and quantification by image analysis of Western blot bands. The expected band for this antibody might be quite wide according to the manufacturer, which is why we developed in-house software to detect the band based on the intensity threshold criterion. Error bars represent the standard deviation of three independent experiments. +Statistically significant difference between FN concentrations (2 and 20 μg/mL). *Conditions with a significant difference. (b) α5 integrins bound to FN are observed after crosslinking and extraction of cellular components. Scale bar=10 μm (P≤0.05).
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[{'image_id': 'fig-2', 'image_file_name': 'fig-2.jpg', 'image_path': '../data/media_files/PMC3776618/fig-2.jpg', 'caption': 'Cell adhesion and focal adhesion kinase (FAK) phosphorylation after 4\u2009h on both substrates. (a) Distribution of focal adhesion protein vinculin and its incorporation into focal contact plaques. Scale bar=50\u2009μm. (b) Representative Western blot bands for FAK and pFAK with quantification by image analysis of Western blot bands. The error bars represent the standard deviation of three independent experiments. +There is a statistically significant difference between FN concentrations (2 and 20\u2009μg/mL). *Conditions with a significant difference (P≤0.05).', 'hash': '76fff6d9e9c0d0c1ba0e85f101cc0ee3214706b9c843152ce33a8bf0a21f7e0c'}, {'image_id': 'fig-5', 'image_file_name': 'fig-5.jpg', 'image_path': '../data/media_files/PMC3776618/fig-5.jpg', 'caption': 'Matrix degradation quantified by gene expression of MMP2 and MMP9. (a) Representative bands of RT-PCR for MMP2 and MMP9 after 4\u2009h and 1 day of culture on the different substrates (PEA, PMA, glass; unlabeled bands follow the same pattern). Gapdh was included as a constitutive gene. (b, c) Quantification of gene expression for MMP2 and MMP9 on the different substrates. The intensity of each band was related to the level of Gapdh under the same conditions, and represented as fold change relative to the values obtained for 2\u2009μg/mL on glass. Error bars represent the standard deviation of three different experiments. *Conditions with a significant difference (P≤0.05). MMP, matrix metalloproteinase.', 'hash': '2ce17d69e996b9ca6c19b3a8048f7562500303a5125e968696d9b3479d4fe615'}, {'image_id': 'fig-4', 'image_file_name': 'fig-4.jpg', 'image_path': '../data/media_files/PMC3776618/fig-4.jpg', 'caption': 'Integrin adhesion quantified by gene expression of α5 and αv integrins. (a) Representative bands of reverse-transcription (RT)-PCR for α5 and αv integrins after 4\u2009h and 1 day of culture on the different substrates (PEA, PMA, glass; unlabeled bands follow the same pattern). Gapdh was included as a constitutive gene. (b, c) Quantification of gene expression for α5 and αv integrins on the different substrates. The intensity of each band was related to the level of Gapdh under the same conditions, and represented as fold change relative to the values obtained for 2\u2009μg/mL on glass. Error bars are represented by the standard deviation of three different experiments. *Conditions with a significant difference for every protein concentration and culture time (P≤0.05).', 'hash': '61c0a9ed37eb8e8d8ceedf57786915e8d6c4dba06e628869f74c47f93846f3fa'}, {'image_id': 'fig-3', 'image_file_name': 'fig-3.jpg', 'image_path': '../data/media_files/PMC3776618/fig-3.jpg', 'caption': 'α5 integrin expression after 4\u2009h on the different substrates. (a) Representative Western blot bands for α5 integrin and quantification by image analysis of Western blot bands. The expected band for this antibody might be quite wide according to the manufacturer, which is why we developed in-house software to detect the band based on the intensity threshold criterion. Error bars represent the standard deviation of three independent experiments. +Statistically significant difference between FN concentrations (2 and 20\u2009μg/mL). *Conditions with a significant difference. (b) α5 integrins bound to FN are observed after crosslinking and extraction of cellular components. Scale bar=10\u2009μm (P≤0.05).', 'hash': '75542365db1e57e4c84e796d710d120488511b0a8e6d43b7707527f16cfab436'}, {'image_id': 'fig-6', 'image_file_name': 'fig-6.jpg', 'image_path': '../data/media_files/PMC3776618/fig-6.jpg', 'caption': 'Matrix degradation quantified by enzymatic activity of MMP2 and MMP9. (a) Representative bands of gelatin zymography for pro-MMP9, MMP9+TIMP-1, pro-MMP9, MMP9, pro-MMP2, MMP2, and pro-MMP13 after 4\u2009h and 1 day of culture on the different substrates (PEA, PMA, Glass). (b) Amplification of representative bands of gelatin zymography for pro-MMP9, MMP9+TIMP-1, pro-MMP9, MMP9, and pro-MMP13 with the different substrates and FN adsorption at 1 day of culture. (c, d, e) Quantification of MMP2, pro-MMP2, and pro-MMP13 activity on the different substrates. Error bars are represented by the standard deviation of three different experiments. *Conditions with a significant difference. TIMP, tissue inhibitors of metalloproteinase (P≤0.05).', 'hash': 'c982dd76a96e18c58a064c39d61b49e1d91da1c4486b713b8f46329b06de14a9'}, {'image_id': 'fig-1', 'image_file_name': 'fig-1.jpg', 'image_path': '../data/media_files/PMC3776618/fig-1.jpg', 'caption': 'Fibronectin adsorption and activity on the different substrates. (a) PEA and PMA chemical structure and fibronectin distribution on the different conditions as observed by the phase magnitude in AFM. The protein was adsorbed for 10\u2009min from different solutions of 2 and 20\u2009μg/mL concentration. (b) FN surface density after adsorption from two solutions with concentrations of 2 and 20\u2009μg/mL. (c) Monoclonal antibody binding for HFN7.1 on the different substrates after FN adsorption from two solution of concentrations 2 and 20\u2009μg/mL. (d) Activity of the adsorbed FN on the different substrates obtained by plotting the monoclonal antibody binding for HFN7.1 relative to the FN surface density calculated in (b). +There is a statistically significant difference between the conditions of 2 and 20\u2009μg/mL; *conditions with a significant difference (P≤0.05). PEA, poly(ethyl acrylate); PMA, poly(methyl acrylate); AFM, atomic force microscopy; FN, fibronectin.', 'hash': '566cef874e30e20076454cdc463cd3bfde2fde19aa15ea9a04859ed9e0269291'}]
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{'fig-1': ['The molecular distribution of FN on the different substrates was previously studied by us using AFM.22\n<xref ref-type="fig" rid="fig-1">Figure 1a</xref> includes the organization of FN on PEA and PMA after adsorption from solutions of different concentrations (2 and 20\u2009μg/mL). For each substrate, FN organization and distribution on the surface depends on the concentration of the initial solution from which the protein is adsorbed. Adsorption from a FN solution of concentration 2\u2009μg/mL leads to isolated FN molecules on the material surface. As the concentration of the FN solution increases, the formation of a FN network occurs on PEA (e.g., material-driven FN fibrillogenesis) but not on PMA. includes the organization of FN on PEA and PMA after adsorption from solutions of different concentrations (2 and 20\u2009μg/mL). For each substrate, FN organization and distribution on the surface depends on the concentration of the initial solution from which the protein is adsorbed. Adsorption from a FN solution of concentration 2\u2009μg/mL leads to isolated FN molecules on the material surface. As the concentration of the FN solution increases, the formation of a FN network occurs on PEA (e.g., material-driven FN fibrillogenesis) but not on PMA.22', 'The surface density of adsorbed FN was quantified indirectly from the amount of protein remaining nonadsorbed in the supernatant using Western blot.23\n<xref ref-type="fig" rid="fig-1">Figure 1b</xref> shows the surface density of adsorbed FN from solutions of concentration 2 and 20\u2009μg/mL. As expected, the amount of adsorbed FN was higher as the concentration of the protein solution increased. In contrast, there was no significant difference for the amount of adsorbed FN between material substrates at all FN concentrations, which remained constant and approximately 47 and 340\u2009ng/cm shows the surface density of adsorbed FN from solutions of concentration 2 and 20\u2009μg/mL. As expected, the amount of adsorbed FN was higher as the concentration of the protein solution increased. In contrast, there was no significant difference for the amount of adsorbed FN between material substrates at all FN concentrations, which remained constant and approximately 47 and 340\u2009ng/cm2 for the 2 and 20\u2009μg/mL FN solutions, respectively.', 'To evaluate the availability of integrin binding domains after FN adsorption, enzyme-linked immunosorbent assay with a monoclonal antibody was used (<xref ref-type="fig" rid="fig-1">Fig. 1c</xref>). This is a well-established method to probe for structural or conformational changes in adsorbed proteins.). This is a well-established method to probe for structural or conformational changes in adsorbed proteins.29,30 HFN7.1 (monoclonal antibody) was directed against the flexible linker between the 9th and 10th type III repeats of FN, a probe for cell adhesion and integrin binding.31 Adsorption from the 2\u2009μg/mL solution resulted in the same FN activity on PEA and PMA. Further increase of the concentration of the protein solution (20\u2009μg/mL) provided higher activity of FN on PEA, but not on PMA. It is interesting to note that the amount of protein on a surface and its biological activity are two independent parameters. In our case, after the amount of protein on the surface was increased ∼10-fold, the activity measured by HFN 7.1 binding was almost the same, which revealed the role of protein–protein interactions in hiding the availability of the integrin binding sequence of FN. Since the same amount of FN is adsorbed on every material for each one of the adsorbing solutions, <xref ref-type="fig" rid="fig-1">Figure 1d</xref> represents the activity of the protein (availability of cell adhesion domains) as a function of the protein surface density, which revealed higher activity of FN on PEA than PMA for the highest FN density. represents the activity of the protein (availability of cell adhesion domains) as a function of the protein surface density, which revealed higher activity of FN on PEA than PMA for the highest FN density.', 'We have previously shown the biological activity of this material-driven FN network in terms of cell adhesion, signaling, cytoskeleton organization, matrix reorganization, and cell differentiation.20–24 Here we address the link between material-driven FN assembly and matrix protein remodeling (degradation) at the cell–material interface, which we hypothesized must be related to integrin-mediated adhesion through the conformation of the adsorbed protein. We investigated two polymer surfaces that consist in a vinyl chain with a slightly different (one carbon) side group: PEA and PMA.22 These surfaces adsorbed the same amount of FN and showed similar wettability (<xref ref-type="fig" rid="fig-1">Fig. 1b</xref>). However, the conformation and distribution of the protein following passive adsorption onto these surfaces are completely different (). However, the conformation and distribution of the protein following passive adsorption onto these surfaces are completely different (<xref ref-type="fig" rid="fig-1">Fig. 1a</xref>). FN was self-organized into fibrils on PEA (). FN was self-organized into fibrils on PEA (<xref ref-type="fig" rid="fig-1">Fig. 1a-iv</xref>) as the so-called material-driven fibrillogenesis, whereas individual globule-like molecules or small molecular aggregates were present on PMA () as the so-called material-driven fibrillogenesis, whereas individual globule-like molecules or small molecular aggregates were present on PMA (<xref ref-type="fig" rid="fig-1">Fig. 1a-iii</xref>). FN fibril formation on PEA was dependent on the FN solution concentration, since lower concentrations (e.g., 2\u2009μg/mL) result in dispersed adsorbed molecules.). FN fibril formation on PEA was dependent on the FN solution concentration, since lower concentrations (e.g., 2\u2009μg/mL) result in dispersed adsorbed molecules.22', 'The amount of FN adsorbed on every surface (PEA, PMA) was approximately the same for both concentrations of the protein solution (<xref ref-type="fig" rid="fig-1">Fig. 1b</xref>). However, the different distribution of the protein revealed by AFM (). However, the different distribution of the protein revealed by AFM (<xref ref-type="fig" rid="fig-1">Fig. 1a</xref>)—dispersed globular aggregates on PMA versus a network of assembled FN fibrils on PEA—also involved different availability of the integrin binding domain in FN: )—dispersed globular aggregates on PMA versus a network of assembled FN fibrils on PEA—also involved different availability of the integrin binding domain in FN: <xref ref-type="fig" rid="fig-1">Figure 1c</xref> shows significantly higher signal for the HFN7.1 antibody against the flexible linker joining the III9 and III10 domains of FN. shows significantly higher signal for the HFN7.1 antibody against the flexible linker joining the III9 and III10 domains of FN.31 Moreover, this higher activity of FN on PEA was very much dependent on the concentration of the protein solution, and it was drastically diminished upon adsorption from the 2\u2009μg/mL FN solution (<xref ref-type="fig" rid="fig-1">Fig. 1c</xref>). To fully assess these results, we have included in ). To fully assess these results, we have included in <xref ref-type="fig" rid="fig-1">Figure 1d a</xref> representation for the availability of HFN7.1 antibody versus the amount of FN adsorbed on every surface. This representation clearly reveals the synergistic effect of the material-driven FN fibrillogenesis on FN activity, and it disregards any influence of the total amount of adsorbed FN in a comparison of results between PEA and PMA. The higher availability of cell adhesion domains for the material-driven FN fibrils on PEA supports previous results for the biological activity of this surface in terms of cell adhesion and differentiation. representation for the availability of HFN7.1 antibody versus the amount of FN adsorbed on every surface. This representation clearly reveals the synergistic effect of the material-driven FN fibrillogenesis on FN activity, and it disregards any influence of the total amount of adsorbed FN in a comparison of results between PEA and PMA. The higher availability of cell adhesion domains for the material-driven FN fibrils on PEA supports previous results for the biological activity of this surface in terms of cell adhesion and differentiation.20–24', 'We hypothesize that the material-driven FN network must also lead to a different proteolytic activity at the material interface, compared with a similar chemistry on which the protein is not organized (<xref ref-type="fig" rid="fig-1">Fig. 1</xref>). The effect of material chemistry on the proteolytic activity of cells has been barely addressed so far. Expression of MMP2 and MMP9 has been observed in cells cultured on tissue culture polystyrene. There are only a few studies that address the effect of synthetic materials on MMP expression and activity.). The effect of material chemistry on the proteolytic activity of cells has been barely addressed so far. Expression of MMP2 and MMP9 has been observed in cells cultured on tissue culture polystyrene. There are only a few studies that address the effect of synthetic materials on MMP expression and activity.14,49–52 On model surfaces with controlled wettability and surface chemistry, the activation of proteolytic routes occurs in an MMP-dependent way. It has been previously suggested a direct relationship between MMP9 and FN activity at the material interface,15 which we also found in our experiments (<xref ref-type="fig" rid="fig-6">Fig. 6b</xref>): FN activity was higher on the assembled FN fibrils (): FN activity was higher on the assembled FN fibrils (<xref ref-type="fig" rid="fig-1">Fig. 1</xref>), as was the qualitative interpretation of the band for MMP9 activity in ), as was the qualitative interpretation of the band for MMP9 activity in <xref ref-type="fig" rid="fig-6">Figure 6b</xref> (without quantification). Moreover, MMP2 and MMP13 activities (zymography) were much higher on the material-assembled FN matrix, after 4\u2009h and 1 day ( (without quantification). Moreover, MMP2 and MMP13 activities (zymography) were much higher on the material-assembled FN matrix, after 4\u2009h and 1 day (<xref ref-type="fig" rid="fig-6">Fig. 6</xref>). MMP2 has FN type II repeats inserted into the catalytic domain,). MMP2 has FN type II repeats inserted into the catalytic domain,11 and it has been found to cleave vitronectin and FN in vivo, leading to enhanced cell migration.11,12 Degradation of the assembled fibrils at the material interface would enhance the exposure of adhesion sites, which would support enhanced integrin (α5) expression and focal adhesion formation in this system. It also supports the proposed relationship between FN activity, the ability to reorganize the underlying layer of proteins at the material interface, and proteolytic cascades: higher MMP activity is required to remodel the provisional matrix when cells are not able to reorganize this layer of proteins at the material interface, as in fact occurs with the material-driven FN network. It is precisely this initial proteolytic activity (after 4\u2009h) that enhanced cellular behavior on the FN fibrils assembled on PEA. This result supports the hypothesis that cells need to rearrange the initial layer of proteins at the material interface, and that this is very much related to the biocompatibility of materials: when the protein–material interaction is so strong that reorganization cannot occur, proteolytic cascades are enhanced to degrade FN, seeking to start microscale movements at the material interface to direct cell function.'], 'fig-2': ['To gain insights into the mechanisms controlling matrix remodeling on material-driven FN networks, we began by examining the organization of proteins involved in the formation of focal adhesion complexes by immunofluorescence. <xref ref-type="fig" rid="fig-2">Figure 2</xref> shows the distribution of vinculin in cells adhering on the different substrates. Poorly developed focal adhesions were observed on both PEA and PMA for the 2\u2009μg/mL FN coating. However, well-defined focal plaques formed on PEA, but not on PMA, for the 20\u2009μg/mL FN coating. Likewise, although the formation of F-actin fibers on PEA and PMA already occurred at the lowest FN concentration, more prominent F-actin cables terminating in well-developed focal adhesion complexes were found on PEA at the highest FN concentration ( shows the distribution of vinculin in cells adhering on the different substrates. Poorly developed focal adhesions were observed on both PEA and PMA for the 2\u2009μg/mL FN coating. However, well-defined focal plaques formed on PEA, but not on PMA, for the 20\u2009μg/mL FN coating. Likewise, although the formation of F-actin fibers on PEA and PMA already occurred at the lowest FN concentration, more prominent F-actin cables terminating in well-developed focal adhesion complexes were found on PEA at the highest FN concentration (<xref ref-type="fig" rid="fig-2">Fig. 2</xref>).).', 'FAK localizes to focal adhesions to activate multiple signaling pathways that regulate cell migration, survival, proliferation, and differentiation.7 The phosphorylation of FAK at Y-397, the autophosphorylation site and a binding site for Src and PI-3 kinases,32 is shown in <xref ref-type="fig" rid="fig-2">Figure 2</xref>. It remained the same for PEA and PMA at the lower FN concentration, and it increased at the highest FN concentration (20\u2009μg/mL). Moreover, higher levels of pFAK were obtained on PEA than PMA, revealing enhanced signaling from the substrate-assembled FN networks.. It remained the same for PEA and PMA at the lower FN concentration, and it increased at the highest FN concentration (20\u2009μg/mL). Moreover, higher levels of pFAK were obtained on PEA than PMA, revealing enhanced signaling from the substrate-assembled FN networks.', 'After adhesion, cells reorganize the adsorbed layer of FN at the material interface and then secrete matrix proteins.9 In this way, cells assemble FN into a network of fibrils. Along the way, FN experience particular conformational changes, that can be limited after adsorption on the material surface.40,41 This may explain the role of the properties of material surfaces (e.g., wettability) in FN matrix formation.9,42 We have previously shown that cell-mediated FN reorganization does not occur on the material-driven FN network assembled on PEA, due to the high strength of interaction between FN fibrils and the underlying surface chemistry.43 The enhanced initial biological response of the material-driven FN network (<xref ref-type="fig" rid="fig-2">Figs. 2</xref>––<xref ref-type="fig" rid="fig-4">4</xref>) can be explained as a consequence of the following phenomena occurring at the cell–material interface after FN assembly into physiological-like fibrils: (i) the availability of the integrin binding sequence () can be explained as a consequence of the following phenomena occurring at the cell–material interface after FN assembly into physiological-like fibrils: (i) the availability of the integrin binding sequence (<xref ref-type="fig" rid="fig-1">Fig. 1</xref>); (ii) α); (ii) α5 integrin expression, which leads to enhanced focal adhesion formation and cytoskeleton development (<xref ref-type="fig" rid="fig-2">Figs. 2</xref>, , <xref ref-type="fig" rid="fig-3">3</xref>); and (iii) enhanced phosphorylation of FAK (); and (iii) enhanced phosphorylation of FAK (<xref ref-type="fig" rid="fig-2">Fig. 2</xref>).).'], 'fig-3': ['To gain insights into the adhesion mechanism on these synthetic FN matrices assembled at the material interface, we examined expression and binding of α5β1 integrin to the adsorbed FN since this receptor provides the primary adhesion mechanism in this cell model.33 First, we examined α5 integrin expression by Western blot (<xref ref-type="fig" rid="fig-3">Fig. 3a</xref>). No difference was found on PEA between the two FN concentrations used, with a similar expression level on PMA with FN at 2\u2009μg/mL. Surprisingly, very low integrin expression was found on PMA after increasing the concentration of the adsorbing solution to 20\u2009μg/mL. Afterwards, integrin binding to FN-coated materials was analyzed via immunostaining following cross-linking of bound integrins to FN and extraction of cellular components (). No difference was found on PEA between the two FN concentrations used, with a similar expression level on PMA with FN at 2\u2009μg/mL. Surprisingly, very low integrin expression was found on PMA after increasing the concentration of the adsorbing solution to 20\u2009μg/mL. Afterwards, integrin binding to FN-coated materials was analyzed via immunostaining following cross-linking of bound integrins to FN and extraction of cellular components (<xref ref-type="fig" rid="fig-3">Fig. 3b</xref>). No significant differences were found among the material surfaces regardless of FN concentration, demonstrating that the substrate-assembled FN network does not alter integrin binding in the presence of serum.). No significant differences were found among the material surfaces regardless of FN concentration, demonstrating that the substrate-assembled FN network does not alter integrin binding in the presence of serum.22', 'Differences in the availability of FN adhesion domains after adsorption on PEA and PMA from a solution of concentration 20\u2009μg/mL influence the initial cell–material interaction, in terms of integrin expression, focal adhesion formation, and F-actin cytoskeleton development. We examined binding of α5β1 integrin to the adsorbed FN since this receptor provides the primary adhesion mechanism in this cell model.33 Immunofluorescence staining following crosslinking of bound integrins to FN and extraction of cellular components revealed no significant differences in integrin binding among FN on different surfaces. To enhance the visualization of integrin clusters, this experiment was performed in the presence of serum, which is known to contain large amounts of FN and vitronectin that might perturb the effect of the provisional FN matrix previously assembled at the material surface. This is why we investigated protein and gene expression in absence of any additional serum, to enable focusing on the sole effect of FN at the material interface. α5 integrin expression was higher for cells seeded on the material-driven FN network (PEA) than on the dispersed globular-like FN matrix adsorbed on PMA (<xref ref-type="fig" rid="fig-3">Fig. 3a</xref>). The same result was obtained at the gene level for the α). The same result was obtained at the gene level for the α5 receptor (<xref ref-type="fig" rid="fig-4">Fig. 4a</xref>). The opposite trend was found for α). The opposite trend was found for αv expression, which was enhanced on PMA compared with PEA. These results reveal that α5 is the main receptor involved during the initial cell interaction with the material-driven FN network, as happens for the interaction of cells with natural physiological matrices.33 α5β1 binding has been related to the simultaneous availability of the synergy and RGD sequences within FN.33 In contrast, when FN is adsorbed as discrete aggregates on PMA, the availability of the whole integrin binding site of FN is reduced (<xref ref-type="fig" rid="fig-1">Fig. 1</xref>) and cell adhesion mostly occurs through the α) and cell adhesion mostly occurs through the αv receptor, which only needs the exposition of the RGD sequence.'], 'fig-4': ['Gene expressions for α5 and αv integrins were obtained by reverse-transcription PCR (<xref ref-type="fig" rid="fig-4">Fig. 4</xref>). α). α5 expression (<xref ref-type="fig" rid="fig-4">Fig. 4b</xref>) decreases with time but increases with FN concentration. At the lowest FN concentration (2\u2009μg/mL), α) decreases with time but increases with FN concentration. At the lowest FN concentration (2\u2009μg/mL), α5 expression was higher on PEA than PMA (<xref ref-type="fig" rid="fig-4">Fig. 4b</xref>), as it was, but with less difference, at the 20\u2009μg/mL FN concentration. In addition, the opposite trend was found for α), as it was, but with less difference, at the 20\u2009μg/mL FN concentration. In addition, the opposite trend was found for αv expression, which remained higher on PMA than PEA at both FN coatings (<xref ref-type="fig" rid="fig-4">Fig. 4c</xref>).).'], 'fig-5': ['Seeking to understand the relationship between FN adsorption, cell adhesion, and matrix remodeling, we next examined MMP2, MMP9, and MMP13 at both protein activity and gene expression levels. <xref ref-type="fig" rid="fig-5">Figure 5</xref> shows gene expression for MMP2 and MMP9 after 4\u2009h and 1 day. The MMP2 level ( shows gene expression for MMP2 and MMP9 after 4\u2009h and 1 day. The MMP2 level (<xref ref-type="fig" rid="fig-5">Fig. 5b</xref>) was lower on PEA than PMA at the highest concentration of the FN coating (20\u2009μg/mL), that is, on the material-assembled FN fibrils. Only at the shorter time (4\u2009h) and lower FN concentration (2\u2009μg/mL) was MMP2 expression similar on PEA and PMA. The opposite trend was found for MMP9 expression () was lower on PEA than PMA at the highest concentration of the FN coating (20\u2009μg/mL), that is, on the material-assembled FN fibrils. Only at the shorter time (4\u2009h) and lower FN concentration (2\u2009μg/mL) was MMP2 expression similar on PEA and PMA. The opposite trend was found for MMP9 expression (<xref ref-type="fig" rid="fig-5">Fig. 5c</xref>): similar levels were found on both PEA and PMA at every time but at 4\u2009h at the lowest FN concentration (2\u2009μg/mL).): similar levels were found on both PEA and PMA at every time but at 4\u2009h at the lowest FN concentration (2\u2009μg/mL).'], 'fig-6': ['Further insights in matrix degradation can be obtained by investigating MMP activity by gelatin zymography (<xref ref-type="fig" rid="fig-6">Fig. 6</xref>). Both the pro-form of the protein and the active one (MMP2) were highly dependent on the underlying material surface and the observation time, with higher level of activity on PEA than PMA (). Both the pro-form of the protein and the active one (MMP2) were highly dependent on the underlying material surface and the observation time, with higher level of activity on PEA than PMA (<xref ref-type="fig" rid="fig-6">Fig. 6c, d</xref>). Increasing the concentration of the FN solution did not significantly alter the activity of the protein. (). Increasing the concentration of the FN solution did not significantly alter the activity of the protein. (<xref ref-type="fig" rid="fig-6">Fig. 6c, d</xref>). The activity of MMP9 and its forms (). The activity of MMP9 and its forms (<xref ref-type="fig" rid="fig-6">Fig. 6b</xref>) could only be observed after 1 day of culture, while the pro-MMP13 behaved as MMP2 for the higher concentration of the FN solution () could only be observed after 1 day of culture, while the pro-MMP13 behaved as MMP2 for the higher concentration of the FN solution (<xref ref-type="fig" rid="fig-6">Fig. 6e</xref>). That is to say, MMP13 was more active on cells seeded on the material-driven FN network after 1 day.). That is to say, MMP13 was more active on cells seeded on the material-driven FN network after 1 day.']}
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Role of Material-Driven Fibronectin Fibrillogenesis in Protein Remodeling
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[
"cellular biology",
"extracellular matrix",
"molecular biology",
"tissue engineering"
] |
Biores Open Access
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1380610800
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None
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other
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PMC3776618
| null | null |
[
""
] |
Biores Open Access. 2013 Oct; 2(5):364-373
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NO-CC CODE
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FBW7 cancer-specific arginine mutants have a dominant-negative phenotype. (A) Western blot of cyclin E and MCL1 levels in FBW7 wild-type, FBW7 heterozygous and FBW7 homozygous knockout HCT116 cells and FBW7 heterozygous knockout HCT116 cells with FBW7 wild-type and arginine mutations expressed. (B) Abnormal anaphases (expressed as a percentage of total anaphases) in asynchronous FBW7 +/− cells expressing wild-type FBW7 and FBW7 arginine mutations. (C) Examples of abnormal anaphases found in HCT116 cells include: normal anaphases (i), lagging chromosomes (ii and iii), anaphase bridges (iv and v), and anaphases containing multiple phenotypes (vi). Multipolar anaphases were also occasionally observed as slides were costained with DAPI (vii) and an antibody for α-tubulin (viii). (ix) Merged image. (*) P < 0.05.
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885fig4
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2
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5dd589b071c5c5792d05277e487097e4f0512c82a6bcaba904cfcdcc1f985f82
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885fig4.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
749,
715
] |
[{'image_id': '885fig1', 'image_file_name': '885fig1.jpg', 'image_path': '../data/media_files/PMC4649658/885fig1.jpg', 'caption': 'BUBR1 as a synthetic lethal candidate for FBW7 knockout cells. (A) Results from a genome-wide shRNA screen using HCT116 wild-type (FBW7 +/+) and FBW7 knockout (FBW7 −/−) cells. Genes considered as candidate negative genetic interactions are shown in dark gray. The data point representing the BUBR1 gene is shown as a solid red circle. (B) Knockdown of BUBR1 by Western blot after infection with shRNA. (C) Validation of the genetic interaction between FBW7 and BUBR1 using a clonogenic assay. The graph shown on the right shows the average of three replicates. (D) Validation that BUBR1 shRNA is more detrimental to FBW7 −/− cells than FBW7 +/+ cells when compared to a control shRNA (luciferase) in a 96-well nuclei-counting assay. (E) Differential growth after knockdown of BUBR1 in another colorectal cell line (HT29) that was engineered to express either control (luciferase) or FBW7 shRNA. Cells were counted in 96 well after nuclei staining. (*) P < 0.05; (**) P < 0.01; (***) P < 0.0001.', 'hash': '29eba9ad5160ad7d6a88a48b38ca71b0e4502d49cc992797df17597584bc7be7'}, {'image_id': '885fig6', 'image_file_name': '885fig6.jpg', 'image_path': '../data/media_files/PMC4649658/885fig6.jpg', 'caption': 'Establishment of sensitivity to SAC knockdown in FBW7 knockout cells requires multiple substrates. (A) Percentages of G1-, S-, and G2/M-phase cells in HCT116 cells overexpressing nondegradable forms of cyclin E and MCL1. (B) Western blot of HCT116 colorectal cell lines overexpressing nondegradable forms of MCL1 and full-length cyclin E or cyclin E lacking the N-terminal 65 residues (Δ65 cyclin E). (C) Percentages of >4 N cells in three independent experiments. (D) Percentages of cells in the sub-G1 range in three independent experiments. (E) HCT116 cells show sensitivity to BUBR1 knockdown only after expression of MCL1 and the Δ65 form of cyclin E. (*) P < 0.05; (**) P < 0.005; (***) P < 0.0005.', 'hash': 'a7ea1977462729c2e1653da155bd8e05a6ce837e3ef66bf497726f98ef09973a'}, {'image_id': '885fig7', 'image_file_name': '885fig7.jpg', 'image_path': '../data/media_files/PMC4649658/885fig7.jpg', 'caption': 'A model for SAC dependence in cells lacking FBW7. (A) FBW7 +/+ cells in prometaphase efficiently align their chromosomes and correctly segregate properly in anaphase with less reliance on the SAC. FBW7 −/− cells have an increase in cyclin E, which causes problems in mitosis and may lead to SAC activation. FBW7 −/− cells can survive prolonged SAC activation in part because of stabilization of MCL1 allowing more time for chromosome alignment. (B) A decrease in SAC activation by knockdown of BUBR1 may significantly shorten the time available for chromosome alignment in FBW7 −/− cells, resulting in improper chromosome segregation and intolerable levels of chromosome instability.', 'hash': '1d8cd923de73ff8cb26c6c5a377dcd37fbddd3632a154ce6b75f0c301da294c5'}, {'image_id': '885fig3', 'image_file_name': '885fig3.jpg', 'image_path': '../data/media_files/PMC4649658/885fig3.jpg', 'caption': 'Knockdown of the SAC increases aneuploidy in FBW7 −/− cells. (A) Flow cytometry profiles of FBW7 +/+ and FBW7 −/− cells stained with propidium iodide and infected with BUBR1 shRNA. (B) Quantification of the >4 N cells as an average over three independent experiments. (C) Western blot of p53 levels after knockdown of BUBR1. (*) P < 0.05.', 'hash': 'c00e35c79860cd366b82524384e37361c935981df6041548355e2b6ae7550c75'}, {'image_id': '885fig4', 'image_file_name': '885fig4.jpg', 'image_path': '../data/media_files/PMC4649658/885fig4.jpg', 'caption': 'FBW7 cancer-specific arginine mutants have a dominant-negative phenotype. (A) Western blot of cyclin E and MCL1 levels in FBW7 wild-type, FBW7 heterozygous and FBW7 homozygous knockout HCT116 cells and FBW7 heterozygous knockout HCT116 cells with FBW7 wild-type and arginine mutations expressed. (B) Abnormal anaphases (expressed as a percentage of total anaphases) in asynchronous FBW7 +/− cells expressing wild-type FBW7 and FBW7 arginine mutations. (C) Examples of abnormal anaphases found in HCT116 cells include: normal anaphases (i), lagging chromosomes (ii and iii), anaphase bridges (iv and v), and anaphases containing multiple phenotypes (vi). Multipolar anaphases were also occasionally observed as slides were costained with DAPI (vii) and an antibody for α-tubulin (viii). (ix) Merged image. (*) P < 0.05.', 'hash': '5dd589b071c5c5792d05277e487097e4f0512c82a6bcaba904cfcdcc1f985f82'}, {'image_id': '885fig5', 'image_file_name': '885fig5.jpg', 'image_path': '../data/media_files/PMC4649658/885fig5.jpg', 'caption': 'Cyclin E is necessary for sensitivity of FBW7 knockout cells to knockdown of the SAC. (A) FBW7 knockout cells infected with BUBR1 shRNA in a 96-well nuclei-counting assay after knockdown of cyclin E1 (CCNE1). (B) Western blot confirming knockdown of cyclin E after shRNA infection. (**) P < 0.001.', 'hash': '2ba76f58d65704e5114d3803431d2c411935c877bd81a62061d1794c243eba2d'}, {'image_id': '885fig2', 'image_file_name': '885fig2.jpg', 'image_path': '../data/media_files/PMC4649658/885fig2.jpg', 'caption': 'FBW7 knockout cells are more sensitive to knockdown of the SAC. (A) Mitotic profiles of FBW7 wild-type and knockout cells expressed as a percentage of the total number of cells in mitosis. (B) Levels of the APC/C targets securin and cyclin B in FBW7 wild-type and knockout cells. (NOC, FBW7 +/+ cells arrested with nocodazole). (C and D) Differential growth between FBW7 +/+ and FBW7 −/− cells using a 96-well nuclei counting assay after infection with shRNA to the SAC components BUB1 (D) and MPS1 (E). (*) P < 0.05; (**) P < 0.001.', 'hash': '4cd3e6f78177ebdf9a51f7e82dea06a2964ded2861b638ae7e48215b75fa3b04'}]
|
{'885fig1': ['To investigate potential genes to target for selective killing in cells lacking FBW7, we used the HCT116 FBW7 −/− line previously generated by Rajagopalan et al. (2004). This line is homozygous null for FBW7 due to a knockout of exon 8 in both alleles of FBW7 and was originally shown to have increases in cyclin E substrate levels and chromosome instability (CIN) (Rajagopalan et al. 2004). The genome-wide screen was performed using a pooled lentiviral shRNA library representing ∼16,000 human genes (Moffat et al. 2006; Marcotte et al. 2012; Vizeacoumar et al. 2013). This library was then infected into a matched pair of cell lines, HCT116 FBW7 +/+ and HCT116 FBW7 −/− to determine which shRNAs and genes were lost from the pool over time. Comparison of the GARP scores (see Materials and Methods) between these two lines revealed 122 potential negative interactions (<xref ref-type="fig" rid="885fig1">Figure 1A</xref>, , Table S3, Table S4, and Table S5). One of the candidate genes, BUBR1, validated well when a subset of shRNAs were further tested (Figure S1).', 'Knockdown of BUBR1 was confirmed as being selectively detrimental to the proliferation of FBW7 −/− cells using independent shRNAs not in the original pooled screen (<xref ref-type="fig" rid="885fig1">Figure 1B</xref>). Both clonogenic assays and high-content microscopy counting showed a significant decrease in proliferation of ). Both clonogenic assays and high-content microscopy counting showed a significant decrease in proliferation of FBW7 −/− cells after BUBR1 knockdown when compared with similarly infected FBW7 +/+ cells (<xref ref-type="fig" rid="885fig1">Figure 1, C and D</xref>). BUBR1 was also shown to be needed to maintain proliferation in another colorectal cell line, HT29, where FBW7 was depleted by shRNA, further establishing a negative genetic interaction between ). BUBR1 was also shown to be needed to maintain proliferation in another colorectal cell line, HT29, where FBW7 was depleted by shRNA, further establishing a negative genetic interaction between FBW7 and BUBR1 (<xref ref-type="fig" rid="885fig1">Figure 1E</xref> and and Figure S2A).'], '885fig2': ['The most-well-known role of BUBR1 is as a component of the mitotic SAC, which, when activated, prevents cells from entering anaphase by blocking the activity of the APC/CCDC20 ubiquitin ligase (Jia et al. 2013). Consistent with this role in maintaining proper chromosome segregation, HCT116 FBW7 −/− mitotic cells have difficulty aligning their chromosomes at the metaphase plate, which leads to increased segregation errors (Rajagopalan et al. 2004). We similarly observed a higher percentage of mitotic cells in prometaphase in FBW7 −/− cells when compared with FBW7 +/+ cells (<xref ref-type="fig" rid="885fig2">Figure 2A</xref>). ). FBW7 −/− cells also had an increase in the levels of the APC/C substrates cyclin B and Securin (<xref ref-type="fig" rid="885fig2">Figure 2B</xref>). This strongly indicates that cells that lack FBW7 depend on an intact SAC.). This strongly indicates that cells that lack FBW7 depend on an intact SAC.', 'To confirm this, we used shRNA to knock down two other proteins with known roles in the SAC, BUB1, and MPS1 (also called TTK). Knockdown of either of these components caused a significant decrease in cell growth in FBW7 −/− cells when compared with FBW7 +/+ cells (<xref ref-type="fig" rid="885fig2">Figure 2, C and D</xref>, and , and Figure S2, B and C). We also found that cells depleted of FBW7 showed little or no resistance to low levels of the spindle poisons nocodazole or paclitaxel, which activate the SAC (Figure S2, D and E). Taken into consideration with previous studies that have shown that FBW7 activity is needed for cell sensitivity to high levels of anti-tubulin chemotherapeutics (Finkin et al. 2008; Wertz et al. 2011), we propose knockdown of the SAC as an alternative anti-mitotic strategy to traditional chemotherapeutics targeting the spindle.', 'FBW7 −/− cells are vulnerable to knockdown not just of BUBR1, but also to two other members of the SAC, BUB1, and MPS1 (<xref ref-type="fig" rid="885fig2">Figure 2, C and D</xref>). These cells show a higher percentage of cells in prometaphase and higher levels of APC/C substrates when compared with wild-type cells (). These cells show a higher percentage of cells in prometaphase and higher levels of APC/C substrates when compared with wild-type cells (<xref ref-type="fig" rid="885fig2">Figure 2, A and B</xref>) suggesting that ) suggesting that FBW7 knockout cells may be more dependent on SAC function. In cancer, SAC mutations are rare, but upregulation of SAC proteins and mRNA are seen and may associate with CIN (Carter et al. 2006; Yuan et al. 2006; Janssen and Medema 2013), suggesting that the requirement of an intact mitotic checkpoint is not limited to FBW7 knockout cells.'], '885fig3': ['To determine the consequences of decreasing SAC function in cells lacking FBW7, we compared the cell-cycle profiles of FBW7 +/+ and FBW7 −/− cells after knockdown of BUBR1 with shRNA (<xref ref-type="fig" rid="885fig3">Figure 3A</xref> and and Figure S3A). FBW7 −/− cells infected with BUBR1 shRNA showed a more than twofold increase in the >4 N DNA over BUBR1 shRNA-infected FBW7 +/+ cells (<xref ref-type="fig" rid="885fig3">Figure 3B</xref>). Western blotting of lysates after BUBR1 knockdown showed increases in the level of p53 (). Western blotting of lysates after BUBR1 knockdown showed increases in the level of p53 (<xref ref-type="fig" rid="885fig3">Figure 3C</xref>), but no cleavage of the apoptotic marker PARP (), but no cleavage of the apoptotic marker PARP (Figure S3B), suggesting that the decrease in cell proliferation is not due to apoptotic cell death. Furthermore, staining of the cells with pH 3 (a marker for mitosis) showed that, as has been shown, knockdown of the SAC with BUBR1 shRNA decreased the percentage of cells in mitosis (Moffat et al. 2006) (Figure S3C). This analysis implies that after knockdown of BUBR1, cells lacking FBW7 do not delay in mitosis at the SAC, but become highly aneuploid and arrest in a p53-dependent manner.'], '885fig4': ['Mutations observed in FBW7 in cancer are often single allele, missense mutations (Davis et al. 2014). To determine how these mutations affect FBW7 substrate levels and phenotypes, we expressed several common mutations (R465C, R479Q, R505C) in HCT116 cells and compared these cells to parental cells expressing vector alone. These three target arginine residues are responsible for the coordination of the substrate phosphate moiety in the binding pocket of FBW7 and, when combined, have a higher frequency of mutation compared with the rest of the FBW7 protein (Rajagopalan et al. 2004; Davis et al. 2014). We determined that R465C, R479Q, and R505C all displayed several dominant-negative phenotypes when expressed in a heterozygous FBW7 background. These phenotypes included changes in cyclin E and MCL1 substrate expression as well as anaphase bridges (<xref ref-type="fig" rid="885fig4">Figure 4</xref> and and Figure S4A). In contrast, in a homozygous null FBW7 background these mutations showed the same phenotypes as the vector control Figure S4, B–D). We also observed that after BUBR1 knockdown, there was a decrease in colony number when cells were infected with cancer-specific mutations as compared to those infected with wild-type FBW7 (Figure S4E).'], '885fig5': ['FBW7 has several substrates that are involved in mitosis that could potentially activate the SAC. In particular, we wanted to investigate the function of the cell-cycle regulator cyclin E in the genetic interaction between FBW7 and BUBR1 given that the CIN phenotype in FBW7 knockout HCT116 cells was previously associated with the deregulation of this FBW7-associated substrate (Rajagopalan et al. 2004). We also wanted to look at the role of the survival protein MCL1, which is degraded by FBW7 only after the SAC is activated and, unlike cyclin E, should not be needed for sensitivity to SAC knockdown (Inuzuka et al. 2011; Wertz et al. 2011). In accordance with this, we found that high levels of cyclin E were necessary for the decrease in proliferation of FBW7 −/− cells after BUBR1 knockdown (<xref ref-type="fig" rid="885fig5">Figure 5</xref>). In contrast, knockdown of MCL1 did not show any rescue of the ). In contrast, knockdown of MCL1 did not show any rescue of the BUBR1/FBW7 genetic interaction (Figure S5, A and B) suggesting that high levels of cyclin E are needed for the genetic interaction between FBW7 and BUBR1 in HCT116 cells.', 'We determined in our experiments that cyclin E was required for cells lacking FBW7 to be sensitized to SAC knockdown (<xref ref-type="fig" rid="885fig5">Figure 5</xref>). High or dysregulated levels of cyclin E are known to cause mitotic defects such as centrosome amplification and increases in chromosome breaks and translocations (). High or dysregulated levels of cyclin E are known to cause mitotic defects such as centrosome amplification and increases in chromosome breaks and translocations (Hinchcliffe et al. 1999; Lacey et al. 1999; Loeb et al. 2005; Bagheri-Yarmand et al. 2010). Notably, cells overexpressing cyclin E can also inhibit APC/CCdh1 activity and increase the APC/C substrates cyclin B1 and Securin (Keck et al. 2007). These cells have increases in the number of cells in prometaphase and unaligned metaphase due to a mitotic delay as chromosomes misalign at the metaphase plate (Keck et al. 2007). Consistent with their ability to dysregulate cyclin E, FBW7 −/− cells show many similar phenotypes to cyclin E-overexpressing cells. They have been shown previously to have increased centrosome number (Rajagopalan et al. 2004). As well, up to 30% of HCT116 FBW7 −/− cells do not align their chromosomes at the metaphase plate during mitosis (Rajagopalan et al. 2004). We also show here that FBW7 −/− cells have an increase in the number of cells in prometaphase and higher levels of APC/C substrates when compared with FBW7 +/+ cells (<xref ref-type="fig" rid="885fig2">Figure 2</xref>). This suggests that defects in cyclin E levels could directly contribute to a prolonged metaphase arrest and SAC activation in ). This suggests that defects in cyclin E levels could directly contribute to a prolonged metaphase arrest and SAC activation in FBW7 −/− cells.'], '885fig6': ['Cells infected to express full-length nondegradable cyclin E produced cell lines that grew much more slowly than wild type and had a higher percentage of cells in S-phase (<xref ref-type="fig" rid="885fig6">Figure 6, A and B</xref>, and , and Figure S5C). This defect in cell-cycle progression is consistent with what has been reported previously (Spruck et al. 1999). Interestingly, expression of Δ65 cyclin E resulted in cell-cycle profiles similar to vector alone cell lines (<xref ref-type="fig" rid="885fig6">Figure 6A</xref> and and Figure S5C) suggesting that LMW cyclin E did not perturb the cell cycle to the extent of full-length cyclin E. Both forms of cyclin E increased the percentage of cells with >4 N DNA (<xref ref-type="fig" rid="885fig6">Figure 6C</xref>) consistent with previous studies that cyclin E overexpression can cause CIN () consistent with previous studies that cyclin E overexpression can cause CIN (Spruck et al. 1999; Akli et al. 2004; Bagheri-Yarmand et al. 2010). Unexpectedly, coexpression of MCL1 with cyclin E isoforms did not significantly increase CIN as measured by flow cytometry. It did, however, decrease the fraction of sub-G1 cells (<xref ref-type="fig" rid="885fig6">Figure 6D</xref>) suggesting that after overexpression of cyclin E, MCL1 has a role in cell survival in this system.) suggesting that after overexpression of cyclin E, MCL1 has a role in cell survival in this system.', 'Knockdown of the SAC using BUBR1 shRNA in these cell lines showed that overexpression of cyclin E in either form was not sufficient to establish dependence of HCT116 cells on the SAC (<xref ref-type="fig" rid="885fig6">Figure 6E</xref>). Instead, overexpression of both Δ65 and MCL1 was required for sensitivity to knockdown of BUBR1. Exactly why the Δ65 isoform, but not full-length cyclin E, was needed to establish sensitivity to BUBR1 knockdown is not known. It is possible that either the slow growth and cell-cycle perturbation of the full-length cyclin E-expressing lines or the consistently lower protein levels of MCL1 in the lines coexpressing full-length cyclin E and MCL1 could be a factor. Together, the results from these cell lines illustrate the complexity that can underlie the mechanisms of CIN in cells and emphasize the need for a better understanding of these mechanisms if we are to target them therapeutically.). Instead, overexpression of both Δ65 and MCL1 was required for sensitivity to knockdown of BUBR1. Exactly why the Δ65 isoform, but not full-length cyclin E, was needed to establish sensitivity to BUBR1 knockdown is not known. It is possible that either the slow growth and cell-cycle perturbation of the full-length cyclin E-expressing lines or the consistently lower protein levels of MCL1 in the lines coexpressing full-length cyclin E and MCL1 could be a factor. Together, the results from these cell lines illustrate the complexity that can underlie the mechanisms of CIN in cells and emphasize the need for a better understanding of these mechanisms if we are to target them therapeutically.', 'As shown in <xref ref-type="fig" rid="885fig6">Figure 6E</xref>, the establishment of SAC sensitivity in wild-type cells required the overexpression of both truncated cyclin E and the prosurvival factor MCL1. This kind of analysis is important for multifunctional tumor suppressors like FBW7 as not all functions associated with the protein may be relevant across all tumor types. In fact, work by , the establishment of SAC sensitivity in wild-type cells required the overexpression of both truncated cyclin E and the prosurvival factor MCL1. This kind of analysis is important for multifunctional tumor suppressors like FBW7 as not all functions associated with the protein may be relevant across all tumor types. In fact, work by Ekholm-Reed et al. (2004) has suggested that while FBW7 mutation can deregulate cyclin E in endometrial tumors, this does not necessarily lead to high cyclin E levels; whether this is due to a more recently identified feedback loop or some other mechanism is unknown (Xu et al. 2010). Similarly, studies in colorectal tumors suggest that the CIN phenotype of FBW7 knockout cells is found in some, but not all, FBW7-mutated tumors (Rajagopalan et al. 2004; Kemp et al. 2005). This suggests that further stratification of FBW7-mutated cells may be warranted when considering SAC inhibition as a potential therapy. As well, the effect of other FBW7 substrates, which converge on the SAC including the crucial mitotic kinases Aurora A and Aurora B, and the regulator of chromatid cohesion Jun B, may require further study (Teng et al. 2012; Perez-Benavente et al. 2013).'], '885fig7': ['Synthetic lethality has been proposed as a therapeutic strategy for treating cancer. An advantage of this approach is that the synthetic lethal drug target is distinct from the somatically mutated cancer gene product, which itself may be undruggable. In this article, we use this strategy to identify potential synthetic lethal candidates for FBW7, a currently undruggable tumor suppressor gene with several oncogenic substrates that are similarly difficult to target. We identified the gene BUBR1 as a synthetic lethal partner and found that FBW7 knockout cells were vulnerable because of a dependence of these cells on the mitotic checkpoint. Cells that lacked FBW7 and the mitotic checkpoint had not only decreased proliferation but also a significant increase in aneuploidy likely due to premature anaphase entry (<xref ref-type="fig" rid="885fig7">Figure 7</xref>).).', 'FBW7 is functionally pleiotropic and its knockout in cells can result in several different observable phenotypes including increased CIN (Rajagopalan et al. 2004). Interestingly, our model presented in <xref ref-type="fig" rid="885fig7">Figure 7</xref> demonstrates that targeting the CIN phenotype in demonstrates that targeting the CIN phenotype in FBW7 −/− cells is a viable synthetic lethal strategy. This is consistent with the hypothesis that there is an optimal or permissible level of CIN that can be tolerated in tumor cells but is not found in normal cells (Komarova and Wodarz 2004; Bakhoum and Compton 2012; Janssen and Medema 2013). It follows then that it may be possible to target the cellular stress of CIN therapeutically as it represents a cancer-specific vulnerability (Luo et al. 2009; Bakhoum and Compton 2012). Support for this hypothesis has been seen in genetic studies in mice where there appears to be a threshold of survivable CIN, but levels of instability beyond this threshold are deleterious to tumor formation (Janssen and Medema 2013; Silk et al. 2013). Our experiments suggest that the stress of CIN in HCT116 FBW7 knockout cells can also be exploited using this strategy.']}
|
Dependence of Human Colorectal Cells Lacking the FBW7 Tumor Suppressor on the Spindle Assembly Checkpoint
|
[
"FBW7",
"BUBR1",
"spindle assembly checkpoint",
"synthetic lethality",
"RNAi"
] |
Genetics
|
1446969600
|
[{'@Label': 'OBJECTIVES', '@NlmCategory': 'OBJECTIVE', '#text': 'Healing in cancellous metaphyseal bone might be different from midshaft fracture healing due to different access to mesenchymal stem cells, and because metaphyseal bone often heals without a cartilaginous phase. Inflammation plays an important role in the healing of a shaft fracture, but if metaphyseal injury is different, it is important to clarify if the role of inflammation is also different. The biology of fracture healing is also influenced by the degree of mechanical stability. It is unclear if inflammation interacts with stability-related factors.'}, {'@Label': 'METHODS', '@NlmCategory': 'METHODS', '#text': 'We investigated the role of inflammation in three different models: a metaphyseal screw pull-out, a shaft fracture with unstable nailing (IM-nail) and a stable external fixation (ExFix) model. For each, half of the animals received dexamethasone to reduce inflammation, and half received control injections. Mechanical and morphometric evaluation was used.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'As expected, dexamethasone had a strong inhibitory effect on the healing of unstable, but also stable, shaft fractures. In contrast, dexamethasone tended to increase the mechanical strength of metaphyseal bone regenerated under stable conditions.'}, {'@Label': 'CONCLUSIONS', '@NlmCategory': 'CONCLUSIONS', '#text': 'It seems that dexamethasone has different effects on metaphyseal and diaphyseal bone healing. This could be explained by the different role of inflammation at different sites of injury. Cite this article: Bone Joint Res 2015;4:170-175.'}]
|
[] |
other
|
PMC4649658
| null |
13
|
[
"{'Citation': 'Sandberg O, Aspenberg P. Different effects of indomethacin on healing of shaft and metaphyseal fractures. Acta Orthop 2015;86:243–247.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4404779'}, {'@IdType': 'pubmed', '#text': '25323801'}]}}",
"{'Citation': 'Aspenberg P, Sandberg O. Distal radial fractures heal by direct woven bone formation. Acta Orthop 2013;84:297–300.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3715812'}, {'@IdType': 'pubmed', '#text': '23570338'}]}}",
"{'Citation': 'Chen WT, Han C, Zhang PX, et al. A special healing pattern in stable metaphyseal fractures. Acta Orthop 2015;86:238–242.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4404778'}, {'@IdType': 'pubmed', '#text': '25582035'}]}}",
"{'Citation': 'Kumagai K, Vasanji A, Drazba JA, Butler RS, Muschler GF. Circulating cells with osteogenic potential are physiologically mobilized into the fracture healing site in the parabiotic mice model. J Orthop Res 2008;26:165–175.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '17729300'}}}",
"{'Citation': 'Bernhardsson M, Sandberg O, Aspenberg P. Experimental models for cancellous bone healing in the rat. Acta Orthop 2015;27:1–6. (Epub ahead of print).', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4750777'}, {'@IdType': 'pubmed', '#text': '26200395'}]}}",
"{'Citation': 'Siclari VA, Zhu J, Akiyama K, et al. Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow. Bone 2013;53:575–586.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3674849'}, {'@IdType': 'pubmed', '#text': '23274348'}]}}",
"{'Citation': 'Tu J, Henneicke H, Zhang Y, et al. Disruption of glucocorticoid signaling in chondrocytes delays metaphyseal fracture healing but does not affect normal cartilage and bone development. Bone 2014;69:12–22.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4284102'}, {'@IdType': 'pubmed', '#text': '25193158'}]}}",
"{'Citation': 'Ton FN, Gunawardene SC, Lee H, Neer RM. Effects of low-dose prednisone on bone metabolism. J Bone Miner Res 2005;20:464–470.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15746991'}}}",
"{'Citation': 'Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab Clin North Am 2012;41:595–611.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3417039'}, {'@IdType': 'pubmed', '#text': '22877431'}]}}",
"{'Citation': 'Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 1998;102:274–282.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC508885'}, {'@IdType': 'pubmed', '#text': '9664068'}]}}",
"{'Citation': 'Haugeberg G, Strand A, Kvien TK, Kirwan JR. Reduced loss of hand bone density with prednisolone in early rheumatoid arthritis: results from a randomized placebo-controlled trial. Arch Intern Med 2005;165:1293–1297.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '15956010'}}}",
"{'Citation': 'Grahnemo L, Jochems C, Andersson A, et al. Possible role of lymphocytes in glucocorticoid-induced increase in trabecular bone mineral density. J Endocrinol 2015;224:97–108.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4254076'}, {'@IdType': 'pubmed', '#text': '25359897'}]}}",
"{'Citation': 'Claes L, Reusch M, Göckelmann M, et al. Metaphyseal fracture healing follows similar biomechanical rules as diaphyseal healing. J Orthop Res 2011;29:425–432.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '20882588'}}}"
] |
Genetics. 2015 Nov 8; 201(3):885-895
|
NO-CC CODE
|
|
Links between Rsx RNA expression and X-inactivation and reactivationa, Rsx clouds (arrowheads) are present in supporting cells (‘s’) but not in meiotic cells (‘m’, labelled with HORMAD1). b, Rsx clouds (arrowheads) colocalise with the XCI marker H3K27me3 which is not observed in meiotic cells. c, RNA FISH for the X-gene Msn shows that while supporting cells undergo XCI (i.e. display a single RNA spot, arrow), meiotic cells have two active Xs. Msn RNA signals are very bright in meiotic cells due to increase in global transcription during this point in germ cell development35. Scale bars 5µm.
|
nihms412084f3
|
2
|
d984b4030f25d7ab5154020c3d553e73ca6f5b8f7ff615e533f118287035a506
|
nihms412084f3.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
503,
1050
] |
[{'image_id': 'nihms412084f1', 'image_file_name': 'nihms412084f1.jpg', 'image_path': '../data/media_files/PMC3484893/nihms412084f1.jpg', 'caption': 'Discovery of a candidate X-inactivating RNA in the opossuma, RNA FISH mapping of the novel gene using BACs; green BACs give RNA FISH cloud signals, red BACs do not. BAC VM18-839J22 gives a cloud signal (green; second panel) identical to Xist RNA (first panel) as seen in mouse brain cells (additional RNA cloud images Supplementary Figure 1). The cloud is still observed with VM18-839J22del (third panel), deleted for Hprt1 (Supplementary Table 1 for recombineering sequences) and VM18-303M7 (fourth panel). The 82kb critical interval is defined by VM18-839J22del and VM18-3O1. The Lnx3 gene which gave rise to Xist3, maps to a locus distinct from Rsx. MB = megabases b, top: RTPCR identification of a female-specific 47kb transcript (green rectangle) within the 82kb critical interval (primers Supplementary Table 1). Transcript limits shown above the X. middle: RNA FISH images showing RNA clouds in female but not male brain cells. bottom: RTPCR using primer pair M3 (black rectangle in first RTPCR image) shows female-specific expression in all tissues. Gapdh is an autosomal control. c, combined VM18-839J22 RNA FISH / H3K27me3 immunostaining shows the inactive X (marked by dotted line in first panel) coated with the novel RNA. d, combined VM18-839J22 RNA and DNA FISH for the novel RNA. No RNA signal is observed from the active X locus (Xa), but an RNA signal colocalizes with the DNA locus on the inactive X (Xi). . Scale bars 5µm.', 'hash': 'ddebd4c3a072abc16650fd18951e97854185cc7e35d913d801ecc675777a1c38'}, {'image_id': 'nihms412084f2', 'image_file_name': 'nihms412084f2.jpg', 'image_path': '../data/media_files/PMC3484893/nihms412084f2.jpg', 'caption': 'Characterisation of the Rsx RNAa, RNA-Seq shows female specific expression of Rsx,while reads mapping to the Phf6 and Hprt1 genes are found in both sexes. Boxed area shows magnification of Rsx locus. Pale red reads are ambiguous, i.e. they hit multiple repeats within the Rsx RNA. Inferred exons and their verification by RTPCR are shown. Dark grey bars along the chromosome co-ordinate axis represent DNA sequence gaps (Online Methods). b, Northern analysis of Rsx. (controls: Supplementary Figure 2, primers for probes: Supplementary Table 1). Top left panel: female-specific expression of an RNA greater than 23kb. Middle panel: size verification of Rsx RNA by comparison with the 17kb Xist RNA. Right two panels: verification of the strandedness of Rsx transcription (sequences in Supplementary Table 1). Bottom panel: multi-tissue blot showing female-specific Rsx expression in all tissues. c, Comparison of repeat organisation with that of Xist by sequence-similarity plots, window size = 28 nucleotides (grey area represents the unsequenced 2.8kb). The 5-prime 12kb stretch of repeats includes two highly conserved 34 and 35mer motifs (Supplementary Figure 3 for predicted 34mer stem-loops). RNA FISH using a repeat probe (green) co-localises with BAC VM18-839J22 (red; antisense probe, Supplementary Table 1). A sense probe generates no signals (data not shown), confirming the transcription orientation of Rsx. d, Female:male adjusted ratios inferred from brain RNA-Seq data identifying Rsx as a candidate XCI RNA (Online Methods). The second highest ranking RNA, MAPK4K, was found by RTPCR not to be female-specific in other tissues, so can be excluded as an XCI candidate. Scale bars 5µm.', 'hash': 'e53328c9bfde55e4da7e331f196f03684327029a30f1e640211d3597a13b8d8d'}, {'image_id': 'nihms412084f4', 'image_file_name': 'nihms412084f4.jpg', 'image_path': '../data/media_files/PMC3484893/nihms412084f4.jpg', 'caption': 'Autosomal gene silencing in mouse ES cells by an Rsx transgenea, in Rsx transgenic female ES cell clone 303.2, the full length Rsx transgene is expressed, as shown by RTPCR spanning all exon-exon boundary and ‘M3’ (see Figure 1) RTPCR. b, Rsx RNA appears as a cloud in 303.2 cells, indicating autosomal coating. RNA FISH for three chromosome 18 genes; Ndfip1, Prrc1 and Synpo, shows that this coating induces gene silencing in differentiated cells (first column), while in others silencing does not occur (middle column). Wild type ES cells show biallelic expression for each chromosome 18 gene (last column). Scale bars 5µm. c, quantitation of gene silencing (n>100 cells / gene) in differentiated 303.2 cells versus controls (PCR primers for chromosome 18 RNA FISH probes, Supplementary Table 1).', 'hash': 'c55ecc0a7101f24b19595d4eca3583a726df78c64b03d23506752e765c4d1156'}, {'image_id': 'nihms412084f3', 'image_file_name': 'nihms412084f3.jpg', 'image_path': '../data/media_files/PMC3484893/nihms412084f3.jpg', 'caption': 'Links between Rsx RNA expression and X-inactivation and reactivationa, Rsx clouds (arrowheads) are present in supporting cells (‘s’) but not in meiotic cells (‘m’, labelled with HORMAD1). b, Rsx clouds (arrowheads) colocalise with the XCI marker H3K27me3 which is not observed in meiotic cells. c, RNA FISH for the X-gene Msn shows that while supporting cells undergo XCI (i.e. display a single RNA spot, arrow), meiotic cells have two active Xs. Msn RNA signals are very bright in meiotic cells due to increase in global transcription during this point in germ cell development35. Scale bars 5µm.', 'hash': 'd984b4030f25d7ab5154020c3d553e73ca6f5b8f7ff615e533f118287035a506'}]
|
{'nihms412084f1': ['While analyzing XCI in the female brain of Monodelphis domestica, the short-tailed opossum, we used RNA FISH to study the expression of the X-gene Hprt1 with a BAC, VM18-839J22, containing Hprt1 plus 49kb of upstream and 82kb of downstream sequence, and in which no other known genes mapped (<xref ref-type="fig" rid="nihms412084f1">Fig 1a</xref>). RNA FISH signals usually appear as pinpoint dots. However the RNA signal detected resembled a cloud (). RNA FISH signals usually appear as pinpoint dots. However the RNA signal detected resembled a cloud (<xref ref-type="fig" rid="nihms412084f1">Fig. 1a</xref>, , Supplementary Figure 1) that was reminiscent of the Xist RNA cloud seen in female mouse (<xref ref-type="fig" rid="nihms412084f1">Fig. 1a</xref>) and human cells) and human cells15. We observed the same RNA cloud using a modified form of the BAC carrying an Hprt1 deletion (<xref ref-type="fig" rid="nihms412084f1">Fig. 1a</xref>). The RNA therefore originated from another, uncharacterised gene located within the genomic region defined by VM18-839J22. RNA FISH using additional BACs narrowed down this region to 82kb downstream of ). The RNA therefore originated from another, uncharacterised gene located within the genomic region defined by VM18-839J22. RNA FISH using additional BACs narrowed down this region to 82kb downstream of Hprt1 (<xref ref-type="fig" rid="nihms412084f1">Fig. 1a</xref>). We identified the RNA using RTPCR on female brain cDNA with primers located along this critical region (). We identified the RNA using RTPCR on female brain cDNA with primers located along this critical region (<xref ref-type="fig" rid="nihms412084f1">Fig. 1b</xref>; ; Supplementary Table 1), revealing a transcription unit spanning 47kb (<xref ref-type="fig" rid="nihms412084f1">Fig. 1b</xref>).).', 'We then investigated whether the RNA exhibited other Xist-like features. First, we looked for evidence of sexually dimorphic expression. No RNA clouds were detected in male opossum brain by VM18-839J22 BAC RNA FISH (<xref ref-type="fig" rid="nihms412084f1">Fig. 1b</xref>), demonstrating that in this tissue expression of the RNA was female-specific. Consistent with this, RTPCR on male brain cDNA revealed no expression of the 47kb transcript previously identified in females (), demonstrating that in this tissue expression of the RNA was female-specific. Consistent with this, RTPCR on male brain cDNA revealed no expression of the 47kb transcript previously identified in females (<xref ref-type="fig" rid="nihms412084f1">Fig. 1b</xref>). RTPCR on a broad array of tissues, representing derivatives of endoderm, mesoderm and ectoderm, from both males and females revealed expression of the RNA in all female but not male tissues examined (). RTPCR on a broad array of tissues, representing derivatives of endoderm, mesoderm and ectoderm, from both males and females revealed expression of the RNA in all female but not male tissues examined (<xref ref-type="fig" rid="nihms412084f1">Fig. 1b</xref>).).', 'Next, we established whether the RNA coats the inactive X. We combined VM18-839J22 RNA FISH on female brain cells with immunostaining for the inactive X marker H3K27me3. We observed colocalization of RNA clouds and H3K27me3 signals (<xref ref-type="fig" rid="nihms412084f1">Fig. 1c</xref>), demonstrating inactive X coating.), demonstrating inactive X coating.', 'To determine if the RNA was expressed from the inactive X, we performed dual RNA/DNA FISH using BAC VM18-839J22. No RNA signal was seen colocalizing with the DNA signal on the active X (<xref ref-type="fig" rid="nihms412084f1">Fig. 1d</xref>). In contrast, an RNA signal was observed colocalizing with the DNA signal on the inactive X (). In contrast, an RNA signal was observed colocalizing with the DNA signal on the inactive X (<xref ref-type="fig" rid="nihms412084f1">Fig. 1d</xref>). This RNA signal was brighter than others in the surrounding cloud, a feature characteristic of a site of nascent RNA synthesis. Thus, the RNA is expressed only from the inactive X. This must be the paternal X, as this chromosome is always chosen for inactivation). This RNA signal was brighter than others in the surrounding cloud, a feature characteristic of a site of nascent RNA synthesis. Thus, the RNA is expressed only from the inactive X. This must be the paternal X, as this chromosome is always chosen for inactivation6. In summary, like Xist, the RNA that we identified is female-specific, coats the inactive X and is transcribed only from the inactive X. We call the RNA Rsx (RNA-on-the-silent X).', 'a, in Rsx transgenic female ES cell clone 303.2, the full length Rsx transgene is expressed, as shown by RTPCR spanning all exon-exon boundary and ‘M3’ (see <xref ref-type="fig" rid="nihms412084f1">Figure 1</xref>) RTPCR. ) RTPCR. b, Rsx RNA appears as a cloud in 303.2 cells, indicating autosomal coating. RNA FISH for three chromosome 18 genes; Ndfip1, Prrc1 and Synpo, shows that this coating induces gene silencing in differentiated cells (first column), while in others silencing does not occur (middle column). Wild type ES cells show biallelic expression for each chromosome 18 gene (last column). Scale bars 5µm. c, quantitation of gene silencing (n>100 cells / gene) in differentiated 303.2 cells versus controls (PCR primers for chromosome 18 RNA FISH probes, Supplementary Table 1).'], 'nihms412084f2': ['To further characterize Rsx, we performed RNA-sequencing (RNA-Seq) on female opossum brain (<xref ref-type="fig" rid="nihms412084f2">Fig. 2a</xref>). This confirmed that the ). This confirmed that the Rsx gene generates a precursor RNA of 47kb (UCSC MonDom5 co-ordinates: chrX 35,605,415-35,651,609) transcribed antisense relative to Hprt1. Split RNA reads implied that Rsx encodes a spliced RNA comprised of four exons: this was confirmed by RTPCR (<xref ref-type="fig" rid="nihms412084f2">Fig. 2a</xref>, , Supplementary Table 1). The RNA-Seq data predicted that the mature Rsx RNA is large, approximating 27kb, with 25kb of sequence deriving from a single exon. Northern blots confirmed that Rsx RNA was large, exceeding the 17kb mouse Xist RNA in size, and validated the strandedness, female-specificity and broadness of Rsx expression (<xref ref-type="fig" rid="nihms412084f2">Fig. 2b</xref>). The level of ). The level of Rsx expression varied between female tissues, an observation also noted for Xist (Supplementary Figure 2). 3’-RACE demonstrated that Rsx transcripts are polyadenylated.', 'Sequence comparisons between Rsx and Xist revealed no significant homology. Nevertheless Rsx exhibited features reminiscent of Xist. Notably, it was highly enriched in tandem repeats biased towards the 5-prime end of the RNA (<xref ref-type="fig" rid="nihms412084f2">Fig. 2c</xref>) and exhibiting high GC content. The ) and exhibiting high GC content. The Rsx repeats included two highly conserved and similar motifs with potential to form stem loop structures (<xref ref-type="fig" rid="nihms412084f2">Fig. 2c</xref>; ; Supplementary Figure 3). RNA FISH using an oligonucleotide probe recognising one of these repeats gave a cloud signal indistinguishable from that seen using VM18-839J22 BAC, confirming that the repeats are included in the RNA that coats the inactive X (<xref ref-type="fig" rid="nihms412084f2">Fig. 2c</xref>). The longest ORF found for ). The longest ORF found for Rsx constituted less than five percent of the total RNA length, and was located in the repeat region, suggesting that Rsx functions as a non-coding RNA. We conclude that the Rsx and Xist RNAs display similar features.', 'RNA-Seq has been used previously to identify novel transcripts17 We speculated that analysis of RNA-Seq data alone would identify Rsx as a candidate XCI RNA. An RNA with a role in XCI would be X-linked and expressed only in females.and so should be evident in a comparison of female and male transcriptomes. To identify X-linked genes with sexually dimorphic expression levels, we compared the numbers of reads mapping to each region of the X in the female with that in the male brain and expressed this as a female:male ratio (Supplementary Table 2; and Methods). When all transcribed regions on the X were examined, Rsx was an outlier, with a female:male ratio exceeding the second-ranked RNA by three-fold (<xref ref-type="fig" rid="nihms412084f2">Fig. 2d</xref>). We repeated this RNA-Seq approach on liver, in which the level of ). We repeated this RNA-Seq approach on liver, in which the level of Rsx expression is low (<xref ref-type="fig" rid="nihms412084f2">Fig. 2b</xref>). In this analysis, ). In this analysis, Rsx appeared second (Supplementary Table 2). Thus, RNA-Seq can be used as a preliminary discovery tool to identify RNAs involved in dosage compensation.', 'RNA seq gives an overall predicted size of the mature Rsx RNA as 26,800 bp. Note that the predicted transcription start site differs when using RNA-Seq or 5’ RACE (co-ordinates in Supplementary Table 1). Split reads spanning the two sequence gaps on the right, implying that these gaps contain only intronic sequence (<xref ref-type="fig" rid="nihms412084f2">Figure 2a</xref>). We sequenced 8kb of the gaps located within this intron, and no RNA-Seq reads mapped to this sequence, suggesting that additional exons have not been missed. A third gap resides in the middle of the third exon of ). We sequenced 8kb of the gaps located within this intron, and no RNA-Seq reads mapped to this sequence, suggesting that additional exons have not been missed. A third gap resides in the middle of the third exon of Rsx. PCR shows that this gap is 5kb, rather than 8kb according to the MonDom5 version of genome assembly. We sequenced 2.2kb of this gap, and encountered a short and highly repetitive unit that precludes sequencing of the remaining 2.8kb.'], 'nihms412084f3': ['To investigate a link between Rsx RNA and XCI, we examined Rsx expression in the female germ line. In mice, Xist is expressed in somatic tissues but is silenced during oocyte development. This is accompanied by loss of H3K27me3 from the inactive X and X-reactivation18, 19, 20. Similar to other somatic cells, supporting cells in the ovary displayed Rsx clouds (<xref ref-type="fig" rid="nihms412084f3">Fig. 3a</xref>) and XCI, as shown by X chromosome H3K27me3 enrichment () and XCI, as shown by X chromosome H3K27me3 enrichment (<xref ref-type="fig" rid="nihms412084f3">Fig. 3b</xref>) and monoallelic expression of the X-gene ) and monoallelic expression of the X-gene Msn (<xref ref-type="fig" rid="nihms412084f3">Fig. 3c</xref>). However, in germ cells, identified by HORMAD1 immunostaining). However, in germ cells, identified by HORMAD1 immunostaining21, Rsx clouds were absent (<xref ref-type="fig" rid="nihms412084f3">Fig. 3a</xref>). Consistent with a relationship between ). Consistent with a relationship between Rsx expression and XCI, most meiotic cells had two active Xs, with no X chromosome H3K27me3 accumulation (<xref ref-type="fig" rid="nihms412084f3">Fig. 3b</xref>), and biallelic ), and biallelic Msn expression (<xref ref-type="fig" rid="nihms412084f3">Fig. 3c</xref>). ). Rsx expression is therefore linked to X-inactivation and reactivation.'], 'nihms412084f4': ['We next carried out experiments to address whether Rsx induces gene silencing. Xist transgenes function as ectopic X-inactivation centres in mouse embryonic stem (ES) cells, with Xist RNA coating the transgenic chromosome and inducing gene silencing in cis22, 23, 24. We generated an XX ES cell line, 303.2, carrying a single-copy chromosome 18-integrated transgene expressing full length Rsx RNA (<xref ref-type="fig" rid="nihms412084f4">Fig. 4a</xref>).).', 'We performed RNA FISH for Rsx and three chromosome 18 genes, Ndfip1, Prrc1 and Synpo, mapping near the transgene integration site, in differentiated 303.2 ES cells. We observed coating of the transgenic chromosome by Rsx RNA (<xref ref-type="fig" rid="nihms412084f4">Fig. 4b</xref>). While ). While Ndfip1, Prrc1 and Synpo were biallelically expressed in control ES cells (<xref ref-type="fig" rid="nihms412084f4">Fig. 4b,c</xref>), all three genes were silenced in more than one half of 303.2 ES cells (), all three genes were silenced in more than one half of 303.2 ES cells (<xref ref-type="fig" rid="nihms412084f4">Fig. 4b,c</xref>). Silencing also occurred in undifferentiated 303.2 cells, albeit in a lower proportion than seen post-differentiation (). Silencing also occurred in undifferentiated 303.2 cells, albeit in a lower proportion than seen post-differentiation (<xref ref-type="fig" rid="nihms412084f4">Fig. 4c</xref>). This finding is reminiscent of that obtained with human XIST transgenes in mouse ES cells). This finding is reminiscent of that obtained with human XIST transgenes in mouse ES cells25. We conclude that Rsx expression can induce gene silencing in cis.']}
|
Rsx Xist , a metatherian RNA with -like properties
| null |
Nature
|
1342076400
|
[{'@Label': 'OBJECTIVE', '@NlmCategory': 'OBJECTIVE', '#text': 'Recent US work identified "metabolically healthy overweight" and "metabolically at risk normal weight" individuals. Less is known for modernizing countries with recent increased obesity.'}, {'@Label': 'DESIGN AND METHODS', '@NlmCategory': 'METHODS', '#text': 'Fasting blood samples, anthropometry and blood pressure from 8,233 adults aged 18-98 in the 2009 nationwide China Health and Nutrition Survey, were used to determine prevalence of overweight (Asian cut point, BMI ≥ 23 kg/m(2) ) and five risk factors (prediabetes/diabetes (hemoglobin A1c ≥ 5.7%) inflammation (high-sensitivity C-reactive protein (hsCRP) ≥ 3 mg/l), prehypertension/hypertension (Systolic blood pressure/diastolic blood pressure ≥ 130/85 mm Hg), high triglycerides (≥ 150 mg/dl), low high-density lipoprotein cholesterol (<40 (men)/ <50 mg/dl (women)). Sex-stratified, logistic, and multinomial logistic regression models estimated concurrent obesity and cardiometabolic risk, with and without abdominal obesity, adjusting for age, smoking, alcohol consumption, physical activity, urbanicity, and income.'}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'Irrespective of urbanicity, 78.3% of the sample had ≥ 1 elevated cardiometabolic risk factor (normal weight: 33.2% had ≥ 1 elevated risk factor; overweight: 5.7% had none). At the age of 18-30 years, 47.4% had no elevated risk factors, which dropped to 6% by the age 70, largely due to age-related increase in hypertension risk (18-30 years: 11%; >70 years: 73%). Abdominal obesity was highly predictive of metabolic risk, irrespective of overweight (e.g., "metabolically at risk overweight" relative to "metabolically healthy normal weight" (men: relative risk ratio (RRR) = 39.06; 95% confidence interval (CI): 23.47, 65.00; women: RRR = 22.26; 95% CI: 17.49, 28.33)).'}, {'@Label': 'CONCLUSION', '@NlmCategory': 'CONCLUSIONS', '#text': 'A large proportion of Chinese adults have metabolic abnormalities. High hypertension risk with age, underlies the low prevalence of metabolically healthy overweight. Screening for cardiometabolic-related outcomes dependent upon overweight will likely miss a large portion of the Chinese at risk population.'}]
|
[
"Adult",
"Age Factors",
"Aged",
"Asian People",
"Blood Pressure",
"Body Mass Index",
"C-Reactive Protein",
"Cardiovascular Diseases",
"China",
"Cholesterol, HDL",
"Confidence Intervals",
"Diabetes Mellitus",
"Female",
"Glycated Hemoglobin",
"Humans",
"Hypertension",
"Inflammation",
"Lipids",
"Logistic Models",
"Male",
"Middle Aged",
"Obesity",
"Obesity, Abdominal",
"Overweight",
"Reference Values",
"Risk Factors",
"Sex Factors",
"Triglycerides"
] |
other
|
PMC3484893
| null |
40
|
[
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] |
Nature. 2012 Jul 12; 487(7406):254-258
|
NO-CC CODE
|
|
Live-cell imaging
of Spinach2 fusion RNAs with different fluorophores.
(a) COS7 cells expressing (CGG)60-Spinach2 in the presence
of either DFHBI or DFHBI-1T. Cells were initially cultured in the
presence of 20 μM DFHBI (top panel), and images were acquired
using a 100 ms exposure. The media was then exchanged with media containing
20 μM DFHBI-1T for 10 min, and images of the same cell nuclei
were obtained using identical image acquisition conditions. Increased
fluorescence was seen in cells cultured with DFHBI-1T (lower panel).
(b) Quantification of average brightness of 10 foci normalized to
brightness of DFHBI. Average and SEM values are shown. (c) Cells expressing
(CGG)60-Spinach2 in the presence of either DFHBI or DFHBI-2T
were imaged using both GFP and YFP filter sets. Scale bar, 10 μm.
|
ja-2013-10819x_0002
|
2
|
c94eb53b686782f199a171ad9eb67b15a7e7e9a486cf533755bda53299e6d0e6
|
ja-2013-10819x_0002.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
666,
864
] |
[{'image_id': 'ja-2013-10819x_0001', 'image_file_name': 'ja-2013-10819x_0001.jpg', 'image_path': '../data/media_files/PMC3929357/ja-2013-10819x_0001.jpg', 'caption': 'Different fluorophores bind Spinach2 and\nform RNA–fluorophore\ncomplexes with unique spectral properties. Excitation (dotted line)\nand emission (solid line) spectra of Spinach2–fluorophore complexes.\nExcitation and emission spectra were collected in the presence of\nexcess Spinach2 (5 μM) in the presence of different fluorophores\n(1 μM) in binding buffer (40 mM HEPES, pH 7.4, 125 mM KCl, 10\nmM MgCl2): (a) DFHBI, (b) DFHBI-1T, and (c) DFHBI-2T. Spectra\nare shown as the percent of the maximum excitation and emission fluorescence\nfor each Spinach2–fluorophore complex. Excitation spectra were\ncollected at the indicated emission maximum wavelength, and emission\nspectra were collected at the indicated excitation-maximum wavelength.\nThe numbering in (a) indicates the atom numbering scheme for the imidazolone\nring.', 'hash': '5e75a247f05ce7152a747fb8b591de51df5fe8b4a0b0ed0433ddc5d226a9fa9f'}, {'image_id': 'ja-2013-10819x_0004', 'image_file_name': 'ja-2013-10819x_0004.jpg', 'image_path': '../data/media_files/PMC3929357/ja-2013-10819x_0004.jpg', 'caption': 'No caption found', 'hash': 'df3bd46a31c4bfbb2ea05aec03df259dc1e0163863ffa723e709389da8846b4f'}, {'image_id': 'ja-2013-10819x_0003', 'image_file_name': 'ja-2013-10819x_0003.jpg', 'image_path': '../data/media_files/PMC3929357/ja-2013-10819x_0003.jpg', 'caption': 'Photophysical and Binding Properties\nof Fluorophore-Spinach2 Complexes', 'hash': 'e3f3037dfadb0f366ed4d01e1304b6ee44a6022b297287c3cef2ba755d148104'}, {'image_id': 'ja-2013-10819x_0002', 'image_file_name': 'ja-2013-10819x_0002.jpg', 'image_path': '../data/media_files/PMC3929357/ja-2013-10819x_0002.jpg', 'caption': 'Live-cell imaging\nof Spinach2 fusion RNAs with different fluorophores.\n(a) COS7 cells expressing (CGG)60-Spinach2 in the presence\nof either DFHBI or DFHBI-1T. Cells were initially cultured in the\npresence of 20 μM DFHBI (top panel), and images were acquired\nusing a 100 ms exposure. The media was then exchanged with media containing\n20 μM DFHBI-1T for 10 min, and images of the same cell nuclei\nwere obtained using identical image acquisition conditions. Increased\nfluorescence was seen in cells cultured with DFHBI-1T (lower panel).\n(b) Quantification of average brightness of 10 foci normalized to\nbrightness of DFHBI. Average and SEM values are shown. (c) Cells expressing\n(CGG)60-Spinach2 in the presence of either DFHBI or DFHBI-2T\nwere imaged using both GFP and YFP filter sets. Scale bar, 10 μm.', 'hash': 'c94eb53b686782f199a171ad9eb67b15a7e7e9a486cf533755bda53299e6d0e6'}]
|
{'ja-2013-10819x_0001': ['We first examined the effects of substitutions at the N-1 position\nof the imidazolone ring. Replacing the methyl substituent in DFHBI\nwith a 1,1,1-trifluoroethyl substituent (DFHBI-1T, (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one) resulted in a Spinach2\ncomplex with a 35 nm red shift in the excitation peak and a slight\nred shift in the emission peak (Figure <xref rid="ja-2013-10819x_0001" ref-type="fig">1</xref>, Table , Table 1). DFHBI-1T also exhibited an overall increase in\nbrightness, which reflects a slight increase in the extinction coefficient\nand an increase in the quantum yield (Table 1).', 'We also\nconsidered the effects of switching the methyl substituent\nat the C-2 position of the imidazolinone ring to a trifluoromethyl\n(DFHBI-2T, (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1-methyl-2-(trifluoromethyl)-1H-imidazol-5(4H)-one). When bound to Spinach2,\nthis molecule exhibited a marked 53 nm red shift in the excitation\nand a 22 nm red shift in the emission maxima, although the overall\nbrightness was somewhat reduced due to a decrease in the quantum yield\n(Figure <xref rid="ja-2013-10819x_0001" ref-type="fig">1</xref>, Table , Table 1).\nThe increase in KD to ∼1.2 μM\n(Figure <xref rid="ja-2013-10819x_0001" ref-type="fig">1</xref>, Table , Table 1)\nsuggests that the bulky trifluoromethyl moiety may exhibit steric\nhindrance with the Spinach2 aptamer.'], 'ja-2013-10819x_0002': ['We next asked if DFHBI-1T and DFHBI-2T can be used to image Spinach2-tagged\nRNA in living cells. For these experiments, we expressed the CGG repeat\ntoxic RNA that causes fragile X ataxia and tremor syndrome.6 This RNA comprises 60 CGG repeats and forms mobile\nintranuclear RNA inclusions in cells. This RNA was tagged with Spinach2\nas described previously.6 Cells were initially\ntreated with media containing 20 μM DFHBI, resulting in the\nexpected green intranuclear foci, which were imaged using a GFP filter\ncube. However, replacing the media with media containing 20 μM\nDFHBI-1T resulted in foci that were ∼60% brighter (Figure <xref rid="ja-2013-10819x_0002" ref-type="fig">2</xref>a,b). The increase in fluorescence is consistent\nwith improved excitation of Spinach2 complexed with DFHBI-1T.a,b). The increase in fluorescence is consistent\nwith improved excitation of Spinach2 complexed with DFHBI-1T.', 'We next sought to characterize\nthe properties of Spinach2 bound\nto DFHBI-2T in living cells. The spectral properties of this complex\ndoes not overlap with the standard GFP filter cube, but are instead\nmore compatible with YFP filter cubes, which typically have an excitation\nbandpass filter transmitting 500 ± 10 nm light, a dichroic mirror\nat 515 nm, and an emission filter that transmits 535 ± 15 nm\nlight. Indeed, cells expressing (CGG)60-Spinach2 exhibited\nreadily detectable intranuclear foci when imaged with the GFP filter\ncube, but only minimal fluorescence when imaged with the YFP filter\ncube (Figure <xref rid="ja-2013-10819x_0002" ref-type="fig">2</xref>c). However, when the media was\nswitched with media DFHBI-2T, fluorescence was markedly reduced when\nimaging with the GFP filter cube but was readily detectable using\nthe YFP filter cube (Figure c). However, when the media was\nswitched with media DFHBI-2T, fluorescence was markedly reduced when\nimaging with the GFP filter cube but was readily detectable using\nthe YFP filter cube (Figure <xref rid="ja-2013-10819x_0002" ref-type="fig">2</xref>c). These data\nindicate that Spinach2 imaged DFHBI-2T results in fluorescence that\nis detectable using the yellow emission channel.c). These data\nindicate that Spinach2 imaged DFHBI-2T results in fluorescence that\nis detectable using the yellow emission channel.']}
|
Plug-and-Play
Fluorophores Extend the Spectral Properties
of Spinach
| null |
J Am Chem Soc
|
1390982400
|
Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with cancer. The underlying mechanisms, however, remain largely unidentified. We have recently demonstrated that KLF8 expression enhances the activity but not expression of matrix metalloproteinase-2 (MMP2), the target substrate of MMP14. Here, we report a novel KLF8 to MMP14 signaling that promotes human breast cancer invasion and metastasis. Using cell lines for inducible expression and knockdown of KLF8, we demonstrate that KLF8 promotes MMP14 expression at the transcriptional level. Knocking down KLF8 expression inhibited the breast cancer cell invasion both in vitro and in vivo as well as the lung metastasis in mice, which could be rescued by ectopic expression of MMP14. Promoter reporter assays and oligonucleotide and chromatin immunoprecipitations determined that KLF8 activates the human MMP14 gene promoter by both directly acting on the promoter and indirectly via promoting the nuclear translocation of β-catenin, the expression of T-cell factor-1 (TCF1) and subsequent activation of the promoter by the β-catenin/TCF1 complex. Inhibition of focal adhesion kinase (FAK) using pharmacological inhibitor, RNA interference or knockout showed that the cell surface presentation of active MMP14 downstream of KLF8 depends on FAK expression and activity. Taken together, this work identified novel signaling mechanisms by which KLF8 and FAK work together to promote the extracellular activity of MMP14 critical for breast cancer metastasis.
|
[
"Animals",
"Breast Neoplasms",
"Cell Line, Tumor",
"Cell Membrane",
"Disease Models, Animal",
"Disease Progression",
"Female",
"Focal Adhesion Kinase 1",
"Gene Expression",
"Hepatocyte Nuclear Factor 1-alpha",
"Heterografts",
"Humans",
"Kruppel-Like Transcription Factors",
"Lung Neoplasms",
"Matrix Metalloproteinase 14",
"Mice",
"Models, Biological",
"Neoplasm Invasiveness",
"Neoplasm Metastasis",
"Protein Binding",
"Repressor Proteins",
"Transcription, Genetic",
"beta Catenin"
] |
other
|
PMC3929357
| null |
32
|
[
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"{'Citation': 'Wang X, Urvalek AM, Liu J, Zhao J. Activation of KLF8 transcription by focal adhesion kinase in human ovarian epithelial and cancer cells. J Biol Chem. 2008 May 16;283(20):13934–42. Epub 2008/03/21. eng.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '18353772'}}}",
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] |
J Am Chem Soc. 2014 Jan 29; 136(4):1198-1201
|
NO-CC CODE
|
|
Establishment of cetuximab-resistant cells in 3D culture.(a) Five thousand cells/ml were cultured in type-1 collagen for 17 days. Fresh medium was added with different concentrations of CTX every 2 days, and colony number was determined using a GelCount plate reader. n=2 independent experiments performed in triplicate. (b) Twelve-day old CC and CR were treated with CTX (10 μg/ml) for 3 days. Representative images from 3 independent experiments are shown. Scale bars, 1000 μm. (c) Left: Representative fluorescence images of GFP signals captured from subcutaneous tumors, generated by injection of CC and CC-CR stably transduced with GFP-expressing lentivirus. Right: Quantification of radiant efficiency from tumors. n=8. **P<0.01 by paired Student's t test. (d) Representative H&E staining of the tumor xenografts from the indicated groups. Scale bar, 100 μm. (e) Quantification of IHC staining in Fig. 1g. n=8 mice. **P<0.01 by Student's t test. (f) CC and CC-CR cells grown on Transwell filters were incubated with Alexa Fluor 488-labeled C225 mAb directed against the extracellular domain of EGFR and then stained for F-actin (Phalloidin) and nuclei (DAPI). Scale bars, 20 μm. Data represent mean ± s.d. in a, c, and e.
|
nihms905237f1
|
2
|
10a2ecf96c1e04c87d488769d94310658472de6a890077629d664dda78ae007f
|
nihms905237f1.jpg
|
multiple
|
multiple panels: images & plots
|
[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
580,
755
] |
[{'image_id': 'nihms905237f13', 'image_file_name': 'nihms905237f13.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f13.jpg', 'caption': "Transcriptome profiling of CC and CC-CR in 3D(a) Left, heatmap of top 50 differentially expressed transcripts in CC-CR versus CC from 3 independent 3D culture experiments. Gene expression values are gene-wise z-transformed and are colored red for high abundance and blue for low abundance, as indicated in the scale bar. Right, miRNA heatmap showing miRNAs altered (>2-fold and FDR<0.01) in CC-CR versus CC. (b) Genomic organization of lncRNA MIR100HG, host gene of miR-100/let-7a-2/miR-125b-1 cluster, on human chromosome 11 (hsa chr11). (c) qRT-PCR showing upregulation of lncRNA MIR100HG, miR-100 and miR-125b in CC-CR compared to CC in 3D. In CC-CR, cells were treated with CTX (CTX+, 3 μg/ml) or normal culture medium (CTX-) for 14 consecutive days in 3D. ACTB or U6 snRNA served as the internal control, respectively. n=3 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by Dunnett's test. (d) Scatter plots of MIR100HG versus miR-100 or miR-125b expression in TCGA CRC data repository. Pearson correlation coefficients (r) and P values are shown. (e) RNA FISH showing high MIR100HG (red) expression in CC-CR mouse tumor xenografts compared to CC xenografts. Concomitantly, high miR-100 (green) and miR-125b (red) signals were observed in CC-CR tumors; the yellow fluorescent signal indicates co-expression of miR-100 and miR-125b. Scale bars, 50 μm. (f) qRT-PCR analysis of MIR100HG, miR-100 and miR-125b expression levels among a panel of 30 CRC cell lines ranked by their responsiveness to cetuximab (see Extended Data Table 3). ACTB or U6 snRNA served as the internal control. Fold changes were normalized to CC. n.d., not detected. n=3 independent experiments performed in triplicate. Data represent mean ± s.d. in c and f.", 'hash': 'cc96a4e5299b26b84fc7f792c1d5dbc08349dfe2af15f78e450c074eda3f1985'}, {'image_id': 'nihms905237f9', 'image_file_name': 'nihms905237f9.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f9.jpg', 'caption': "A double-negative feedback loop between MIR100HG/miR-125b and GATA6. (a) Immunoblots of GATA6 expression in CC transfected with 2 independent siRNAs against GATA6 or control siRNA (siCTL). (b) Immunoblots of GATA6 expression in CC-CR transfected with either pcDNA3.1-GATA6 (WT GATA6), or pcDNA3.1-mutant GATA6 (MUT GATA6), or empty vector (CTL). (c) Luciferase reporter assays were performed in HuTu80 by co-transfection of pGL3-MIR100HG promoter luciferase reporter with increasing concentrations of pcDNA3.1-GATA6, and a Renilla control. Luciferase activity was normalized to Renilla values. n=3 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by Dunnett's test. (d) The luciferase vector pGL3 driven by either wild-type, deletion, or mutant (MUT) promoter was transfected in HuTu80, and luciferase activity was measured. n=3 independent experiments. *P<0.05, **P<0.01 by Student's t test. (e) ChIP assays were performed with anti-GATA6 antibody or control IgG in CC-CR overexpressing either WT GATA6, MUT GATA6, or CTL. The abundance of DNA within the MIR100HG promoter region was assessed by qRT-PCR with a primer pair spanning the GATA-binding site 2. A primer pair 6.4 kb distal to the MIR100HG promoter (Distal) was used as control. Data are presented as relative enrichment normalized to control IgG. **P<0.01 by one-way ANOVA followed by LSD post-hoc test. (f) EMSA using nuclear extracts from CC and the indicated probes. Ab, antibody. Representative of 3 independent experiments. (g) Luciferase reporter analysis of a wild-type (WT) or MUT GATA6 3′ UTR activity upon addition of synthetic miR-125b in Caco-2. n=2 independent experiments. **P<0.01 by Student's t test. (h) Immunoblots of GATA6 in stable miR-125b-transduced Caco-2 and 125b-Sp-transduced HuTu80. (i) Box plots showing MIR100HG expression in the lower (<25%) and the higher (>75%) quartiles of GATA6 expression from GEO CRC datasets GSE14333 and GSE39582. **P<0.01 by Mann–Whitney U test. (j)\nMET genomic status detected by FISH assay. There was no obvious change in MET copy number in 10 paired tumor specimens pre- and post-cetuximab treatment. Representative images are shown. Red, MET locus; green, chromosome 7 centromere (CEP7). Scale bar, 20 μm. Data represent mean ± s.d. in c-e and g.", 'hash': '29141c342e04503d4c4f5347cb4606912343cf233ed7bc9a8da0a8b0a25a2b83'}, {'image_id': 'nihms905237f14', 'image_file_name': 'nihms905237f14.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f14.jpg', 'caption': "Cooperativity of miR-100 and miR-125b in CTX resistance(a, b) Indicated cells were grown in 3D in normal medium (CTL) or treated with CTX (3 μg/ml) in 3D. The resultant colonies were counted after 18 days. Sponge (Sp). n=3 independent experiments performed in triplicate. *P<0.05, **P<0.01 by one-way ANOVA followed by Dunnett's test compared with CTL-Sp or miR-CTL. (c, d) Left: indicated cells were cultured in 3D for 12 days and CTX (10 μg/ml) was added for 24 h before cells were fixed, stained for Cleaved Casp-3 (cyan) and Ki-67 (magenta). Scale bars, 50 μm. Right: quantification of the morphological changes among indicated cell lines. n=4 independent experiments. (e, f) Left: indicated cells were injected subcutaneously into nude mice (n=8). After tumor size reached approximately 100 mm3, the mice received CTX treatment (1 mg/mouse, i.p. injection every 3 days). Representative fluorescent images of GFP signals captured from subcutaneous tumors are shown. Middle: growth curve of tumors in nude mice (n=8) injected with cells as indicated. **P<0.01 by repeated-measures ANOVA test followed by Dunnett's test. Right: tumors (n=8) were isolated on day 28 after treatment and tumor weight was calculated. **P<0.01 by one-way ANOVA followed by Dunnett's test compared with CTL-Sp or miR-CTL. Data represent mean ± s.d. n.s., not significant.", 'hash': '0045b57d18225ef2f419d1096ea96dedb78781fa7b686234a4018648b04efeab'}, {'image_id': 'nihms905237f7', 'image_file_name': 'nihms905237f7.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f7.jpg', 'caption': "Effects of differential modulation of miR-100 and/or miR-125b on nuclear β-catenin expression levels.(a) Immunoblots for β-catenin from nuclear fractions in the CC and Caco-2 cells overexpressing miR-100 and/or miR-125b, or CC-CR, DLD-1 and CTX-R7 cells expressing miR-100 and/or miR-125b sponges. Lamin A/C served as the control for nuclear fractions. Representative of 2 independent experiments. (b) Representative IHC of β-catenin in the indicated xenografts (n=8). Scale bars: 50 μm. Quantification of nuclear β-catenin positive cells is shown. Data represent mean ± s.d. **P<0.01 by Student's t test.", 'hash': '5b127bc7d0ada00ffe8ef82e8b9c33abec878f1862d2829119b0ff6d835f6a62'}, {'image_id': 'nihms905237t10', 'image_file_name': 'nihms905237t10.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t10.jpg', 'caption': 'No caption found', 'hash': 'dc9be36436a4dc97e9bf376021f85bc37ee965a391924ba521bc54c73019d9e7'}, {'image_id': 'nihms905237t5', 'image_file_name': 'nihms905237t5.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t5.jpg', 'caption': 'No caption found', 'hash': '4acb6aee2f865f838789a1c38e007e0026ba45c7416e9364b9119e3be9c9d67d'}, {'image_id': 'nihms905237t2', 'image_file_name': 'nihms905237t2.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t2.jpg', 'caption': 'No caption found', 'hash': '413cd1a79248edbb349144e7c9d3c0bb8425e14571eba7b28dc7eebfb9fc8767'}, {'image_id': 'nihms905237f11b', 'image_file_name': 'nihms905237f11b.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11b.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': 'c3af033b1e6c5640308eedc4d487b15c32519524f5787b96fe67d1d9481b473b'}, {'image_id': 'nihms905237f11e', 'image_file_name': 'nihms905237f11e.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11e.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': '5dae2dcb17f42e8f3cefa2aeb31dd32de84349f30b45d7d03040eff7d556487e'}, {'image_id': 'nihms905237f1', 'image_file_name': 'nihms905237f1.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f1.jpg', 'caption': "Establishment of cetuximab-resistant cells in 3D culture.(a) Five thousand cells/ml were cultured in type-1 collagen for 17 days. Fresh medium was added with different concentrations of CTX every 2 days, and colony number was determined using a GelCount plate reader. n=2 independent experiments performed in triplicate. (b) Twelve-day old CC and CR were treated with CTX (10 μg/ml) for 3 days. Representative images from 3 independent experiments are shown. Scale bars, 1000 μm. (c) Left: Representative fluorescence images of GFP signals captured from subcutaneous tumors, generated by injection of CC and CC-CR stably transduced with GFP-expressing lentivirus. Right: Quantification of radiant efficiency from tumors. n=8. **P<0.01 by paired Student's t test. (d) Representative H&E staining of the tumor xenografts from the indicated groups. Scale bar, 100 μm. (e) Quantification of IHC staining in Fig. 1g. n=8 mice. **P<0.01 by Student's t test. (f) CC and CC-CR cells grown on Transwell filters were incubated with Alexa Fluor 488-labeled C225 mAb directed against the extracellular domain of EGFR and then stained for F-actin (Phalloidin) and nuclei (DAPI). Scale bars, 20 μm. Data represent mean ± s.d. in a, c, and e.", 'hash': '10a2ecf96c1e04c87d488769d94310658472de6a890077629d664dda78ae007f'}, {'image_id': 'nihms905237f6', 'image_file_name': 'nihms905237f6.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f6.jpg', 'caption': "miR-100/125b coordinately represses five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling.(a) Left: Wnt activation in CC and CC-CR cells was measured by the 64 Wnt/β -catenin target genes (Wnt signature score). **P<0.01 by Student's t test. Right: Scatter plots of MIR100HG expression versus 64-gene Wnt signature score on 458 CRC. Pearson correlation coefficients (r) and P values are shown. (b) Immunoblots of DKK1, DKK3, ZNRF3, RNF43, and APC2 levels from 3D cell lysates of CC and CC-CR. In CC-CR, cells were treated with CTX (CTX+, 3 μg/ml) or normal culture medium (CTX-) for 14 days in 3D before protein extraction. Representative of 3 independent experiments. (c) Top : representative IHC images of DKK1, DKK3, ZNRF3, RNF43, and APC2 in CC and CC-CR xenografts (n=8). Bottom: measurement of protein expression, **P<0.01 by Mann-Whitney U test. (d) Dual luciferase assays of genes predicted to be regulated by miR-100 or miR-125b in Caco-2. Renilla luciferase activity was normalized to firefly activity. n=2 independent experiments. **P<0.01 by Student's t test. (e) Immunoblots of indicated proteins in stable miRNA-transduced Caco-2 and sponge (Sp)-transduced HuTu80. Representative of 2 independent experiments. (f) Immunoblot of nuclear and cytoplasmic extracts for β-catenin and p-β-catenin (S552). Loading controls were GAPDH for cytoplasmic fractions and Lamin A/C for nuclear fractions. (g) CC and CC-CR in 3D were treated with CTX (10 μg/ml) and/or Wnt3a (100 ng/ml). Immunoblots of indicated proteins after 48 h of treatment are shown. Representative of 3 independent experiments. (h) qRT-PCR analysis of Wnt targets CCND1, CD44, FOSL1, and NKD1 mRNAs at indicated time points following CTX (10 μg/ml) treatment in 3D. n=2 independent experiments performed in triplicate. **P<0.01 by two-way ANOVA test. Data represent mean ± s.d. in a, c, d, and h.", 'hash': '28852d73c3eebc9c5b7e98364a1f25546651576e6b03d5aeb5df9aed425a6d2f'}, {'image_id': 'nihms905237f8', 'image_file_name': 'nihms905237f8.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f8.jpg', 'caption': "Blockade of Wnt signaling restores cetuximab responsiveness to cetuximab-resistant cells.(a, b) Left: CC-CR doxycycline (Dox)-on DKK1 or DKK3 cells were cultured in the presence or absence of Dox (1 μg/ml) and harvested at 48 h. Total cell lysates and conditioned media were harvested and subjected to immunoblot analysis. Right: indicated cells were grown in 3D in normal medium or treated with CTX (3 μg/ml). The resultant colonies were counted after 18 days. n=3 experiments performed in triplicate. **P<0.01 by Student's t test. (c) CC-CR cells were grown in 3D in normal medium (CTL), treated with CTX (3 μg/ml) or in combination with recombinant DKK1 (rDKK1) and DKK3 (rDKK3) in 3D every 2 days. The resultant colonies were stained after 18 days for Cleaved Casp-3 (green) and Ki-67 (red). Scale bar, 50 μm. Quantification was shown. n=3 independent experiments. (d) Immunoblots for β-catenin from nuclear and cytoplasmic fractions of indicated cells upon CTX (10 μg/ml) treatment. Loading controls were GAPDH for cytoplasmic fractions and Lamin A/C for nuclear fractions. (e) CC-CR were treated with CTX (3 μg/ml), and/or XAV-939 (1, 5, 10 μM), and/or ICG-001 (1, 2.5, 5 μM) in 3D for 18 days, and colony number was determined. n=3 experiments performed in triplicate. (f) DLD-1 and HCT8 cells were treated with CTX (200 μg/ml) and/or ICG-001 (4 μM) for 14 days in 3D, and colony number was determined. n=2 independent experiments performed in triplicate. (g) Quantification of radiant efficiency from tumors (n=6) represented on Fig. 4i. **P<0.01 by paired Student's t test. (h) Representative IHC images and quantification of Ki-67 and Cleaved Casp-3 from CC-CR xenografts (n=6) treated with control saline (CTL), or CTX (1 mg/mouse, i.p. injection, every 3 days), and/or ICG-001 (150 mg/kg, i.p. injection, daily). Scale bar, 50 μm. *P<0.05, **P<0.01 by one-way ANOVA followed by Dunnett's test in c, e, and h, and one-way ANOVA followed by LSD post-hoc test in f. Data represent mean ± s.d. in a-c and e-h. n.s., not significant.", 'hash': 'f7068c770dba2cdc8d81c2fa3ece151b3770f47385d26199a3b692e04ba175da'}, {'image_id': 'nihms905237f15', 'image_file_name': 'nihms905237f15.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f15.jpg', 'caption': "miR-100 and miR-125b augment Wnt signaling by repressing multiple Wnt negative regulators(a) Predicted miR-100 (red) and miR-125b (blue) binding sites in 3′ untranslated regions (3′ UTRs) of human DKK1, DKK3, ZNRF3, RNF43, and APC2. CDS, coding sequence. (b, c) Dual luciferase assays of candidates predicted to be regulated by miR-100 or miR-125b. Renilla luciferase activity was normalized to firefly activity and presented as relative luciferase activity. n=2 independent experiments. **P<0.01 by Student's t test. (d) Immunoblots of indicated proteins in stable miRNA-transduced CC and sponge (Sp)-transduced CC-CR. Representative of 3 independent experiments. (e) Immunofluorescence of p-β-catenin (Y489). Scale bars, 50 μm. Right, quantification of 4 independent experiments. **P<0.01 by Student's t test. (f) Representative IHC of β-catenin in CC and CC-CR xenografts (n=8). Scale bars: 50 μm (main); 20 μm (inset). Quantification of nuclear β-catenin-positive cells is shown. **P<0.01 by Student's t test. (g) qRT-PCR analysis of Wnt target genes in CC and CC-CR cells. n=3 independent experiments performed in triplicate. *P<0.05, **P<0.01 by Student's t test. (h) qRT-PCR analysis of Wnt target genes in the indicated stable miRNA-transduced CC cells. n=3 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by Dunnett's test. (i) CC-CR were injected subcutaneously into nude mice (n=6). When tumor size reached around 100 mm3, mice were treated with control saline, or CTX (1 mg/mouse, i.p. injection every 3 days) and/or ICG-001 (150 mg/kg i.p. injection daily). Representative in vivo fluorescent images are shown. (j) Growth curve of tumors in nude mice (n=6) treated with different compounds. **P<0.01 by repeated-measures ANOVA test followed by LSD post-hoc test. (k) Tumors (n=6) were isolated on day 28 after treatment and tumor weight was measured. **P<0.01 by one-way ANOVA followed by LSD post-hoc test. Data represent mean ± s.d.", 'hash': '00433fb6b6adbb254f46e1824358def2692749ad9264acbac7cc869e02580963'}, {'image_id': 'nihms905237f12', 'image_file_name': 'nihms905237f12.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f12.jpg', 'caption': "Characterization of cetuximab-resistant CC (CC-CR) in 3D(a) Schematic of experimental approach to establish cetuximab (CTX)-resistant cells in 3D. In the presence of CTX (3 μg/ml) in 3D type-1 collagen culture, greater than 95% of CC colonies die. Residual colonies were isolated and iteratively passaged in 2D and 3D in the continued presence of CTX over approximately 4 months. These colonies were designated CC-CR. (b) Top: differential interference contrast (DIC) and confocal images of representative CC and CC-CR in 2D and 3D. F-actin was stained with phalloidin (red). Scale bars: 400, 1000, 200, 50 μm, respectively (from left to right). Bottom: left, number of nuclei in the midplane of each colony; right, the morphology of colonies was divided into those with luminal, multi-layered, or solid morphology. n=4 independent experiments, *P<0.05 by Student's t test. (c) CC and CC-CR were cultured in 3D in the presence or absence of CTX (3 μg/ml) and colonies were counted after 18 days. n=3 independent experiments performed in triplicate. **P<0.01 by Student's t test. (d) CC and CC-CR cells were cultured in 3D for 12 days and treated with CTX (10 μg/ml) for 24 h. Ki-67 (red) and Cleaved Caspase-3 (Cleaved Casp-3, green) staining were imaged by confocal microscopy. Representative of 4 independent experiments. Scale bar, 50 μm. Quantification is shown on the right (n=4). **P<0.01 by Student's t test. (e) Immunoblots of 3D cell lysates from CC and CC-CR treated with CTX (10 μg/ml) for indicated time. β-actin served as the loading control. A representative blot from 3 independent experiments is shown. (f) Nude mice (n=8) bearing subcutaneous tumors were treated with control saline or CTX at a dose of 1 mg/mouse, intraperitoneal (i.p.) injection, every 3 days. Tumor volumes were measured every 3 days using calipers. **P<0.01 by repeated-measures ANOVA test followed by LSD post-hoc test. (g) Representative immunohistochemical images of Ki-67 and Cleaved Casp-3 from CC and CC-CR xenografts before and after CTX treatment. Scale bar: 50 μm. Data represent mean ± s.d. in b-d and f. n.s., not significant.", 'hash': '71b363a2ce79acb3a8a4c4978de784a86b3e229bb115b03ccb66320edb7adf64'}, {'image_id': 'nihms905237f11d', 'image_file_name': 'nihms905237f11d.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11d.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': '6ffa984ac4dbc484e25c3da1e8eabf23ad70976fec10ceb9ec26cf73c6abcb2f'}, {'image_id': 'nihms905237f11c', 'image_file_name': 'nihms905237f11c.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11c.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': '5923f99faef89c6e3809af87434e509f740555ebed484e0add0cf1a0101dd186'}, {'image_id': 'nihms905237t3', 'image_file_name': 'nihms905237t3.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t3.jpg', 'caption': 'No caption found', 'hash': '9931e4246b056187f6032adb0ec88655f7d37c94997af5668bd9a795a04a72ad'}, {'image_id': 'nihms905237t4', 'image_file_name': 'nihms905237t4.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t4.jpg', 'caption': 'No caption found', 'hash': '9c977f88e94f00b156ad1000916400a63dca49ad1a52fc4e59466c700bd6b5cc'}, {'image_id': 'nihms905237t11', 'image_file_name': 'nihms905237t11.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t11.jpg', 'caption': 'No caption found', 'hash': 'e8569fe881c7ae8413a288e64fda5d820d05fbffb2b0ee3725b3464acd6c3003'}, {'image_id': 'nihms905237t9', 'image_file_name': 'nihms905237t9.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t9.jpg', 'caption': 'No caption found', 'hash': '041a025ef171429be87de7962abb868ec317407653f1ec60a670f27f3e1d15c0'}, {'image_id': 'nihms905237f11g', 'image_file_name': 'nihms905237f11g.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11g.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': '6954f6c20e5c157e06aac37a0d8bec8bf6dc02be5d885f52c3196b119777240b'}, {'image_id': 'nihms905237t7', 'image_file_name': 'nihms905237t7.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t7.jpg', 'caption': 'No caption found', 'hash': '16414b326f739a08c376b232392955a3639dce27ff1e9dc13557a0a53ada3d3a'}, {'image_id': 'nihms905237f5', 'image_file_name': 'nihms905237f5.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f5.jpg', 'caption': "Effects of differential modulation of miR-100 and/or miR-125b on cetuximab responsiveness in CC and CC-CR in vivo.(a, b) Quantification of radiant efficiency from tumors (n=8) represented on Fig. 3e and f. **P<0.01 by paired Student's t test. (c-f) Representative IHC images and quantification of Ki-67 and Cleaved Casp-3 from indicated xenografts (n=8) treated with CTX. Scale bars, 50 μm. **P<0.01 by one-way ANOVA followed by Dunnett's test in e and f. Data represent mean ± s.d. in a, b, e, and f. n.s., not significant.", 'hash': 'f4e1814f1af353f7200c2e7d205973610bb847dde16d5fe94fbc59b3123d2ac7'}, {'image_id': 'nihms905237f2', 'image_file_name': 'nihms905237f2.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f2.jpg', 'caption': "MIR100HG and miR-100/125b overexpression in cetuximab-resistant colorectal cancer cell lines.(a) qRT-PCR showing upregulation of pri-miR-100 and pri-miR-125b-1 in CC-CR compared to CC grown in 3D. In CC-CR, cells were treated with CTX (CTX+, 3 μg/ml) or normal culture medium (CTX-) for consecutive 14 days in 3D. (b) qRT-PCR showing upregulation of pri-let-7a-2 expression in CC-CR but unchanged expression of mature let-7a between the 2 cell lines. n=3 independent experiments performed in triplicate in a and b. Data represent mean ± s.d. **P<0.01 by one-way ANOVA followed by Dunnett's test compared with CC. (c) Left: a schematic diagram showing the PCR primers used in the 5′ RACE. Right: MIR100HG TSS was validated by 5′ RACE nested PCR in CC-CR with subsequent sequencing of the cloned fragments. Arrow indicates band of expected size. M, DNA marker. (d) Scatter plots of MIR100HG versus let-7a expression in TCGA CRC data repository. No correlation was found between those 2 molecules. (e, f) Expression of MIR100HG and miR-100/125b negatively correlates with cetuximab growth inhibition regardless of KRAS/BRAF mutational status. (e) Scatter plot of MIR100HG and miR-100/125b expression versus cetuximab inhibition rate in a panel of 30 CRC cell lines. (f) Twenty-one cell lines harbor KRAS or BRAF mutation, and 9 cell lines are KRAS/BRAF wild-type (WT). Pearson correlation coefficients (r) and P values are shown.", 'hash': 'c8c486231065c82aa81c2e319a50bfa0dcdfb117c8f73c30693c93e43ea4e101'}, {'image_id': 'nihms905237f16', 'image_file_name': 'nihms905237f16.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f16.jpg', 'caption': "GATA6 transcriptionally represses MIR100HG and is targeted by miR-125b in a double-negative feedback loop(a) Immunoblot of GATA6 in CC and CC-CR cells cultured in 3D. In CC-CR, cells were treated with CTX (CTX+, 3 μg/ml) or normal culture medium (CTX-) for consecutive 14 days in 3D before protein extraction. Representative of 3 independent experiments. (b) Immunofluorescence of GATA6 (green) and nuclei (blue). Scale bar, 50 μm. (c) Representative IHC of GATA6 in CC and CC-CR xenografts (n=8). (d) qRT-PCR analysis of MIR100HG and GATA6 expression at indicated time points following CTX treatment (10 μg/ml) in CC cultured in 3D. n=3 independent experiments. (e) CC cells were transfected with two independent siRNAs against GATA6 or control (siCTL), treated with CTX (10 μg/ml) and subjected to qRT-PCR analysis. n=2 independent experiments performed in triplicate. **P<0.01 by Student's t test. (f) Luciferase reporter assays were performed by co-transfection of pGL3-MIR100HG promoter luciferase reporter with increasing concentrations of pcDNA3.1-GATA6 plasmid or empty vector control (CTL), along with a Renilla luciferase reporter. Luciferase activity was measured 36 h post-transfection and normalized to Renilla values. n=3 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by Dunnett's test. (g) A schematic representation of consecutive deletion and mutation constructs spanning the -2000∼+500 region of MIR100HG promoter. The putative GATA6-binding sites within MIR100HG promoter are shown in black boxes. (h) The luciferase vector pGL3 driven by either wild-type, deletion or mutant (MUT) promoter was transfected in CC-CR, and luciferase activity was measured. n=3 independent experiments. *P<0.05, **P<0.01 by Student's t test. (i) Luciferase reporter analysis of a wild-type (WT) or mutant (MUT) GATA6 3′ UTR activity upon addition of either synthetic miR-125b or a negative control miR-CTL. **P<0.01 by Student's t test. (j) Immunoblots of GATA6 in stable miR-125b-transduced CC and 125b-Sp-transduced CC-CR. Representative of 3 independent experiments. (k) Box plots showing expression of GATA6 (left) and MIR100HG (middle) by stage from the TCGA CRC data repository. Right panel depicts MIR100HG expression in the lower (<25%) and the higher (>75%) quartile of GATA6 expression. *P<0.05, **P<0.01 by Mann-Whitney U test. n.s., not significant. Data represent mean ± s.d. in d-f, h, and i.", 'hash': 'ac2fce8a1319de599ea05643a9efdfa06740a4923baad8a2fd1dfa2d3f2f804d'}, {'image_id': 'nihms905237t1', 'image_file_name': 'nihms905237t1.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t1.jpg', 'caption': 'No caption found', 'hash': '002d0189bb1a3095dd20ce1f743b0ca54ec3e37ce3a77c7587c20d39912f936a'}, {'image_id': 'nihms905237t6', 'image_file_name': 'nihms905237t6.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t6.jpg', 'caption': 'No caption found', 'hash': 'c06f369c86481335f9824327d0204efca2e34bba72bb8ac162290e36a0801f72'}, {'image_id': 'nihms905237t8', 'image_file_name': 'nihms905237t8.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237t8.jpg', 'caption': 'No caption found', 'hash': '740538508d1983e1c84a1ee658d9cdb82503844b26909899ce79823b6a4b0ae0'}, {'image_id': 'nihms905237f11f', 'image_file_name': 'nihms905237f11f.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11f.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': '514d8d1227aeaec2323ebc16f8075c65ae2a22ff0d102afe900f747ac050c21e'}, {'image_id': 'nihms905237f11a', 'image_file_name': 'nihms905237f11a.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f11a.jpg', 'caption': 'Original images of immunoblots with molecular weight standards.', 'hash': '11b239eab05910f3b699a49d15e4e24336457f5a826c17a076b18684978f995c'}, {'image_id': 'nihms905237f17', 'image_file_name': 'nihms905237f17.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f17.jpg', 'caption': 'Increased MIR100HG and miR-100/125b are found in CRC patient specimens at time of progression on cetuximab(a) qRT-PCR of miR-100 and miR-125b levels in 10 pairs of matched human CRC specimens pre- and post-cetuximab resistance. Each symbol represents mean value of an individual patient. *P<0.05 by Wilcoxon matched-pairs signed rank test. (b, c) Frequency of nuclear β-catenin-positive cells (b) and GATA6-positive cells (c) in 10 pairs of matched human CRC specimens pre- and post-cetuximab resistance. *P<0.05 by Wilcoxon matched-pairs signed rank test. (d) Representative FISH images of MIR100HG, miR-100/125b and corresponding IHC images of β-catenin and GATA6 staining in representative three paired human CRC specimens obtained pre- and post-cetuximab resistance. Scale bars, 50 μm (main); 500 μm (inset).', 'hash': 'cb5068de76c1be37cd3d1746e41455ef095fef34a4667e8fcee644f5b2dfe1ec'}, {'image_id': 'nihms905237f10', 'image_file_name': 'nihms905237f10.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f10.jpg', 'caption': 'Model of a new mode of acquired and de novo cetuximab resistance. We propose a complex circuitry in which the lncRNA MIR100HG through embedded miR-100 and miR-125b confers cetuximab resistance by targeting and decreasing expression of five negative regulators of Wnt signaling, DKK1, DKK3, ZNRF3, RNF43, and APC2. This results in increased Wnt signaling and cetuximab resistance; this resistance can be overcome by blockade of Wnt signaling. We present evidence that GATA6 represses MIR100HG expression, but that miR-125b targets GATA6 to relieve this repression.', 'hash': '5a5fa277b49eed69a4fd9c74794e08a1bf3e38ac4c750a162f0a20364d843593'}, {'image_id': 'nihms905237f3', 'image_file_name': 'nihms905237f3.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f3.jpg', 'caption': "MIR100HG and miR-100/125b expression in head and neck squamous cell cancer cell lines and modulation of miR-100 and/or miR-125b in CC and CC-CR cells.(a) qRT-PCR analysis of MIR100HG, miR-100, and miR-125b expression among the CTX-sensitive head and neck squamous cell carcinoma (HNSCC) cell line SCC25 and its derived CTX-resistant sublines (CTX-R1, R3, R4, R5, R7, and R8) upon continuous exposure to cetuximab, as well as UNC10, a de novo CTX-resistant cell line. n=3 independent experiments performed in triplicate. *P<0.05, **P<0.01 by one-way ANOVA followed by Dunnett's test compared with SCC25. (b) qRT-PCR of indicated miRNA expression in CC stably overexpressing miR-100, miR-125b, or Bicistron. (c) qRT-PCR of indicated miRNA expression in CC-CR stably expressing miR-100 sponge (100-Sp), miR-125b sponge (125b-Sp), or bicistron sponge (Bicistron-Sp). Values were normalized to U6 snRNA. n=3 experiments performed in triplicate. **P<0.01 by Student's t test. (d, e) Quantification of Ki-67 and Cleaved Casp-3 in Fig. 3c and d. n=4 independent experiments. *P<0.05, **P<0.01 by Student's t test. Data represent mean ± s.d. n.s., not significant.", 'hash': 'b72522617a32cc05c1cac5669ccdb3154a91b62662d51ccd2fe1b795c43aa933'}, {'image_id': 'nihms905237f4', 'image_file_name': 'nihms905237f4.jpg', 'image_path': '../data/media_files/PMC5961502/nihms905237f4.jpg', 'caption': "miR-100 and miR-125b cooperativity drives cetuximab resistance in colorectal cancer and head and neck squamous cell cancer cell lines.(a) Caco-2 cells stably overexpressing Bicistron or control (miR-CTL) were cultured in 3D for 5 days and treated with CTX (50 μg/ml) for 24 h. Immunofluorescence was performed for Cleaved Casp-3 (cyan) and Ki-67 (magenta) with quantification shown on the right. Scale bar, 50 μm. n=3 independent experiments. *P<0.05, **P<0.01 by Student's t test. (b) DLD-1 cells stably expressing Bicistron-Sp or control (CTL-Sp) were cultured in 3D for 10 days and treated with CTX (200 μg/ml) for 24 h. Staining of Cleaved Casp-3 (cyan) and Ki-67 (magenta) were shown. Scale bars, 50 μm. Quantification is shown on the right. n=3 independent experiments. *P<0.05, **P<0.01 by Student's t test. (c, d) Indicated cells were grown in 3D in normal medium (CTL) or treated with CTX (50 μg/ml for Caco-2, and 200 μg/ml for DLD-1) in 3D. The resultant colonies were counted. n=2 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by Dunnett's test compared with miR-CTL or CTL-Sp. (e) Left: CTX-R7 cells stably expressing miR-100 and/or miR-125b sponges were grown in normal medium (CTL) or treated with CTX (30 μg/ml). Cell viability was measured by cell counting kit-8 (CCK-8) assays after 72 h. n=3 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by Dunnett's test compared with CTL-Sp. Middle: qRT-PCR analysis of Wnt target genes in the stable bicistron sponge-transduced CTX-R7 cells. n=2 independent experiments performed in triplicate. *P<0.05, **P<0.01 by Student's t test. Right: CTX-R7 cells were treated with CTX (30 μg/ml) and/or ICG-001 (2 μM) for 72 h, and cell viability was measured by CCK-8 assays. n=2 independent experiments performed in triplicate. **P<0.01 by one-way ANOVA followed by by LSD post-hoc test. Data represent mean ± s.d. n.s., not significant.", 'hash': 'e480fd93df8d34ee39b37d0a6607043aca3515d47750548d6279f82d742f3524'}]
|
{'nihms905237f12': ['By placing single cells from a human KRAS/NRAS/BRAF wild-type, microsatellite unstable CRC cell line, HCA-7, into 3D culture in type-1 collagen, a line was derived from colonies with cystic morphology and designated cystic colonies (CC) 14,15. Proliferation of CC was inhibited by cetuximab in 3D culture but not in 2D plastic culture14. Upon continuous exposure to cetuximab in 3D culture for approximately 4 months, a line was generated and designated CC-cetuximab resistant (CC-CR) (<xref rid="nihms905237f12" ref-type="fig">Fig. 1a</xref>). In 2D culture, CC and CC-CR were morphologically indistinguishable. In 3D, however, CC formed hollow cysts with a central lumen lined by a monolayer of polarized cells, whereas CC-CR formed solid disorganized colonies (). In 2D culture, CC and CC-CR were morphologically indistinguishable. In 3D, however, CC formed hollow cysts with a central lumen lined by a monolayer of polarized cells, whereas CC-CR formed solid disorganized colonies (<xref rid="nihms905237f12" ref-type="fig">Fig. 1b</xref>). As expected, cetuximab inhibited CC growth in 3D, while CC-CR remained refractory to cetuximab up to 200 μg/ml (). As expected, cetuximab inhibited CC growth in 3D, while CC-CR remained refractory to cetuximab up to 200 μg/ml (<xref rid="nihms905237f12" ref-type="fig">Fig. 1c</xref>; ; <xref rid="nihms905237f1" ref-type="fig">Extended Data Fig. 1a and b</xref>). Decreased expression of the proliferative marker, Ki-67, and increased expression of the apoptotic marker, cleaved Caspase-3, were observed in CC 24 h after cetuximab treatment, but these indices were unaffected in CC-CR (). Decreased expression of the proliferative marker, Ki-67, and increased expression of the apoptotic marker, cleaved Caspase-3, were observed in CC 24 h after cetuximab treatment, but these indices were unaffected in CC-CR (<xref rid="nihms905237f12" ref-type="fig">Fig. 1d</xref>). In cetuximab-treated CC, we observed reduced levels of p-EGFR, p-ERK1/2, p-AKT and Cyclin D1, as well as increased cleaved Caspase-3 and the pro-apoptotic marker, BIM; these markers were largely unaffected in cetuximab-treated CC-CR (). In cetuximab-treated CC, we observed reduced levels of p-EGFR, p-ERK1/2, p-AKT and Cyclin D1, as well as increased cleaved Caspase-3 and the pro-apoptotic marker, BIM; these markers were largely unaffected in cetuximab-treated CC-CR (<xref rid="nihms905237f12" ref-type="fig">Fig. 1e</xref>). Next, CC and CC-CR were stably transduced with a green fluorescent protein (GFP)-expressing lentiviral vector and injected subcutaneously into athymic nude mice. CC tumors were well differentiated and regressed upon administration of cetuximab. In contrast, CC-CR tumors were poorly differentiated and continued to grow in the presence of cetuximab, although not to the extent of untreated tumors (). Next, CC and CC-CR were stably transduced with a green fluorescent protein (GFP)-expressing lentiviral vector and injected subcutaneously into athymic nude mice. CC tumors were well differentiated and regressed upon administration of cetuximab. In contrast, CC-CR tumors were poorly differentiated and continued to grow in the presence of cetuximab, although not to the extent of untreated tumors (<xref rid="nihms905237f12" ref-type="fig">Fig. 1f and g</xref>; ; <xref rid="nihms905237f1" ref-type="fig">Extended Data Fig. 1c-e</xref>).).', '(a) Five thousand cells/ml were cultured in type-1 collagen for 17 days. Fresh medium was added with different concentrations of CTX every 2 days, and colony number was determined using a GelCount plate reader. n=2 independent experiments performed in triplicate. (b) Twelve-day old CC and CR were treated with CTX (10 μg/ml) for 3 days. Representative images from 3 independent experiments are shown. Scale bars, 1000 μm. (c) Left: Representative fluorescence images of GFP signals captured from subcutaneous tumors, generated by injection of CC and CC-CR stably transduced with GFP-expressing lentivirus. Right: Quantification of radiant efficiency from tumors. n=8. **P<0.01 by paired Student\'s t test. (d) Representative H&E staining of the tumor xenografts from the indicated groups. Scale bar, 100 μm. (e) Quantification of IHC staining in <xref rid="nihms905237f12" ref-type="fig">Fig. 1g</xref>. n=8 mice. **. n=8 mice. **P<0.01 by Student\'s t test. (f) CC and CC-CR cells grown on Transwell filters were incubated with Alexa Fluor 488-labeled C225 mAb directed against the extracellular domain of EGFR and then stained for F-actin (Phalloidin) and nuclei (DAPI). Scale bars, 20 μm. Data represent mean ± s.d. in a, c, and e.'], 'nihms905237f1': ['We first considered known mechanisms of cetuximab resistance in this 3D model. By whole exome sequencing and RNA Sequencing (RNA-Seq), no known genetic events linked to cetuximab resistance were found, including all reported gene mutations, copy number changes and gene fusion events (Extended Data Table 1). By RNA-Seq, we found 141 transcripts upregulated and 220 transcripts downregulated in CC-CR compared to CC (fold change>2 and false-discovery rate, FDR<0.01). Expression levels of ERBB1-4, the 7 EGFR ligands, and MET were comparable between CC and CC-CR (Extended Data Table 2). Immunofluorescence also showed equivalent cell-surface EGFR staining in CC and CC-CR (<xref rid="nihms905237f1" ref-type="fig">Extended Data Fig. 1f</xref>). Small RNA-Seq detected 7 miRNAs upregulated and 24 miRNAs downregulated in CC-CR compared to CC (fold change>2 and FDR<0.01). Of note, the most upregulated transcript in CC-CR was lncRNA MIR100HG, and the two most upregulated miRNAs were miR-125b and miR-100 (). Small RNA-Seq detected 7 miRNAs upregulated and 24 miRNAs downregulated in CC-CR compared to CC (fold change>2 and FDR<0.01). Of note, the most upregulated transcript in CC-CR was lncRNA MIR100HG, and the two most upregulated miRNAs were miR-125b and miR-100 (<xref rid="nihms905237f13" ref-type="fig">Fig. 2a</xref>).).'], 'nihms905237f13': ['MIR100HG is the host gene of the miR-100/let-7a-2/miR-125b-1 cluster on chromosome 11 (<xref rid="nihms905237f13" ref-type="fig">Fig. 2b</xref>). qRT-PCR analysis confirmed upregulation of endogenous MIR100HG expression in CC-CR in the presence or absence of cetuximab (). qRT-PCR analysis confirmed upregulation of endogenous MIR100HG expression in CC-CR in the presence or absence of cetuximab (<xref rid="nihms905237f13" ref-type="fig">Fig. 2c</xref>). pri-miR-100, pri-miR-125b-1, and their corresponding mature miRNA, miR-100 and miR-125b, were also enriched in CC-CR (). pri-miR-100, pri-miR-125b-1, and their corresponding mature miRNA, miR-100 and miR-125b, were also enriched in CC-CR (<xref rid="nihms905237f13" ref-type="fig">Fig. 2c</xref> and and <xref rid="nihms905237f2" ref-type="fig">Extended Data Fig. 2a</xref>). Although pri-let-7a-2 was upregulated in CC-CR, mature let-7a was unchanged compared to CC (). Although pri-let-7a-2 was upregulated in CC-CR, mature let-7a was unchanged compared to CC (<xref rid="nihms905237f2" ref-type="fig">Extended Data Fig. 2b</xref>). The transcriptional start site (TSS) of MIR100HG was confirmed by 5′ RACE-PCR (). The transcriptional start site (TSS) of MIR100HG was confirmed by 5′ RACE-PCR (<xref rid="nihms905237f2" ref-type="fig">Extended Data Fig. 2c</xref>). Analysis of The Cancer Genome Atlas (TCGA) CRC data repository revealed that miR-100 and miR-125b expression is tightly correlated with MIR100HG expression (). Analysis of The Cancer Genome Atlas (TCGA) CRC data repository revealed that miR-100 and miR-125b expression is tightly correlated with MIR100HG expression (<xref rid="nihms905237f13" ref-type="fig">Fig. 2d</xref>). RNA fluorescence ). RNA fluorescence in situ hybridization (FISH) showed highly enriched MIR100HG and miR-100/125b expression in CC-CR tumor xenografts (<xref rid="nihms905237f13" ref-type="fig">Fig. 2e</xref>). In contrast, let-7a expression did not correlate with that of MIR100HG (). In contrast, let-7a expression did not correlate with that of MIR100HG (<xref rid="nihms905237f2" ref-type="fig">Extended Data Fig. 2d</xref>).).', 'To assess whether MIR100HG and miR-100/125b overexpression extended beyond this one cell line, we examined their expression in a panel of 30 CRC cell lines placed upon a continuum of cetuximab sensitivity and resistance based upon published reports16,17 (Extended Data Table 3). Expression of MIR100HG and miR-100/125b were enriched in more cetuximab-resistant lines compared to the more sensitive lines (<xref rid="nihms905237f13" ref-type="fig">Fig. 2f</xref>). Their expression inversely correlated with cetuximab resistance, regardless of ). Their expression inversely correlated with cetuximab resistance, regardless of KRAS/BRAF mutational status (<xref rid="nihms905237f2" ref-type="fig">Extended Data Fig. 2e and f</xref>). For example, two of the cetuximab-sensitive lines (GEO and SW403) expressed low levels of MIR100HG and miR-100/125b despite harboring mutant ). For example, two of the cetuximab-sensitive lines (GEO and SW403) expressed low levels of MIR100HG and miR-100/125b despite harboring mutant KRAS. In addition, we also observed upregulation of MIR100HG and miR-100/125b in the setting of cetuximab resistance in HNSCC cell lines (<xref rid="nihms905237f3" ref-type="fig">Extended Data Fig. 3a</xref>). Thus, MIR100HG and miR-100/125b are upregulated in the setting of cetuximab resistance in CRC and HNSCC cell lines and this phenomenon occurs in both acquired and ). Thus, MIR100HG and miR-100/125b are upregulated in the setting of cetuximab resistance in CRC and HNSCC cell lines and this phenomenon occurs in both acquired and de novo resistance. These findings led us to further explore the function of MIR100HG and miR-100/125b in cetuximab resistance.', 'To understand how miR-100 and miR-125b influence cetuximab responsiveness, we considered the most downregulated genes in CC-CR to be potential targets of these miRNAs. Two negative regulators of Wnt signaling, DKK1 and DKK3, were decreased over 30-fold in CC-CR compared to CC (<xref rid="nihms905237f13" ref-type="fig">Fig. 2a</xref>). Meanwhile, Wnt activity was enhanced in CC-CR by analysis of 64 consensus β-catenin target genes). Meanwhile, Wnt activity was enhanced in CC-CR by analysis of 64 consensus β-catenin target genes19 (<xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6a</xref>). Analysis of a large human CRC dataset (n=458) also revealed that MIR100HG expression levels positively correlated with the Wnt score). Analysis of a large human CRC dataset (n=458) also revealed that MIR100HG expression levels positively correlated with the Wnt score19, whereas no correlation was observed between MIR100HG and the Ras-Az score20, which measures MEK activation as a downstream index of RAS signaling (<xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6a</xref> and data not shown). Functional enrichment analysis further identified Wnt pathway enrichment in miR-100 and miR-125b putative targets ( and data not shown). Functional enrichment analysis further identified Wnt pathway enrichment in miR-100 and miR-125b putative targets (Extended Data Table 4). We thus considered whether miR-100 and miR-125b might target components of Wnt signaling. Through computational target prediction, we found that 3′ UTRs of DKK1 and DKK3 contain binding sites for miR-100 and miR-125b, respectively (<xref rid="nihms905237f15" ref-type="fig">Fig. 4a</xref>). Since clustered miRNAs are co-expressed and often coordinately regulate molecular pathways by targeting different components of the same pathway). Since clustered miRNAs are co-expressed and often coordinately regulate molecular pathways by targeting different components of the same pathway21, we searched for other negative regulators of Wnt signaling that contain putative binding sites for miR-100 or miR-125b, and identified zinc and ring finger 3 (ZNRF3), ring finger protein 43 (RNF43), and APC2 as potential targets of miR-100 or miR-125b alone or in combination (<xref rid="nihms905237f15" ref-type="fig">Fig. 4a</xref> and and Extended Data Table 5). Decreased protein levels of these five Wnt negative regulators in CC-CR compared to CC were confirmed by both immunoblots in cell lines and immunostaining in xenografts (<xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6b and c</xref>). Using 3′ UTR luciferase reporter assays, we confirmed these five candidates are direct targets of miR-100 and/or miR-125b in both CC and Caco-2 cells; repression of these genes was rescued by mutations in the corresponding binding sites (). Using 3′ UTR luciferase reporter assays, we confirmed these five candidates are direct targets of miR-100 and/or miR-125b in both CC and Caco-2 cells; repression of these genes was rescued by mutations in the corresponding binding sites (<xref rid="nihms905237f15" ref-type="fig">Fig. 4b and c</xref>; ; <xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6d</xref>). Immunoblots confirmed the regulation of the predicted targets by miR-100 and miR-125b alone or in combination in CC and CC-CR (). Immunoblots confirmed the regulation of the predicted targets by miR-100 and miR-125b alone or in combination in CC and CC-CR (<xref rid="nihms905237f15" ref-type="fig">Fig. 4d</xref>). Consistently, this regulation was also observed in Caco-2 cells (low endogenous miR-100/125b expression) and HuTu80 cells (high endogenous miR-100/125b expression) (). Consistently, this regulation was also observed in Caco-2 cells (low endogenous miR-100/125b expression) and HuTu80 cells (high endogenous miR-100/125b expression) (<xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6e</xref>).).', 'To explore mechanism(s) by which miR-100 and miR-125b are upregulated in CC-CR, we investigated transcriptional regulation of the host gene, MIR100HG. Possible transcription factors containing binding sites within the 2.5 kb promoter of MIR100HG were mapped in silico using the Match program (version 1.0)25 and cross-referenced with the RNA-Seq dataset (Extended Data Table 6). Among these transcription factors, we focused on the zinc-finger transcription factor GATA6, which was downregulated at both the mRNA and protein level in CC-CR in 3D culture and in nude mouse xenografts (<xref rid="nihms905237f13" ref-type="fig">Fig. 2a</xref> and and <xref rid="nihms905237f16" ref-type="fig">Fig. 5a-c</xref>).).', 'Mutational status of 30 CRC cell lines used in <xref rid="nihms905237f13" ref-type="fig">Fig. 2f</xref> and their response to cetuximab and their response to cetuximab'], 'nihms905237f3': ['Since a major role of certain lncRNAs is production of embedded miRNAs10,18, we asked whether cetuximab resistance is mediated by miR-100 and miR-125b overexpression. To this end, we delivered lentiviral-based overexpression or sponge constructs into CC and CC-CR, respectively, to generate stable cell lines expressing each miRNA, the miR-100/125b bicistron, or their corresponding sponges (<xref rid="nihms905237f3" ref-type="fig">Extended Data Fig. 3b and c</xref>). Although the miR-100 sponge had no significant effect on colony number in CC-CR in 3D culture, both the miR-125b and bicistron sponges significantly reduced colony number (). Although the miR-100 sponge had no significant effect on colony number in CC-CR in 3D culture, both the miR-125b and bicistron sponges significantly reduced colony number (<xref rid="nihms905237f14" ref-type="fig">Fig. 3a</xref>). In the presence of cetuximab, the miR-100 sponge modestly reduced colony number, whereas the reduction in colony number was more pronounced with the miR-125b sponge and the bicistron sponge (). In the presence of cetuximab, the miR-100 sponge modestly reduced colony number, whereas the reduction in colony number was more pronounced with the miR-125b sponge and the bicistron sponge (<xref rid="nihms905237f14" ref-type="fig">Fig. 3a</xref>). In contrast, opposite effects were observed in CC upon overexpressing miR-100 and miR-125b individually and together. The miR-100/125b bicistron, but not individual miRNAs, increased colony number in CC (). In contrast, opposite effects were observed in CC upon overexpressing miR-100 and miR-125b individually and together. The miR-100/125b bicistron, but not individual miRNAs, increased colony number in CC (<xref rid="nihms905237f14" ref-type="fig">Fig. 3b</xref>). Upon cetuximab treatment, the miR-100/125b bicistron conferred the strongest pro-survival effect; when introduced individually, miR-125b had a greater effect than miR-100 (). Upon cetuximab treatment, the miR-100/125b bicistron conferred the strongest pro-survival effect; when introduced individually, miR-125b had a greater effect than miR-100 (<xref rid="nihms905237f14" ref-type="fig">Fig. 3b</xref>). Similar opposing effects were observed in morphological changes, as well as Ki-67 and cleaved Caspase-3 staining upon expressing the different sponges in CC-CR and the different miRNAs in CC (). Similar opposing effects were observed in morphological changes, as well as Ki-67 and cleaved Caspase-3 staining upon expressing the different sponges in CC-CR and the different miRNAs in CC (<xref rid="nihms905237f14" ref-type="fig">Fig. 3c and d</xref>; ; <xref rid="nihms905237f3" ref-type="fig">Extended Data Fig. 3d and e</xref>). Additionally, overexpression of the miR-100/125b bicistron in Caco-2 cells (low endogenous miR-100/125b expression) rendered cells less responsive to cetuximab, whereas inhibition of the miR-100/125b bicistron in DLD-1 cells and HNSCC SCC25-derived CTX-R7 cells (both with high endogenous miR-100/125b expression) restored cetuximab responsiveness (). Additionally, overexpression of the miR-100/125b bicistron in Caco-2 cells (low endogenous miR-100/125b expression) rendered cells less responsive to cetuximab, whereas inhibition of the miR-100/125b bicistron in DLD-1 cells and HNSCC SCC25-derived CTX-R7 cells (both with high endogenous miR-100/125b expression) restored cetuximab responsiveness (<xref rid="nihms905237f4" ref-type="fig">Extended Data Fig. 4</xref>). Similar results were observed when CC-CR and CC cells with the differing manipulations were established as subcutaneous xenografts in nude mice and treated with cetuximab (). Similar results were observed when CC-CR and CC cells with the differing manipulations were established as subcutaneous xenografts in nude mice and treated with cetuximab (<xref rid="nihms905237f14" ref-type="fig">Fig. 3e and f</xref>; ; <xref rid="nihms905237f5" ref-type="fig">Extended Data Fig. 5</xref>). Together, these results are consistent with a model in which miR-100 and miR-125b cooperate to confer cetuximab resistance.). Together, these results are consistent with a model in which miR-100 and miR-125b cooperate to confer cetuximab resistance.'], 'nihms905237f15': ['We next examined whether miR-100/125b-induced downregulation of these Wnt negative regulators resulted in increased Wnt signaling. Although total β-catenin levels were not significantly altered, CC-CR exhibited increased active tyrosine phosphorylated p-Y489 β-catenin and increased nuclear β-catenin compared to CC in 3D (<xref rid="nihms905237f15" ref-type="fig">Fig. 4e</xref> and and <xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6f</xref>). Consistently, β-catenin was largely confined to the plasma membrane in CC xenografts, whereas it was largely nuclear in CC-CR xenografts (). Consistently, β-catenin was largely confined to the plasma membrane in CC xenografts, whereas it was largely nuclear in CC-CR xenografts (<xref rid="nihms905237f15" ref-type="fig">Fig. 4f</xref>). Moreover, mRNA expression of a panel of Wnt target genes was significantly enriched in CC-CR versus CC (). Moreover, mRNA expression of a panel of Wnt target genes was significantly enriched in CC-CR versus CC (<xref rid="nihms905237f15" ref-type="fig">Fig. 4g</xref>). Cetuximab blocked Wnt3a-induced Wnt activation in CC, but had no obvious effect on Wnt3a-induced Wnt signaling in CC-CR (). Cetuximab blocked Wnt3a-induced Wnt activation in CC, but had no obvious effect on Wnt3a-induced Wnt signaling in CC-CR (<xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6g</xref>). Cetuximab also led to a marked and persistent decrease in Wnt target genes in CC over 48 h, whereas expression of those genes in CC-CR was only modestly decreased at early time points after treatment before rebounding at later time points (). Cetuximab also led to a marked and persistent decrease in Wnt target genes in CC over 48 h, whereas expression of those genes in CC-CR was only modestly decreased at early time points after treatment before rebounding at later time points (<xref rid="nihms905237f6" ref-type="fig">Extended Data Fig. 6h</xref>). Furthermore, nuclear β-catenin levels increased in CC and Caco-2 cells stably overexpressing either miR-100 or miR-125b, and the increase was greater in cells expressing the miR-100/125b bicistron (). Furthermore, nuclear β-catenin levels increased in CC and Caco-2 cells stably overexpressing either miR-100 or miR-125b, and the increase was greater in cells expressing the miR-100/125b bicistron (<xref rid="nihms905237f7" ref-type="fig">Extended Data Fig. 7a</xref>). In contrast, nuclear β-catenin levels were reduced upon overexpressing the miR-100/125b bicistron sponge in CC-CR, DLD-1, and CTX-R7 cells (). In contrast, nuclear β-catenin levels were reduced upon overexpressing the miR-100/125b bicistron sponge in CC-CR, DLD-1, and CTX-R7 cells (<xref rid="nihms905237f7" ref-type="fig">Extended Data Fig. 7a</xref>). Corresponding changes of Wnt target genes were also observed (). Corresponding changes of Wnt target genes were also observed (<xref rid="nihms905237f15" ref-type="fig">Fig. 4h</xref> and and <xref rid="nihms905237f4" ref-type="fig">Extended Data Fig. 4e</xref>). Consistent with these findings, nuclear β-catenin immunoreactivity increased in CC nude mouse xenografts expressing the miR-100/125b bicistron and decreased in their CC-CR counterparts expressing the bicistronic sponge (). Consistent with these findings, nuclear β-catenin immunoreactivity increased in CC nude mouse xenografts expressing the miR-100/125b bicistron and decreased in their CC-CR counterparts expressing the bicistronic sponge (<xref rid="nihms905237f7" ref-type="fig">Extended Data Fig. 7b</xref>).).', '(a, b) Left: CC-CR doxycycline (Dox)-on DKK1 or DKK3 cells were cultured in the presence or absence of Dox (1 μg/ml) and harvested at 48 h. Total cell lysates and conditioned media were harvested and subjected to immunoblot analysis. Right: indicated cells were grown in 3D in normal medium or treated with CTX (3 μg/ml). The resultant colonies were counted after 18 days. n=3 experiments performed in triplicate. **P<0.01 by Student\'s t test. (c) CC-CR cells were grown in 3D in normal medium (CTL), treated with CTX (3 μg/ml) or in combination with recombinant DKK1 (rDKK1) and DKK3 (rDKK3) in 3D every 2 days. The resultant colonies were stained after 18 days for Cleaved Casp-3 (green) and Ki-67 (red). Scale bar, 50 μm. Quantification was shown. n=3 independent experiments. (d) Immunoblots for β-catenin from nuclear and cytoplasmic fractions of indicated cells upon CTX (10 μg/ml) treatment. Loading controls were GAPDH for cytoplasmic fractions and Lamin A/C for nuclear fractions. (e) CC-CR were treated with CTX (3 μg/ml), and/or XAV-939 (1, 5, 10 μM), and/or ICG-001 (1, 2.5, 5 μM) in 3D for 18 days, and colony number was determined. n=3 experiments performed in triplicate. (f) DLD-1 and HCT8 cells were treated with CTX (200 μg/ml) and/or ICG-001 (4 μM) for 14 days in 3D, and colony number was determined. n=2 independent experiments performed in triplicate. (g) Quantification of radiant efficiency from tumors (n=6) represented on <xref rid="nihms905237f15" ref-type="fig">Fig. 4i</xref>. **. **P<0.01 by paired Student\'s t test. (h) Representative IHC images and quantification of Ki-67 and Cleaved Casp-3 from CC-CR xenografts (n=6) treated with control saline (CTL), or CTX (1 mg/mouse, i.p. injection, every 3 days), and/or ICG-001 (150 mg/kg, i.p. injection, daily). Scale bar, 50 μm. *P<0.05, **P<0.01 by one-way ANOVA followed by Dunnett\'s test in c, e, and h, and one-way ANOVA followed by LSD post-hoc test in f. Data represent mean ± s.d. in a-c and e-h. n.s., not significant.'], 'nihms905237f8': ['Based on our findings that Wnt signaling is increased in CC-CR, we hypothesized that cetuximab responsiveness may be restored by suppression of Wnt signaling. Since DKK1 and DKK3 are secreted Wnt antagonists and among the most downregulated genes in CC-CR, we tested whether their overexpression could overcome cetuximab resistance using a doxycycline-inducible lentiviral system22. Although induction of DKK1 or DKK3 resulted in a slight reduction in colony number, this effect was augmented with addition of cetuximab (<xref rid="nihms905237f8" ref-type="fig">Extended Data Fig. 8a and b</xref>). Moreover, administration of recombinant DKK1 and DKK3 enhanced the ability of cetuximab to decrease proliferation and increase apoptosis (). Moreover, administration of recombinant DKK1 and DKK3 enhanced the ability of cetuximab to decrease proliferation and increase apoptosis (<xref rid="nihms905237f8" ref-type="fig">Extended Data Fig. 8c</xref>). Furthermore, nuclear β-catenin expression decreased when DKK1 or DKK3 was inducibly expressed in CC-CR in the presence of cetuximab (). Furthermore, nuclear β-catenin expression decreased when DKK1 or DKK3 was inducibly expressed in CC-CR in the presence of cetuximab (<xref rid="nihms905237f8" ref-type="fig">Extended Data Fig. 8d</xref>). We next tested whether pharmacological inhibition of Wnt activity sensitized CC-CR to cetuximab using a tankyrase inhibitor, XAV-939). We next tested whether pharmacological inhibition of Wnt activity sensitized CC-CR to cetuximab using a tankyrase inhibitor, XAV-93923, and a β-catenin/CBP inhibitor, ICG-00124. Both compounds caused a concentration-dependent reduction in colony number, and cetuximab growth inhibition was enhanced by their addition (<xref rid="nihms905237f8" ref-type="fig">Extended Data Fig. 8e</xref>). ICG-001 also enhanced the growth inhibitory effects of cetuximab in other CRC and HNSCC cell lines with high expression of MIR100HG (). ICG-001 also enhanced the growth inhibitory effects of cetuximab in other CRC and HNSCC cell lines with high expression of MIR100HG (<xref rid="nihms905237f8" ref-type="fig">Extended Data Fig. 8f</xref> and and <xref rid="nihms905237f15" ref-type="fig">4e</xref>). In CC-CR nude mouse xenografts, administration of cetuximab and ICG-001 individually only slowed tumor growth; however, combined treatment resulted in tumor regression (). In CC-CR nude mouse xenografts, administration of cetuximab and ICG-001 individually only slowed tumor growth; however, combined treatment resulted in tumor regression (<xref rid="nihms905237f15" ref-type="fig">Fig. 4i-k</xref>; ; <xref rid="nihms905237f8" ref-type="fig">Extended Data Fig. 8g and h</xref>). Thus, blockade of Wnt signaling, either upstream or downstream of the APC/β-catenin degradation complex, restores cetuximab responsiveness to cetuximab-resistant cells.). Thus, blockade of Wnt signaling, either upstream or downstream of the APC/β-catenin degradation complex, restores cetuximab responsiveness to cetuximab-resistant cells.'], 'nihms905237f16': ['GATA6 is critical for gut endoderm development, and it both promotes and suppresses gastrointestinal and pancreatic neoplasia26-29. We found that MIR100HG expression decreased in cetuximab-treated CC, while GATA6 mRNA progressively increased over 48 h (<xref rid="nihms905237f16" ref-type="fig">Fig. 5d</xref>); however, this phenomenon did not occur in CC-CR (data not shown). GATA6 knockdown in CC (); however, this phenomenon did not occur in CC-CR (data not shown). GATA6 knockdown in CC (<xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9a</xref> and and <xref rid="nihms905237f16" ref-type="fig">Fig. 5e</xref>, top) caused MIR100HG upregulation and its expression no longer decreased upon cetuximab treatment (, top) caused MIR100HG upregulation and its expression no longer decreased upon cetuximab treatment (<xref rid="nihms905237f16" ref-type="fig">Fig. 5e</xref>, bottom), suggesting a repressive effect of GATA6 on MIR100HG. Luciferase reporter assays showed overexpression of GATA6 (, bottom), suggesting a repressive effect of GATA6 on MIR100HG. Luciferase reporter assays showed overexpression of GATA6 (<xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9b</xref>) resulted in a concentration-dependent inhibition of MIR100HG promoter activity () resulted in a concentration-dependent inhibition of MIR100HG promoter activity (<xref rid="nihms905237f16" ref-type="fig">Fig. 5f</xref>). Four putative GATA binding sites (G/A)GATA(A/T) were identified in the MIR100HG promoter region (). Four putative GATA binding sites (G/A)GATA(A/T) were identified in the MIR100HG promoter region (<xref rid="nihms905237f16" ref-type="fig">Fig. 5g</xref>). Sequential deletions and mutations of these binding sites revealed that GATA binding site 2 (-1198 upstream of the TSS) is the major site for GATA6 repression of MIR100HG transcriptional activity (). Sequential deletions and mutations of these binding sites revealed that GATA binding site 2 (-1198 upstream of the TSS) is the major site for GATA6 repression of MIR100HG transcriptional activity (<xref rid="nihms905237f16" ref-type="fig">Fig. 5h</xref>). GATA6 repression of MIR100HG was also validated in HuTu80 cells with low expression of GATA6 and high expression of MIR100HG (). GATA6 repression of MIR100HG was also validated in HuTu80 cells with low expression of GATA6 and high expression of MIR100HG (<xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9c and d</xref>). Chromatin occupancy of GATA6 at GATA-binding site 2 was confirmed by chromatin immunoprecipitation (ChIP) and electromobility shift assay (EMSA) using nuclear extracts from CC cells (). Chromatin occupancy of GATA6 at GATA-binding site 2 was confirmed by chromatin immunoprecipitation (ChIP) and electromobility shift assay (EMSA) using nuclear extracts from CC cells (<xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9e and f</xref>).).', 'Of interest, we found that the 3′ UTR of GATA6 harbors a putative binding site for miR-125b (Extended Data Table 5). In both CC and Caco-2 cells, introduction of miR-125b reduced luciferase activity of the wild-type 3′ UTR reporter construct, but not when the miR-125b site was mutated (<xref rid="nihms905237f16" ref-type="fig">Fig. 5i</xref> and and <xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9g</xref>). As predicted, GATA6 levels were reduced in CC and Caco-2 cells stably expressing miR-125b, and conversely increased in CC-CR and HuTu80 cells expressing the miR-125b sponge (). As predicted, GATA6 levels were reduced in CC and Caco-2 cells stably expressing miR-125b, and conversely increased in CC-CR and HuTu80 cells expressing the miR-125b sponge (<xref rid="nihms905237f16" ref-type="fig">Fig. 5j</xref> and and <xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9h</xref>). Further analysis of the TCGA data repository indicated that GATA6 is significantly downregulated, whereas MIR100HG is significantly upregulated in stage IV CRC patients (). Further analysis of the TCGA data repository indicated that GATA6 is significantly downregulated, whereas MIR100HG is significantly upregulated in stage IV CRC patients (<xref rid="nihms905237f16" ref-type="fig">Fig. 5k</xref>). Also, CRCs with lower quartile expression of GATA6 tend to have higher expression of MIR100HG in the TCGA data repository (). Also, CRCs with lower quartile expression of GATA6 tend to have higher expression of MIR100HG in the TCGA data repository (<xref rid="nihms905237f16" ref-type="fig">Fig. 5k</xref>), as well as in two additional CRC datasets (), as well as in two additional CRC datasets (<xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9i</xref>). Together, these findings suggest a double-negative regulatory circuit between GATA6 and MIR100HG/miR-125b underlies cetuximab resistance.). Together, these findings suggest a double-negative regulatory circuit between GATA6 and MIR100HG/miR-125b underlies cetuximab resistance.'], 'nihms905237f17': ['To examine whether this mode of cetuximab resistance occurs in human CRC, we obtained paired tumor specimens from ten individuals prior to the start of cetuximab treatment and at the time of tumor progression (Extended Data Table 7). KRAS/NRAS/BRAF mutations had been excluded in tumor specimens obtained prior to treatment with cetuximab. qRT-PCR showed that miR-100 and miR-125b were coordinately overexpressed (rs=0.842, P<0.01) in tumors that progressed on treatment compared to pre-treatment levels (P<0.05, <xref rid="nihms905237f17" ref-type="fig">Fig. 6a</xref>). In addition, nuclear β-catenin immunoreactivity was significantly higher in tumors that progressed on cetuximab (). In addition, nuclear β-catenin immunoreactivity was significantly higher in tumors that progressed on cetuximab (<xref rid="nihms905237f17" ref-type="fig">Fig. 6b</xref>). miR-125b expression directly correlated with nuclear β-catenin staining (). miR-125b expression directly correlated with nuclear β-catenin staining (rs=0.636, P<0.05); the correlation between miR-100 expression and nuclear β-catenin staining did not reach statistical significance (rs=0.612, P=0.06). Conversely and consistent with our pre-clinical findings, there was reduced nuclear GATA6 expression in tumors that advanced on cetuximab (<xref rid="nihms905237f17" ref-type="fig">Fig. 6c</xref>). However, we did not find a significant inverse correlation between miR-100 and GATA6 (). However, we did not find a significant inverse correlation between miR-100 and GATA6 (rs=-0.455, P=0.187), or miR-125b and GATA6 (rs=-0.515, P=0.128). By FISH analysis, the MIR100HG, miR-100, and miR-125b signals increased in tumors that progressed on treatment. In these same samples, there was increased β-catenin staining and reduced GATA6 staining (<xref rid="nihms905237f17" ref-type="fig">Fig. 6d</xref>). We excluded ). We excluded MET amplification by FISH in all ten paired specimens and sequenced the post-treatment tumors for mutations in KRAS/NRAS/BRAF (<xref rid="nihms905237f9" ref-type="fig">Extended Data Fig. 9j</xref> and and Extended Data Table 8). NRAS and KRAS mutations were detected in 2 cases, respectively; we confirmed that these were likely acquired events by re-sequencing the pre-treatment DNA. In both cases, MIR100HG and miR-100/125b were increased. In the remaining 8 cases that lacked genetic resistance events, 5 cases exhibited upregulated MIR100HG and miR-100/125b in the tumors post-treatment. These clinical data support our pre-clinical findings and demonstrate that upregulation of MIR100HG and miR-100/125b occur in the setting of acquired cetuximab resistance in CRC patients, and this upregulation may both coincide with and be independent of genetic mutations associated with cetuximab resistance.', 'Cases 1, 2, 3 in <xref rid="nihms905237f17" ref-type="fig">Fig. 6d</xref> denote subject No. 2, 3, 5 in this table. denote subject No. 2, 3, 5 in this table.', 'Cases 1, 2, 3 in <xref rid="nihms905237f17" ref-type="fig">Fig. 6d</xref> denote Subjects No. 2, 3, 5 in this table. denote Subjects No. 2, 3, 5 in this table.'], 'nihms905237f10': ['In summary, we have identified a complex circuitry underlying cetuximab resistance by upregulation of MIR100HG and its embedded miRNAs (see <xref rid="nihms905237f10" ref-type="fig">Extended Data Fig. 10</xref>). miR-100 and miR-125b coordinately activate Wnt signaling by reducing expression of five negative regulators of Wnt signaling. miR-125b reinforces upregulation of MIR100HG by inhibiting GATA6 expression, which normally suppresses MIR100HG. We show that inhibition of Wnt signaling can overcome this mode of cetuximab resistance, underscoring the potential clinical relevance of the interactions between EGFR and Wnt signaling.). miR-100 and miR-125b coordinately activate Wnt signaling by reducing expression of five negative regulators of Wnt signaling. miR-125b reinforces upregulation of MIR100HG by inhibiting GATA6 expression, which normally suppresses MIR100HG. We show that inhibition of Wnt signaling can overcome this mode of cetuximab resistance, underscoring the potential clinical relevance of the interactions between EGFR and Wnt signaling.'], 'nihms905237f14': ['(a) qRT-PCR analysis of MIR100HG, miR-100, and miR-125b expression among the CTX-sensitive head and neck squamous cell carcinoma (HNSCC) cell line SCC25 and its derived CTX-resistant sublines (CTX-R1, R3, R4, R5, R7, and R8) upon continuous exposure to cetuximab, as well as UNC10, a de novo CTX-resistant cell line. n=3 independent experiments performed in triplicate. *P<0.05, **P<0.01 by one-way ANOVA followed by Dunnett\'s test compared with SCC25. (b) qRT-PCR of indicated miRNA expression in CC stably overexpressing miR-100, miR-125b, or Bicistron. (c) qRT-PCR of indicated miRNA expression in CC-CR stably expressing miR-100 sponge (100-Sp), miR-125b sponge (125b-Sp), or bicistron sponge (Bicistron-Sp). Values were normalized to U6 snRNA. n=3 experiments performed in triplicate. **P<0.01 by Student\'s t test. (d, e) Quantification of Ki-67 and Cleaved Casp-3 in <xref rid="nihms905237f14" ref-type="fig">Fig. 3c and d</xref>. n=4 independent experiments. *. n=4 independent experiments. *P<0.05, **P<0.01 by Student\'s t test. Data represent mean ± s.d. n.s., not significant.', '(a, b) Quantification of radiant efficiency from tumors (n=8) represented on <xref rid="nihms905237f14" ref-type="fig">Fig. 3e and f</xref>. **. **P<0.01 by paired Student\'s t test. (c-f) Representative IHC images and quantification of Ki-67 and Cleaved Casp-3 from indicated xenografts (n=8) treated with CTX. Scale bars, 50 μm. **P<0.01 by one-way ANOVA followed by Dunnett\'s test in e and f. Data represent mean ± s.d. in a, b, e, and f. n.s., not significant.']}
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lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/β-catenin signaling
| null |
Nat Med
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1510819200
|
National performance measurement needs clinical data that track the performance of multi disciplinary teams across episodes of care. Clinical registries are ideal platforms for this work due to their capture of structured, specific data across specialties. Because registries collect data at a national level, and registry data are captured in a consistent structure and format within each registry, registry data are useful for measurement and analysis "out of the box". Registry business models are hampered by the cost of collecting data from EHRs and other source systems and abstracting or mapping them to fit registry data models. The National Quality Registry Network (NQRN) has launched Registries on FHIR, an initiative to lower barriers to achieving semantic interoperability between registries and source data systems. In 2017 Registries on FHIR conducted an information gathering campaign to learn where registries want better interoperability, and how to go about improving it.
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[] |
other
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PMC5961502
| null |
15
|
[
"{'Citation': 'PCPI. [Online] [cited 2017 September 20]. Available from: http://www.thepcpi.org.'}",
"{'Citation': 'NQRN. [Online] [cited 2017 September 20]; Available from: http://www.thepcpi.org/programs-initiatives/national-quality-registry-network.'}",
"{'Citation': 'Gliklich R DNLMe., editor. Registries for Evaluating Patient Outcomes: A User’s Guide. Third edition. Two volumes. (Prepared by the Outcome DEcIDE Center [Outcome Sciences, Inc., a Quintiles company] under Contract No. 290 2005 00351 TO7.) MD: Agency for Healthcare Research and Quality; (3rd ed. Rockville) 2014'}",
"{'Citation': 'D. R. PCPI. [Online] 2015. [cited 2017 September 20]. Available from: http://www.thepcpi.org/education/conferences/past-conferences/2015-conference-presentations.'}",
"{'Citation': 'CMS. Quality Payment Program. [Online] 2017. [cited 2017 September 20]. Available from: https://qpp.cms.gov/about/resource-library.'}",
"{'Citation': 'The Pew Charitable Trusts. [Online]. 2016. [cited 2017 September 20]. Available from: http://www.pewtrusts.org/en/research-and-analysis/fact-sheets/2016/11/next-steps-to-encourage-adoption-of-data-standards-for-clinical-registries.'}",
"{'Citation': 'Goossen WT. Detailed clinical models: representing knowledge, data and semantics in healthcare information technology. Healthcare informatics research. 2014;20(3):163–172.', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC4141130'}, {'@IdType': 'pubmed', '#text': '25152829'}]}}",
"{'Citation': 'Health Level Seven International. [Online]. 2017. [cited 2017 September 21]. Available from: http://www.hl7.org/implement/standards/product_brief.cfm?product_id=463.'}",
"{'Citation': 'Health Level Seven International. [Online] [cited 2017 September 21]. Available from: http://www.hl7.org/events/ciic.cfm?ref=nav.'}",
"{'Citation': 'Health Level Seven International. [Online] [cited 2017 September 21]. Available from: http://www.hl7.org/fhir/?ref=learnmore.'}",
"{'Citation': 'Integrating the Healthcare Enterprise. [Online] [cited 2017 September 21]. Available from: https://www.ihe.net/Profiles/'}",
"{'Citation': 'Healthcare Information and Management Systems Society. [Online] 2013. [cited 2017 September 21]. Available from: http://www.himss.org/librarv/interoperability-standards/what-is-interoperability.'}",
"{'Citation': 'American Hospital Association. [Online] 2017. [cited 2017 September 21]. Available from: http://www.aha.org/research/rc/stat-studies/fast-facts.shtml.'}",
"{'Citation': 'Green M. 50 things to know about the EHR market’s top vendors. Becker’s Health IT & CIO Review. 2015 Jul'}",
"{'Citation': 'Birkhead GS KMSN. Uses of electronic health records for public health surveillance to advance public health. Annual review of public health. 2015 Mar;(36):345–59.', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '25581157'}}}"
] |
Nat Med. 2017 Nov 16; 23(11):1331-1341
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NO-CC CODE
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Downregulation of Rac1 weakens the protective effect of Ezrin silence. (A, B) TEER and FITC-BSA permeability changed after Rac1 inhibition. (C) Simultaneous transfection of shEzrin and shRac1 can promote intracellular F-actin remodeling and cortactin redistribution.
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gr9_lrg
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2
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12530f9c242b9facf2809c785429bb3d52f2adcbf014f9710cd023210892f4dd
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gr9_lrg.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
711,
652
] |
[{'image_id': 'gr6_lrg', 'image_file_name': 'gr6_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr6_lrg.jpg', 'caption': 'The effects of the downregulation of Ezrin on the rearrangement of F-actin and cortactin distribution. (A) In control shRNA cells, F-actin was distributed in a uniform, thin filamentous pattern, primarily around the cell periphery. Cortactin was mainly distributed in the cytoplasm and a small part of the membrane. (B) The same situation was found in the cells of shEzrin group. (C) After 2\xa0h of TNF-α stimulation in the control-shRNA group, F-actin gathered and transferred to the cell center; stress fibers were formed in the cytoplasm, and the distribution of cortactin around the cells was also decreased. (D) This phenomenon can be partially reversed by shEzrin.', 'hash': 'e88988d8da4edae44eb112308290be46b9462df3c0e96ca83a546e3c692994fa'}, {'image_id': 'gr2_lrg', 'image_file_name': 'gr2_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr2_lrg.jpg', 'caption': 'TNF-α induces time-dependent changes in the threonine phosphorylation of Ezrin in PMVECs. (A, B) Time-dependent changes in threonine phosphorylation (A) and relative expressions of Ezrin (B) induced by 100\xa0ng/ml TNF-α. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, *p\xa0<\xa00.01 vs. the beginning time.', 'hash': '957cb6ac51869bedf7033609fde30dc82813821c39c5ba13de3b51145240c535'}, {'image_id': 'gr7_lrg', 'image_file_name': 'gr7_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr7_lrg.jpg', 'caption': 'The effect of shEzrin on the distribution of cortactin in cells. (A) TNF-α could reduce the expression of cortactin on the cell membrane, while shEzrin could reverse the effect of TNF-α. The related results were analyzed by Western blot (B).', 'hash': 'd9ac93265bb7b26b1542ca406975b4d48875a3ef5583d290b8fe5944298e9236'}, {'image_id': 'gr3_lrg', 'image_file_name': 'gr3_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr3_lrg.jpg', 'caption': 'TNF-α inactivates Rac1 in a time-dependent manner. (A) Time-dependent decrease in Rac1-GTP induced by 100\xa0ng/ml TNF-α. (B) Relative expressions of Rac1after treating cells with 100\xa0ng/ml TNF-α. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, **p\xa0<\xa00.001 vs. the beginning time.', 'hash': '07f3b0d0e060a8decab421a6245f8c5c98795516440fa5b1c5e3c91bdd879fe6'}, {'image_id': 'gr9_lrg', 'image_file_name': 'gr9_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr9_lrg.jpg', 'caption': 'Downregulation of Rac1 weakens the protective effect of Ezrin silence. (A, B) TEER and FITC-BSA permeability changed after Rac1 inhibition. (C) Simultaneous transfection of shEzrin and shRac1 can promote intracellular F-actin remodeling and cortactin redistribution.', 'hash': '12530f9c242b9facf2809c785429bb3d52f2adcbf014f9710cd023210892f4dd'}, {'image_id': 'gr5_lrg', 'image_file_name': 'gr5_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr5_lrg.jpg', 'caption': 'Ezrin silencing can reduce the increase of PMVECs permeability induced by TNF-α. (A, B, C, D) The expression changes of Ezrin, Moesin and Radixin in PMVECs transfected with shRNA transfection by Western blotting. (E) Ezrin silencing can reduce the decrease of TEER-induced by TNF-α. (F) Ezrin silencing can partially reverse the increase of FITC-BSA permeability induced by TNF-α. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, *p\xa0<\xa00.01, **p\xa0<\xa00.001.', 'hash': 'c9eeebabeb450e50a0dbbbe76c8f43bb77e8ff0a4e5b644040c613b1b111e8b4'}, {'image_id': 'gr1_lrg', 'image_file_name': 'gr1_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr1_lrg.jpg', 'caption': 'TNF-α increases the MVECs permeability in a concentration-and time-dependent manner. (A) PMVECs were treated with mouse TNF-α (0, 1, 10, 100\xa0ng/ml), and the cell activity of each group was measured using a CCK8 kit. (B–D) TEER (B, C) and FITC-BSA (D) analysis after treating PMVECs with TNF-α for 2\xa0h. Results were expressed as mean\xa0±\xa0standard deviation. N\xa0=\xa06, **p\xa0<\xa00.001 vs untreated control.', 'hash': '161e7229fe9095272b760e5157661aeffba1b5d0e4ebd1b81e49727f3625201a'}, {'image_id': 'gr4_lrg', 'image_file_name': 'gr4_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr4_lrg.jpg', 'caption': 'Rac1 inhibition and TNF-α stimulation (100\xa0ng/ml) together increase threonine phosphorylation of Ezrin in PMVECs. (A, B) After the transfection of PMVECs with shRNA targeting Rac1, the expression of Rac1 was significantly inhibited, and relative expression was assessed by Western blotting. (C, D) The expressions of p-Ezrin and normal Ezrin in different groups were assayed by Western Blotting. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, *p\xa0<\xa00.01, **p\xa0<\xa00.001.', 'hash': '9c5c0dad1fee2ae1da66dfc1565e89e074a02d1f94388f6bb2a4eea26e57514e'}, {'image_id': 'gr8_lrg', 'image_file_name': 'gr8_lrg.jpg', 'image_path': '../data/media_files/PMC7525657/gr8_lrg.jpg', 'caption': 'Ezrin inhibition increases the Rac1 activity in PMVECs. (A) O-Me-cAMP can reduce the decrease of TEER induced by TNF-α. (B) O-Me-cAMP can reduce the increase of FITC-BSA permeability induced by TNF-α. (C) Pull-down assay and Western blot were used to analyze the Rac1 activity after TNF-α stimulation and Ezrin inhibition and relative expressions as assessed by Western blotting (D).', 'hash': 'b0e673dac289184349416b3a21fbf15ca7775ba14816dc7aa1ee5e24652e764d'}]
|
{'gr1_lrg': ['In the present study, PMVECs were identified by binding with lectin from BSI (FITC-BSI) staining and cell purity was >90% (Supplemental Fig. S1). We confirmed previous results that TNF-α reduced the TEER of PMVECs in a time-dependent and dose-dependent manner without affecting cell viability (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>A). Next, we measured the TEER of cells in each group under specific intervention conditions. Compared to the control group, 1–100\xa0ng/ml TNF-α decreased the TEER in PMVECs in a time-dependent manner (A). Next, we measured the TEER of cells in each group under specific intervention conditions. Compared to the control group, 1–100\xa0ng/ml TNF-α decreased the TEER in PMVECs in a time-dependent manner (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>B and C). Furthermore, B and C). Furthermore, FITC-BSA flux increased in a concentration-dependent manner (<xref rid="gr1_lrg" ref-type="fig">Fig. 1</xref>D). These data indicated that the TNF-α could damage the barrier and increase the permeability of PMVECs. The most significant effect was observed at 100\xa0ng/ml TNF-α, which was selected for further experiments.D). These data indicated that the TNF-α could damage the barrier and increase the permeability of PMVECs. The most significant effect was observed at 100\xa0ng/ml TNF-α, which was selected for further experiments.Fig. 1TNF-α increases the MVECs permeability in a concentration-and time-dependent manner. (A) PMVECs were treated with mouse TNF-α (0, 1, 10, 100\xa0ng/ml), and the cell activity of each group was measured using a CCK8 kit. (B–D) TEER (B, C) and FITC-BSA (D) analysis after treating PMVECs with TNF-α for 2\xa0h. Results were expressed as mean\xa0±\xa0standard deviation. N\xa0=\xa06, **p\xa0<\xa00.001 vs untreated control.Fig. 1'], 'gr2_lrg': ['To elucidate the effect of TNF-α on phosphorylation of Ezrin at its critical COOH-terminal threonine, phospho-specific Ezrin antibody (Thr567) was utilized to evaluate threonine phosphorylation of Ezrin by Western Blot. Treatment with 100\xa0ng/mL TNF-α induced a significant increase in Ezrin phosphorylation in a time-dependent manner without affecting the total Ezrin expression, which increased after 15\xa0min, reached maximum levels by 2\xa0h, and remained elevated for at least 8\xa0h (<xref rid="gr2_lrg" ref-type="fig">Fig. 2</xref>\n).\n).Fig. 2TNF-α induces time-dependent changes in the threonine phosphorylation of Ezrin in PMVECs. (A, B) Time-dependent changes in threonine phosphorylation (A) and relative expressions of Ezrin (B) induced by 100\xa0ng/ml TNF-α. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, *p\xa0<\xa00.01 vs. the beginning time.Fig. 2'], 'gr3_lrg': ['Rac1 maintains cellular barrier function through a variety of mechanisms. To investigate the effect of TNF-α on Rac1 expression, PMVECs were stimulated with TNF-α (100\xa0ng/ml), and the expression of Rac1 in cells at different time points was determined by western blot. As shown in <xref rid="gr3_lrg" ref-type="fig">Fig. 3</xref>\n, the TNF-α decreased Rac1 activity in a time-dependent manner.\n, the TNF-α decreased Rac1 activity in a time-dependent manner.Fig. 3TNF-α inactivates Rac1 in a time-dependent manner. (A) Time-dependent decrease in Rac1-GTP induced by 100\xa0ng/ml TNF-α. (B) Relative expressions of Rac1after treating cells with 100\xa0ng/ml TNF-α. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, **p\xa0<\xa00.001 vs. the beginning time.Fig. 3'], 'gr4_lrg': ['To explore the signaling mechanism of Ezrin phosphorylation, we used Rac1-specific shRNA (shRac1). The results showed that the threonine phosphorylation of Ezrin was increased after transfecting PMVECs with shRac1 alone. ShRac1 combined with TNF-αsignificantly increased Ezrin threonine phosphorylation in PMVECs (<xref rid="gr4_lrg" ref-type="fig">Fig. 4</xref>\n).\n).Fig. 4Rac1 inhibition and TNF-α stimulation (100\xa0ng/ml) together increase threonine phosphorylation of Ezrin in PMVECs. (A, B) After the transfection of PMVECs with shRNA targeting Rac1, the expression of Rac1 was significantly inhibited, and relative expression was assessed by Western blotting. (C, D) The expressions of p-Ezrin and normal Ezrin in different groups were assayed by Western Blotting. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, *p\xa0<\xa00.01, **p\xa0<\xa00.001.Fig. 4'], 'gr5_lrg': ['The following experiments were used to determine the role of Ezrin in TNF-α-induced RPMVECs barrier destruction. ShRNA targeting Ezrin was used to inhibit the expression of Ezrin (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>A and B) but not the expression of Moesin or Radixin (A and B) but not the expression of Moesin or Radixin (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>C and D), after which changes in TEER and FITC-BSA permeability of cells were measured. The results showed that Ezrin silencing could partially recover the decrease of TEER and the increase of FITC-BSA permeability induced by TNF-α exposure (C and D), after which changes in TEER and FITC-BSA permeability of cells were measured. The results showed that Ezrin silencing could partially recover the decrease of TEER and the increase of FITC-BSA permeability induced by TNF-α exposure (<xref rid="gr5_lrg" ref-type="fig">Fig. 5</xref>E and F), which indicated that Ezrin silencing could reduce the damage of PMVECs barrier induced by TNF-α.E and F), which indicated that Ezrin silencing could reduce the damage of PMVECs barrier induced by TNF-α.Fig. 5Ezrin silencing can reduce the increase of PMVECs permeability induced by TNF-α. (A, B, C, D) The expression changes of Ezrin, Moesin and Radixin in PMVECs transfected with shRNA transfection by Western blotting. (E) Ezrin silencing can reduce the decrease of TEER-induced by TNF-α. (F) Ezrin silencing can partially reverse the increase of FITC-BSA permeability induced by TNF-α. Values were presented as the mean\xa0±\xa0SD. N\xa0=\xa06, *p\xa0<\xa00.01, **p\xa0<\xa00.001.Fig. 5'], 'gr6_lrg': ['The distribution of F-actin in the cortical region and cortactin on the membrane is one of the key factors for maintaining the endothelial barrier. Next, we examined the effect of Ezrin on cytoskeleton rearrangement. As shown in <xref rid="gr6_lrg" ref-type="fig">Fig. 6</xref>\n, after transfecting cells with control-shRNA or shEzrin, F-actin was evenly arranged and distributed all over the cell (more on the cell membrane), while cortactin was mainly found in the cytoplasm. When PMVECs were exposed to TNF-α, a large number of F-actin gathered and formed stress fibers in the cytoplasm, and the level of cortactin on the cell membrane decreased. PMVECs pretreated with shEzrin reversed F-actin rearrangement and cortactin redistribution induced by TNF-α exposure (\n, after transfecting cells with control-shRNA or shEzrin, F-actin was evenly arranged and distributed all over the cell (more on the cell membrane), while cortactin was mainly found in the cytoplasm. When PMVECs were exposed to TNF-α, a large number of F-actin gathered and formed stress fibers in the cytoplasm, and the level of cortactin on the cell membrane decreased. PMVECs pretreated with shEzrin reversed F-actin rearrangement and cortactin redistribution induced by TNF-α exposure (<xref rid="gr7_lrg" ref-type="fig">Fig. 7</xref>\n). These results were consistent with the immunofluorescence analysis. Therefore, we inferred that Ezrin was essential in the destruction of the PMVEC barrier mediated by TNF-α partly by inducing cytoskeleton remodeling.\n). These results were consistent with the immunofluorescence analysis. Therefore, we inferred that Ezrin was essential in the destruction of the PMVEC barrier mediated by TNF-α partly by inducing cytoskeleton remodeling.Fig. 6The effects of the downregulation of Ezrin on the rearrangement of F-actin and cortactin distribution. (A) In control shRNA cells, F-actin was distributed in a uniform, thin filamentous pattern, primarily around the cell periphery. Cortactin was mainly distributed in the cytoplasm and a small part of the membrane. (B) The same situation was found in the cells of shEzrin group. (C) After 2\xa0h of TNF-α stimulation in the control-shRNA group, F-actin gathered and transferred to the cell center; stress fibers were formed in the cytoplasm, and the distribution of cortactin around the cells was also decreased. (D) This phenomenon can be partially reversed by shEzrin.Fig. 6Fig. 7The effect of shEzrin on the distribution of cortactin in cells. (A) TNF-α could reduce the expression of cortactin on the cell membrane, while shEzrin could reverse the effect of TNF-α. The related results were analyzed by Western blot (B).Fig. 7'], 'gr8_lrg': ['Rac1 is an important regulator of actin cytoskeleton dynamics that has a key role in maintaining endothelial integrity. After pre-incubation with O-me-cAMP, a specific agonist of Rac1, we found that O-me-cAMP partially restored the decrease of TEER and the increase of FITC-BSA permeability induced by TNF-α (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>A and B), and decreased the vascular permeability induced by TNF-α. Moreover, a Western blot analysis indicated that Ezrin inhibition promotes the expression of active rac1 and significantly reduces the decrease of rac1-GTP expression induced by TNF-α exposure (A and B), and decreased the vascular permeability induced by TNF-α. Moreover, a Western blot analysis indicated that Ezrin inhibition promotes the expression of active rac1 and significantly reduces the decrease of rac1-GTP expression induced by TNF-α exposure (<xref rid="gr8_lrg" ref-type="fig">Fig. 8</xref>C and D). These results suggested that both TNF-α and Ezrin could inhibit the activity of Rac1.C and D). These results suggested that both TNF-α and Ezrin could inhibit the activity of Rac1.Fig. 8Ezrin inhibition increases the Rac1 activity in PMVECs. (A) O-Me-cAMP can reduce the decrease of TEER induced by TNF-α. (B) O-Me-cAMP can reduce the increase of FITC-BSA permeability induced by TNF-α. (C) Pull-down assay and Western blot were used to analyze the Rac1 activity after TNF-α stimulation and Ezrin inhibition and relative expressions as assessed by Western blotting (D).Fig. 8'], 'gr9_lrg': ['To confirm whether Rac1 activity was directly related to Ezrin-induced barrier injury, we co-transfected RPMVECs with shEzrin and shRac1, then treated with TNF-α, and finally evaluated changes in cell permeability. As shown in <xref rid="gr9_lrg" ref-type="fig">Fig. 9</xref>\n, Ezrin silencing reduced the damage of the PMVEC barrier caused by TNF-α, while the addition of shRac1 could partially resist shEzrin\'s barrier protection function, which was manifested by the decrease of TEER and the increase of FITC-BSA permeability. In addition, shRac1 aggravated the reshaping of the cytoskeleton and the loss of cortex cortactin.\n, Ezrin silencing reduced the damage of the PMVEC barrier caused by TNF-α, while the addition of shRac1 could partially resist shEzrin\'s barrier protection function, which was manifested by the decrease of TEER and the increase of FITC-BSA permeability. In addition, shRac1 aggravated the reshaping of the cytoskeleton and the loss of cortex cortactin.Fig. 9Downregulation of Rac1 weakens the protective effect of Ezrin silence. (A, B) TEER and FITC-BSA permeability changed after Rac1 inhibition. (C) Simultaneous transfection of shEzrin and shRac1 can promote intracellular F-actin remodeling and cortactin redistribution.Fig. 9']}
|
Tumor necrosis factor-α requires Ezrin to regulate the cytoskeleton and cause pulmonary microvascular endothelial barrier damage
|
[
"PMVEC",
"Ezrin",
"Permeability",
"Rac1",
"Cytoskeleton"
] |
Microvasc Res
|
1611993600
|
The COVID-19 pandemic resulted in more than 4.3 million confirmed cases and more than 2,90,000 deaths worldwide. It has also given rise to fears of an imminent economic crisis and recession. Social distance, self-isolation, and travel restrictions have led to a reduction in the workforce across all economical sectors and have led to a loss of many jobs. Schools have closed down, and the need for commodities and manufactured goods has decreased. On the other hand, the need for medical supplies has increased significantly. The food sector is also facing increased demand as a result of panic buying and storing food products. In response to this global outbreak, we summarize the socioeconomic effects of COVID-19 on the various aspects of the world economy. In Malaysia, the COVID-19 epidemic has checked the resilience of the agriculture sector. Especially the Malaysian paddy industry as country imports 30% of its overall consumption from different parts of the world. The real price of rice triplicating for the consumers, which was alarming for nations in this pandemic situation. The Government of Malaysia introduced the National Agrofood Policy 2011-2020 (NAP4) in 2010 as a guidance document for the implementation of agricultural sector development programs and projects in Malaysia. The NAP4 's 10-year term is to be finished by the end of 2020. Several sectors demonstrate substantial success after approximately 8 years of introduction, while the other classes often lag behind the goal and progress quite slowly. Agricultural sector performance is affected by many problems and challenges. In acknowledgment of the poor success of this field, the Ministry of Agriculture and Agri-Based Industry has launched new approaches, policies, and programs that can change the agricultural sector more rapidly. The new direction is aimed at ensuring national food security and boosting farm and revenues. The authorized government agency needs to revise the policy formulation where Malaysia needs to set stages to revolutionize and modernize the rice farming to address the problem faced by the paddy sector in this pandemic situation to adopt GF. In this study, the researcher focuses on the improvisation of the policy to increase the paddy production sustainably.
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other
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PMC7525657
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161
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[
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] |
Microvasc Res. 2021 Jan 30; 133:104093
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NO-CC CODE
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Requirements for RMMAI recruitment and PAR axis remodeling.(a) Quantification of REC114, ANKRD31, MEI4, and IHO1 foci along unsynapsed axes in leptotene/early zygotene spermatocytes. Error bars: means ± SD. Comparisons to wild type are indicated (two-sided Student’s t test): * = p<0.02, ** = p≤10−7, ns = not significant (p>0.05); exact p values are in Data File S2. Representative micrographs of REC114 staining are shown; other proteins are in Extended Data Fig. 6a. Presence of mo-2 associated blobs (arrowheads) is indicated in the bottom panel. Scale bars: 2 μm. (b) Genetic requirements for PAR loop–axis organization (length of REC114 and mo-2 FISH signals along the PAR axis and axis-orthogonal extension of mo-2). Error bars: means ± SD. (c) Representative SIM images of Y-PAR loop–axis structure in each mutant at late zygonema. Scale bar: 1 μm.
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nihms-1587845-f0003
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2
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bcdad53a4b09f65028c7e1038c6aae8a43c0bcb12c3f33b2f45127ed35079400
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nihms-1587845-f0003.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] |
[
"fluorescence microscopy",
"bar plot",
"immunoblot"
] |
[
927,
1050
] |
[{'image_id': 'nihms-1587845-f0004', 'image_file_name': 'nihms-1587845-f0004.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0004.jpg', 'caption': 'PAR-like structural reorganization and DSB formation on autosomal mo-2 arrays.(a) The mo-2 region of chr9 undergoes axis elongation and splitting similar to PARs (SIM image of a wild-type zygotene spermatocyte). Scale bar: 1 μm. (b) ANKRD31 is required for high-level DSB formation in mo-2 regions and XY pairing. Immuno-FISH for RPA2 and mo-2 was used to detect DSBs. Illustration from Extended Data Fig. 7c. (c) PAR-like DSB formation near autosomal mo-2 regions. Excerpt from Extended Data Fig. 8a. SSDS coverage7,20 is shown for the Y PAR (left) and the mo-2-adjacent region of chr9 (right). Positions of mo-2 repeats are shown below. (d) Early pachytene XY oocyte showing bright RPA2 focus in the PAR. Scale bar: 2 μm.', 'hash': '39538d013e119fde1f091891bc1a63a8a58b7e5e382b326e37d459a6291836fe'}, {'image_id': 'nihms-1587845-f0003', 'image_file_name': 'nihms-1587845-f0003.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0003.jpg', 'caption': 'Requirements for RMMAI recruitment and PAR axis remodeling.(a) Quantification of REC114, ANKRD31, MEI4, and IHO1 foci along unsynapsed axes in leptotene/early zygotene spermatocytes. Error bars: means ± SD. Comparisons to wild type are indicated (two-sided Student’s t test): * = p<0.02, ** = p≤10−7, ns = not significant (p>0.05); exact p values are in Data File S2. Representative micrographs of REC114 staining are shown; other proteins are in Extended Data Fig. 6a. Presence of mo-2 associated blobs (arrowheads) is indicated in the bottom panel. Scale bars: 2 μm. (b) Genetic requirements for PAR loop–axis organization (length of REC114 and mo-2 FISH signals along the PAR axis and axis-orthogonal extension of mo-2). Error bars: means ± SD. (c) Representative SIM images of Y-PAR loop–axis structure in each mutant at late zygonema. Scale bar: 1 μm.', 'hash': 'bcdad53a4b09f65028c7e1038c6aae8a43c0bcb12c3f33b2f45127ed35079400'}, {'image_id': 'nihms-1587845-f0012', 'image_file_name': 'nihms-1587845-f0012.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0012.jpg', 'caption': 'DSB maps on the PAR and autosomal mo-2 regions.(a) SSDS sequence coverage (data from refs. 7,20) is shown for the X PAR (shown previously in different form in ref. 20), the Y PAR, and the mo-2-adjacent regions of chr9 and chr13. The dashed segments indicate gaps in the mm10 genome assembly. We did not assess chr4 because available assemblies are too incomplete. (b) Regions adjacent to the mo-2 region on chr9 show SSDS signal that is reproducibly elevated relative to chr9 average in wild-type testis samples but not in maps from Ankrd31−/− testes or wild-type ovaries. Two of the SSDS browser tracks are reproduced from panel a. The bar graph shows enrichment values from individual SSDS maps (T1–T9 are maps from wild-type testes; O1 and O2 are from wild-type ovaries31). Enrichment values are defined as coverage across the indicated coordinates relative to mean coverage for chr9 (see Methods for details). Note that ovary sample O1 and the Ankrd31−/− adult sample are known to have poorer signal:noise ratios than the other samples20,31. For all SSDS coverage tracks, reads mapping to multiple locations are included after random assignment to one of their mapped positions. However, the same conclusions are reached about ANKRD31-dependence and PRDM9-independence of signal on chr9 and chr13 if only uniquely mapped reads are used. (c) Oocytes incur substantially less DSB formation than spermatocytes near the mo-2 region on chr9. SSDS signal is from ref. 31 (samples T1 and O2). The X-PAR is shown for comparison (previously shown to be essentially devoid of DSBs in ovary samples31). See panel b for quantification.', 'hash': 'dbe8c24ea1eb6ff03d86a1446715a7c4e3fbd0aa3183bc8e16b4a3d7c2152f3a'}, {'image_id': 'nihms-1587845-f0002', 'image_file_name': 'nihms-1587845-f0002.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0002.jpg', 'caption': 'Arrays of the mo-2 minisatellite are sites of RMMAI protein enrichment in the PAR and on autosomes.(a) Left panel: Self alignment of the PARb FISH probe. The circled block is a 20-kb mo-2 cluster. Right panel: Schematic showing the non-centromeric chromosome ends identified by BLAST search using the mo-2 consensus sequence. (b) Colocalization of REC114 blobs with mo-2 oligonucleotide FISH signal (zygotene spermatocyte). Scale bar: 2 μm. (c) PAR enrichment for ANKRD31 and RPA2 correlates with mo-2 copy number. Top panels: late zygotene spermatocyte from F1 hybrid from crosses of B6 × MSM. Scale bars: 1 μm. Bottom panels: PAR-associated signals (A.U., arbitrary units) on B6-derived (XB) and MSM-derived chromosomes (YM) from the indicated number of spermatocytes (N). Red lines: means ± SD. Differences between X and Y PAR intensities are significant for both proteins and for mo-2 FISH (p < 10−6, paired t-test; exact two-sided p values are in Data File S1).', 'hash': '5e4be50e4001d8e0f09681be7272e76a0a5eb38623faf155f1376744446e1a14'}, {'image_id': 'nihms-1587845-f0005', 'image_file_name': 'nihms-1587845-f0005.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0005.jpg', 'caption': 'PAR axis thickening and accumulation of RMMAI proteins.(a) Axis thickening (SYCP3 and HORMAD2 staining) on the PAR (arrowhead) in a late zygotene spermatocyte. Scale bar: 2 μm. HORMAD2 staining in the PAR at late zygonema mimics SYCP3 staining in all late zygonema spermatocytes analyzed (N>20) in three mice. (b) Image adapted under Creative Commons CC-BY license from ref.45 showing enrichment of HORMAD1 on the thick PAR axis of the Y chromosome. (c) Colocalization of ANKRD31 and MEI4, REC114, IHO1, and MEI1. Representative zygotene spermatocytes are shown. Arrowheads indicate densely staining blobs. Areas indicated by dashed boxes are shown at higher magnification. The graphs show the total number of foci colocalized in leptotene/zygotene spermatocytes (error bars are mean ± SD). N.D., not determined: The low immunofluorescence signal for MEI1 did not allow us to quantify the colocalization with ANKRD31, although MEI1 showed clear colocalization with ANKRD31 in the blobs and at least some autosomal foci (insets). Scale bars: 2 μm. Underlying data for all graphs are in Data Files S1–4. Further evidence for extensive colocalization with ANKRD31 is documented in separate studies20,21. (d) PARb FISH probe colocalizes with REC114 blobs. Two blobs are on PAR, as judged by chromosome morphology and bright fluorescence in situ hybridization (FISH) with a PAR boundary probe (PARb) and others highlight specific autosome ends. Scale bar: 2 μm. The colocalization between REC114 blobs and PARb FISH signals has been observed in all spermatocytes analyzed (N>60), from pre-leptonema to early pachynema, in more than three mice. (e) ANKRD31, REC114, and MEI1 immunostaining starts to appear in pre-leptonema. Seminiferous tubules were cultured with 5-ethynyl-3-deoxyuridine (EdU) to label replicating cells, then chromosome spreads were stained for SYCP3 and either MEI1 plus REC114 or ANKRD31 plus PARb FISH. Colocalized foci appear in pre-leptonema (EdU-positive cells that are weakly SYCP3-positive), as previously shown for MEI4 and IHO117,22. Because we can already detect ANKRD31 accumulation at sites of PARb-hybridization, we infer that the stronger sites of accumulation of MEI1 and REC114 also include PARs. Scale bars: 2 μm. PARb colocalized with ANKRD31 blobs (top panel) and MEI1 with REC114 (bottom panel) in all pre-leptotene spermatocytes analyzed (N>20) in one mouse. (f) REC114 is not detected in the mo-2 regions in spermatogonia. Seminiferous tubules were cultured with EdU, and chromosome spreads were stained for DMRT1 (a marker of spermatogonia46) and REC114 plus mo-2 FISH. REC114 blobs colocalized with mo-2 FISH signals in the preleptotene spermatocyte (bottom) but were not apparent in the DMRT1-positive spermatogonium (top). Both cells shown were captured in a single microscopic field. Scale bar: 2 μm. Mo-2 FISH signals do not colocalize with REC114 signal in all the spermatogonia analyzed (N>20) in one mouse. (g) Candidate ANKRD31 interacting proteins. To identify other PAR-associated proteins, ANKRD31 was immunoprecipitated from extracts made from whole testes of 12-dpp-old mice using two different polyclonal antibodies. This table shows a subset of proteins that were identified by mass spectrometry in immunoprecipitates from wild-type testes but not from Ankrd31−/−animals, and not from immunoprecipitates using an irrelevant antibody (anti-Cyclin B3). Full results are in Data File S3. LFQ, label-free quantification. REC114, MEI4, and MEI1 were recovered, confirming specificity. REC114 is known to interact directly with ANKRD3120 and MEI4 is a direct partner of REC11416,47. MEI1 colocalizes with ANKRD31 on chromatin (panel c). We also identified ZMYM3 and PTIP. ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) is a component of LSD1-containing transcription repressor complexes48 and has incompletely understood functions in DNA repair in somatic cells49. Mutation of Zmym3 results in adult male infertility from unknown causes50. However, the spermatocyte metaphase I arrest in this mutant50 may be consistent with presence of achiasmate chromosomes, possibly including X and Y. PTIP (Pax transactivation domain interacting protein; also known as PAXIP1) contains multiple BRCT (BRCA1 C-terminal) domains and regulates gene transcription, class switch recombination, and DNA damage responses in somatic cells51–53. Conditional knockout of Ptip causes spermatogenic arrest, but the function of PTIP during meiosis remains unclear54. Neither ZMYM3 nor PTIP was implicated previously in sex chromosome recombination. (h) Enrichment of ZMYM3 (top) and PTIP (bottom) on the PAR. Sex chromosomes of representative early pachytene spermatocytes are shown. Scale bars: 1 μm. ZMYM3 and PTIP were enriched in the PAR in all spermatocytes analyzed (N>20) in three mice. (i) Yeast two-hybrid assays testing interaction of full-length ANKRD31 fused to the Gal4 activating domain (AD) with either full-length PTIP or the C-terminal 191 amino acids of ZMYM3 fused to the Gal4 DNA binding domain (BD). (Full-length ZMYM3 autoactivates in this assay.) DDO (double dropout) medium selects for presence of both the AD and BD vectors (positive control for growth); QDO (quadruple dropout) and QXA (QDO plus X-α-gal and aureobasidin A) media select for a productive two-hybrid interaction at lower and higher stringency, respectively. Image is representative of two experiments using the same yeast strains.', 'hash': 'cab620b9c626c16c3ad5e55c3cb14b2995a02d9ba38f64f2126d7d5882d1d21d'}, {'image_id': 'nihms-1587845-f0014', 'image_file_name': 'nihms-1587845-f0014.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0014.jpg', 'caption': 'Summary of PAR ultrastructure and molecular determinants of axis remodeling and DSB formation.Schematic representation of the meiotic Y chromosome loop/axis structure before X–Y pairing/synapsis at the transition between zygonema and pachynema. The chromosome axis comprises the meiosis-specific axial proteins SYCP2, SYCP3, HORMAD1, and HORMAD2; cohesin subunits (only REC8 is represented); and the RMMAI proteins (REC114, MEI4, MEI1, ANKRD31, and IHO1). On the non-PAR portion of the Y chromosome axis (left), RMMAI protein loading and DSB formation are partly dependent on HORMAD1 and ANKRD31, and strictly dependent on MEI4, REC11419, IHO121, and presumably MEI118. The DNA is organized into large loops, with a low number of axis-associated RMMAI foci. By contrast, in the PAR (right), the hyper-accumulation of RMMAI proteins at mo-2 minisatellites (possibly spreading into adjacent chromatin) promotes the elongation and subsequent splitting of the PAR sister chromatid axes. Short mo-2-containing chromatin loops stretch along this extended PAR axis, increasing the physical distance between the PAR boundary and the distal PAR sequences, including the telomere. The degree of RMMAI protein loading, PAR axis differentiation, and DSB formation are proportional to the mo-2 FISH signal (which we interpret as reflecting mo-2 copy number), and depend on MEI4, ANKRD31, and presumably REC114.', 'hash': 'a5062ecf41e8cc4377613907e7580d009ab7042e9e4c0b329be5f100640e0835'}, {'image_id': 'nihms-1587845-f0013', 'image_file_name': 'nihms-1587845-f0013.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0013.jpg', 'caption': 'RMMAI accumulation and low-level DSB formation on mo-2 regions in oocytes.(a) Examples of zygotene oocytes showing the colocalization between blobs of IHO1 and REC114, MEI4 and MEI1, or ANKRD31 and mo-2 FISH signal (arrowheads). Scale bars: 2μm. RMMAI blobs colocalized with mo-2 FISH signals in all zygotene oocytes analyzed (N>30) from at least three mice. (b) PAR ultrastructure in oocytes, quantified as in Extended Data Fig. 3b. Late zygotene cells with PAR synapsis are compiled separately from other zygotene cells. Error bars: means ± SD. Scale bar: 1 μm. (c) Examples of zygotene oocytes showing colocalization of ANKRD31 blobs with enrichment for heterochromatin factors. Scale bars: 2 μm. ANKRD31 colocalized with heterochromatin factors blobs in all zygotene oocytes analyzed (N>20) from one mouse. (d) Representative SIM image of a wild-type late zygotene oocyte showing neither detectable splitting of the PAR axis nor REC8 enrichment. Scale bar: 2 μm. The absence of spermatocyte-like differentiation of the PAR axis was observed (N>30 zygotene oocytes) in more than three mice. A modest degree of differentiation was observed in a minority of oocytes (5/45) analyzed by SIM, but this did not resemble the typical PAR axis splitting found in spermatocytes. (e) Prolonged asynapsis does not allow axis splitting to occur in oocytes. Because synapsis appears sufficient to trigger collapse of PAR ultrastructure in spermatocytes (Extended Data Fig. 3b), we asked if preventing synapsis (i.e., in a Syce1−/− mutant) could reveal a cryptic tendency toward axis splitting in oocytes. However, whereas axis splitting was clearly observed by SIM in Syce1−/− mutant spermatocytes, PAR axes were not detectably split in oocytes. Scale bars: 2 μm for main micrograph, 1 μm for insets. Axis splitting of chr9 was observed by SIM in multiple (N>20) Syce1−/− spermatocytes from three different mice. The chr13 or chr4 centromere-distal axes were also occasionally seen to be split, but we did not quantify this for these chromosomes. In males, the differentiation of the PAR or the chr9 axes becomes hardly detectable at later stages in some pachytene-like spermatocytes as cells enter apoptosis, similar to Spo11−/− or Hormad1−/− mice. However, in Syce1−/− oocytes, no significant axis differentiation or splitting was observed by conventional microscopy or by SIM in multiple spermatocytes (N>30) from three different mice, similar to what we observed in wild-type oocytes. (f,h) Delaying synapsis promotes PAR DSB formation in oocytes. Top panels: representative micrographs of pachytene XY (f) and Syce1−/− XX oocytes (h). Middle panels: RPA2 fluorescence intensity at the border of mo-2 FISH signals from PAR, chr9, and chr13. Bottom panels: Percentage of oocytes with RPA2 focus colocalizing with mo-2 regions on PAR, chr9, and chr13. Graphs show data only for pachytene oocytes in which PARs are synapsed (two mice of each genotype). Error bars: means ± SD. Scale bars: 2 μm. (g) Percentage of pachytene oocytes with one or more RPA2 foci colocalizing with mo-2 FISH signal from PAR, chr9 and chr13 in XY pachytene oocytes that had unsynapsed X and Y chromosomes. Scale bar: 2 μm, inset: 1 μm.', 'hash': 'b6496b4c6fb7427ceb3ae0970ffeb5c01cd2db86a8bc439b265b690a7355d147'}, {'image_id': 'nihms-1587845-f0010', 'image_file_name': 'nihms-1587845-f0010.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0010.jpg', 'caption': 'Genetic requirements for RMMAI assembly on chromosomes and for PAR loop–axis organization.(a) Representative micrographs of ANKRD31, MEI4, IHO1 and MEI1 staining in wild type and the indicated mutants (quantification is in Fig. 3a). Scale bars: 2 μm. (b) Measurements of PAR loop–axis organization, as in Fig. 3b, on two additional males. Data from mouse 1 are reproduced from Fig. 3b to facilitate comparison. Means of each measurement for each mouse at each stage are given below, along with the means across all three mice. Means are rounded to two significant figures; the grand means were calculated using unrounded values from individual mice. The number of cells of each stage from each mouse is given (N). (c) REC8 is dispensable for splitting apart of PAR sister chromatid axes, but is required to maintain the connection between sisters at the distal tip of the chromosome. A representative SIM image is shown of a Y chromosome from a late zygotene Rec8−/− spermatocyte. The SYCP3-labeled axes adopt an open-fork configuration. Note that the distal FISH probe (PARd) shows that there are clearly disjoined sisters whereas the PAR boundary (PARb) shows only a single compact signal comparable to wild type. The disposition of the probes and SYCP3 further rules out the crozier configuration as an explanation for split PAR axes. Scale bar: 1 μm. The Y or X PAR structure was resolved by SIM as “fork-shaped” in all spermatocytes analyzed (N>20) from three mice. (d) Quantification of REC114 and MEI4 foci in two additional pairs of wild-type and Ankrd31−/− mice. Horizontal lines indicate means. Fewer foci were observed in the Ankrd31−/− mutant (two-sided Student’s t tests for each comparison of mutant to wild type: p = 5.6 × 10−6 (2nd set, REC114); p = 1.1 × 10−5 (2nd set, MEI4); p = 2.1 × 10−6 (3rd set, REC114); p = 0.017 (3rd, MEI4)). (e) Reduced REC114-staining intensity of axis-associated foci in Ankrd31−/− mutants. To rigorously control for slide-to-slide and within-slide variation in immunostaining, we mixed together wild-type and Ankrd31−/− testis cell suspensions before preparing chromosome spreads. A representative image is shown of a region from a single microscopic field containing two wild-type zygotene spermatocytes (left) and two Ankrd31−/− spermatocytes of equivalent stage (right). Note the diminished intensity of REC114 foci in the Ankrd31−/− spermatocytes. Scale bar: 2 μm. REC114 (non-blob) foci showed lower fluorescence intensity in Ankrd31−/− compared to wild type in all pairs of spermatocytes captured in the same imaging field (N=8 pairs), from one pair of mice. (f) PAR enrichment of heterochromatin-associated factors is independent of ANKRD31. Representative images of the Y chromosome at late zygonema/early pachynema showing colocalization between the decompacted mo-2 chromatin and the indicated proteins. Note that both the FISH and immunofluorescence signals are localized mostly off the axis. Compare with the same signals in absence of SPO11 (Extended Data Fig. 5d). Scale bar: 1 μm. Mo-2 FISH signal colocalized off the axis with the heterochromatin factors in Ankrd31−/− mice in all spermatocytes analyzed (N>30) in more than three mice for CHD3/4 and at least one mouse for ATRX, HP1β, HP1γ, macroH2A1.2, H3K9me3, and H4K20me3.', 'hash': '35f0ff25eb0859e1a27bcc5443cee420d455959340f44b21bb6a3c06048a5f05'}, {'image_id': 'nihms-1587845-f0008', 'image_file_name': 'nihms-1587845-f0008.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0008.jpg', 'caption': 'RMMAI enrichment at mo-2 minisatellite arrays in the PAR and on specific autosomes.(a) Top panel: Self alignment of the PARb FISH probe (reproduced from Fig. 2a). The circled block is a 20-kb mo-2 cluster. Bottom panel: Schematic depicting the last 1.4 Mb of the non-centromeric ends of the indicated chromosomes, showing the positions of mo-2 repeats (green) adjacent to assembly gaps (mm10); mo-2 repeats were identified by BLAST search using the mo-2 consensus sequence. Mo-2 repeats also appear at the distal end of chr4 in the Celera assembly (Mm_Celera, 2009/03/04). PARb and PARd BAC clones are indicated. (b) Confirmation that autosomal mo-2 FISH signals match the chromosomal locations indicated by mm10 or Celera genome assemblies. FISH was performed using an oligonucleotide probe containing the mo-2 consensus sequence in combination with BAC probes for adjacent segments of chromosomes 13, 9 and 4, as indicated. Magenta arrows point to concordant FISH signals. The chr9 BAC probe also hybridizes to the PAR. Scale bars: 2μm. The colocalization of mo-2 and the three autosomal FISH signals was observed in two mice (N>20 spermatocytes). (c) Comparison of mo-2 FISH with REC114 localization relative to the PAR boundary (PARb FISH probe) and the distal PAR (PARd probe). In mid zygonema, the mo-2 FISH signal colocalizes well with REC114 staining in between the PARb and PARd FISH signals. In late zygonema, mo-2 and REC114 are similar to one another and are elongated along the thickened SYCP3 staining of the PAR axis. From early to mid pachynema, REC114 progressively disappears, whereas the mo-2 FISH signal becomes largely extended away from the PAR axes. Note that the relative positions of the PARb and PARd probes reinforce the conclusion that the PAR does not adopt a crozier configuration. Scale bar: 1 μm. The different positioning of PARb and PARd FISH signals compared to mo-2 or REC114 signals was observed in more than 30 spermatocytes in at least three mice. (d) Illustration of the compact organization of the PAR chromatin (mo-2 FISH signal) compared to a whole-Y-chromosome paint probe. Scale bar: 2 μm. The costaining of mo-2 and full chrY probe was evaluated in one mouse (N>20 spermatocytes). (e) Lower mo-2 copy number in the M. m. molossinus subspecies correlates with lower REC114 staining in mo-2 regions. The left panels compare MSM and B6 mice for the colocalization between REC114 immunostaining and mo-2 FISH in leptotene spermatocytes. The REC114 and SYCP3 channels are shown at equivalent exposure for the two strains, whereas a longer exposure is shown for the mo-2 FISH signal in the MSM spermatocyte. Note that the mo-2-associated REC114 blobs are much brighter relative to the smaller dispersed REC114 foci in the B6 spermatocyte than in MSM. The right panel shows representative pachytene spermatocytes to confirm the locations of mo-2 clusters at autosome ends and the PAR in the MSM background. Scale bars: 2 μm. The lower intensity of REC114 blobs in MSM compared to B6 was observed in N>30 spermatocytes in three different pairs of mice. (f) PAR enrichment for ANKRD31 and RPA2 correlates with mo-2 copy number. Top panel: late zygotene spermatocytes from MSM x B6 F1 hybrid. Scale bar: 1 μm. Bottom panel: PAR-associated signals (A.U., arbitrary units) on B6-derived (YB) and MSM-derived chromosomes (XM) from the indicated number of spermatocytes (N). Red lines: means ± SD. Differences between X and Y PAR intensities are significant for both proteins and for mo-2 FISH in both F1 hybrids (p < 10−13, paired t-test; exact two-sided p values are in Data File S5). (g) Representative micrographs of late zygotene spermatocytes from reciprocal F1 hybrid males from crosses of B6 (high mo-2 copy number) and MSM (low mo-2 copy number) parents. Scale bar: 1 μm. (h) Frequency of paired X and Y at late zygonema and mid pachynema analyzed in three MSM and three B6 males. Differences between strains were not statistically significant at either stage (p = 0.241 for late zygonema and p = 0.136 for mid pachynema; two-sided Student’s t test). Note also that MSM X and Y are late-pairing chromosomes, as in the B6 background. The similar pairing kinetics indicates that the lower intensity of RMMAI staining on the MSM PAR is not attributable to earlier PAR pairing and synapsis in this strain. The number of spermatocytes analyzed is indicated (N).', 'hash': 'c971ef64f3d70e7e064fd4c32080b2270cfb80dc8259e7d426ab58149fbabe73'}, {'image_id': 'nihms-1587845-f0006', 'image_file_name': 'nihms-1587845-f0006.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0006.jpg', 'caption': 'PAR ultrastructure.(a) Comparison of conventional microscopy and SIM, showing that the thickened PAR axis in conventional microscopy is resolved as separated axial cores (arrowheads). Scale bars: 2 μm. The thickening of the PAR axis in conventional microscopy and the splitting of the PAR axis in SIM was observed in more than 60 spermatocytes at late zygonema in at least three mice. (b) Ultrastructure of axis proteins SYCP2, SYCP3, and HORMAD2 in the PAR. Scale bars: 1 μm. SYCP2 (left) and HORMAD2 (right) staining mimic SYCP3 staining in late zygonema by conventional microscopy in all cells analyzed (N>30) in at least three mice, and by SIM (N=5, one mouse) (except that HORMAD2 appears rather depleted at the telomeres compared to SYCP3 and SYCP2). (c-d) Ruling out a crozier configuration. In principle, sister chromatid axes could be split apart or the PAR could adopt a crozier configuration in which a single conjoined axis for both sister chromatids is folded back on itself. A crozier (cartooned in c) was ruled out because the telomere binding protein TRF155 decorates the tip of the PAR bubble (d) and FISH signal for the PARb probe is arrayed relatively symmetrically on both axial cores (e), consistent with separated sister chromatid axes (a bubble configuration). Scale bars: 1 μm. We conclude that each axis is a sister chromatid, with a “bubble” from near the PAR boundary almost to the telomere. The presence of TRF1 at the distal tip of the PAR was observed in all spermatocytes analyzed, in one mouse (by conventional microscopy, N>20; by SIM, N=3). PARb FISH signals were relatively symmetrically arranged along the split PAR axes (by conventional microscopy, N>100 in at least three mice, or by SIM, N=9 in three mice). (f) Schematic of PAR ultrastructure and distribution of axis and RMMAI proteins at late zygonema. (g, h) Paired PARs with elongated and split axes occur in late zygonema to early pachynema. Shown are electron micrographs adapted with permission from ref.56 in comparison with SIM immunofluorescence images of spermatocytes at early pachynema (panel g) or late zygonema (panel h; cyan arrowheads indicate examples of incomplete autosomal synapsis). The spermatocytes in the electron micrographs were originally considered to be in mid-to-late pachynema56. However, in our SIM experiments, we can only detect this structure (paired X and Y with elongated and split axes, resembling a crocodile’s jaws) around the zygotene-to-pachytene transition, when RMMAI proteins are still highly abundant on the PAR axes, and when most or all autosomes are completely synapsed. Moreover, other published electron micrographs from mid-to-late pachytene spermatocytes show diagnostic ultrastructural features that are not present in the electron micrographs reproduced here, including a short PAR axis length, multi-stranded stretches of axis on non-PAR portions of the X and Y chromosomes with excrescence of axial elements, and a clear thickening of autosomal telomeres15,57. These observations allow us to conclude definitively that the elongation and splitting of PAR axes are a hallmark of cells from late zygonema into early pachynema. Scale bars in SIM images: 1 μm in panel g, 2 μm in panel h. Extended and split PAR axes were observed by SIM (N>30 spermatocytes) around the zygonema-pachynema transition in more than three mice. (i) REC114 enrichment and axis splitting occurs in the absence of SPO11, thus neither is provoked by DSB formation. Scale bar: 1 μm. PAR axis splitting and extension of the RMMAI signal were observed by SIM in Spo11−/− mice in more than 20 late zygotene-like spermatocytes in more than three mice. The differentiation of the PAR axis became hardly detectable at later stages in some pachytene-like spermatocytes as cells entered apoptosis.', 'hash': '2f570da949b4ad6ab35ff1bf778ef2b961377c7ed931a78fa45bf77a888e7694'}, {'image_id': 'nihms-1587845-f0001', 'image_file_name': 'nihms-1587845-f0001.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0001.jpg', 'caption': 'Ultrastructure of the PAR during male meiosis.(a) Axis thickening (SYCP2 and SYCP3) and ANKRD31 accumulation on X and Y PARs (arrowheads) in late zygonema. The asterisk shows an autosomal ANKRD31 blob. Scale bar: 2 μm. (b) Ultrastructure of the PAR before and after synapsis (montage of representative SIM images). Dashed lines indicate where chromosomes are cropped. SIM: Structured Illumination Microscopy. Scale bar: 1 μm. (c) RMMAI enrichment along split PAR axes in late zygonema. Scale bar: 1 μm. (d) Schematic showing the dynamic remodeling of the PAR loop–axis ensemble during prophase I. See measurements in Extended Data Fig. 3b. Scale bar: 1 μm.', 'hash': 'baab8578a70265a220e817976f5a992f67dea8f8e1d5116460dc3415cbcaf166'}, {'image_id': 'nihms-1587845-f0011', 'image_file_name': 'nihms-1587845-f0011.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0011.jpg', 'caption': 'PAR-associated RPA2 foci.(a) Loop-axis organization of the mo-2 region of chr9 in late zygonema. Compare with the PAR (Fig. 3b). Scale bars: 1 μm. Error bars: means ± SD. (b) Low mo-2 copy number correlates with less loop–axis reorganization (SIM images of late-zygotene F1-hybrid spermatocytes). Scale bars: 1 μm. The differentiation of the B6 PAR was observed in both hybrids B6 × MSM and MSM × B6 in 3 and 4 spermatocytes, respectively by SIM (1 mouse for each) and in more than 20 spermatocytes by conventional microscopy in two mice of each genotype. (c,d,e) Immuno-FISH for RPA2 and mo-2 was used to detect DSBs cytologically in wild type and the indicated mutants. To analyze Rec8 and Hormad1 mutations, we compared to mutants lacking SYCE1 (a synaptonemal complex central element component62) because Syce1−/− mutants show similar meiotic progression defects without defective RMMAI recruitment. Panel c shows representative images. Scale bars: 2 μm, inset 1 μm. Panel d shows the global counts of RPA2 foci for zygotene (zyg) or zygotene-like cells and for pachytene (pach) or pachytene-like cells. Panel e shows, for each cell, the fraction of mo-2 regions that had a colocalized RPA2 focus. Red lines: means ± SD. Statistical significance is indicated in panels c and d for comparisons (two-sided Student’s t tests) of wild type to Ankrd31−/− or of Syce1−/− to either Rec8−/− or Hormad1−/− for matched stages. Exact p values are in Data File S7. Note that the number of discretely scorable mo-2 regions in panel e varied from cell to cell depending on pairing status. (f) Frequent DSB formation at mo-2 regions in the PARs and on autosomes does not require HORMAD1. Micrograph at left shows two adjacent spermatocytes (boundary indicated by dashed line). Scale bar: 2 μm. Insets at right show higher magnification views of the numbered mo-2 regions, all of which are associated with RPA2 immunostaining of varying intensity. This picture illustrates the preferential RPA2 focus formation in mo-2 regions in a Hormad1−/− mouse; quantification is in panel e. (g) Autosomal mo-2 regions often form DSBs late. Immuno-FISH for RPA2, mo-2, and PARb was used to detect DSBs cytologically in wild type from leptonema to mid-pachynema, and to distinguish the X or Y PAR from chromosomes 9 and 13. Chr4 was not assayed because the mo-2 FISH signal was often barely detectable. The top panel shows the global number of RPA2 foci per cell. Black lines are means ± SD. The bottom panel shows the percentage of spermatocytes with an RPA2 focus overlapping the PAR (X, Y, or both) or overlapping chr9 or chr13. A representative image of an early pachytene spermatocyte is shown. Note that, as previously shown for the PAR2, autosomal mo-2 regions continue to accumulate RPA2 foci beyond the time when global RPA2 foci have largely or completely ceased accumulating. Scale bar: 2 μm. (h) X–Y pairing status, quantified by immuno-FISH for SYCP3 and the PARd probe. (i) Montage of SIM images from a B6 male showing that multiple, distinct RPA2 foci can be detected from late zygonema to mid pachynema, suggesting that multiple PAR DSBs can be formed during one meiosis (see also ref. 2 for further discussion). Scale bar: 1μm. The presence of multiple RPA2 foci in the PAR was observed by SIM in more than 20 spermatocytes from late zygonema to mid pachynema in one mouse. (j) Percentage of spermatocytes at the zygotene-pachytene transition with no (0), 1, 2 or 3 distinguishable RPA2 foci on the unsynapsed Y chromosome PAR of MSM and B6 mice. The difference between the strains is statistically significant (negative binomial regression, p = 7.2 × 10−5). N indicates the number of spermatocytes analyzed. A representative picture is shown for each genotype, with one RPA2 focus on the MSM PAR and two apparent sites of RPA2 accumulation on the B6 PAR. The detection of multiple foci is consistent with reported double crossovers6. Scale bar: 1 μm.', 'hash': '66f9a76c1bde89c79f94af1c6a8dbf6373091369362c9fee5db1095978c47d13'}, {'image_id': 'nihms-1587845-f0007', 'image_file_name': 'nihms-1587845-f0007.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0007.jpg', 'caption': 'Time course of the spatial organization of the PAR loop–axis ensemble.(a) Time course of REC8 and ANKRD31 immunostaining along the PAR axis from pre-leptonema (preL, left) to mid pachynema (right). A montage of representative SIM images is shown. Chromosomes a–e are presumptive X or Y, but could be the distal end of chr9. Chromosomes at later stages were unambiguously identified by morphology. Chromosomes i–k show examples where the initial pairing (probably synaptic) contact between X and Y is (i) centromere-proximal (that is, closer to the PAR boundary), (k) distal (closer to the telomere), or (j) interstitial. Scale bar: 1 μm. The preferential enrichment of REC8 at the border of the PAR split axes was observed in more than 30 zygotene spermatocytes by SIM in more than three mice. (b) We collected three measurements of conventional immuno-FISH images from leptonema through mid-pachynema: length of the REC114 signal along the PAR axis; maximal distance from the PARb FISH signal to the distal end of the SYCP3-defined axis; and axis-orthogonal extension of FISH signal for the PARb probe (a proxy for loop sizes). Data were collected on three males. Insets show examples of each type of measurement at each stage. Horizontal black lines indicate means. Means of each measurement for each mouse at each stage are given below, along with the means across all three mice. Means are rounded to two significant figures; the grand means were calculated using unrounded values from individual mice. The number of cells of each stage from each mouse is given (N). Modest variability in the apparent dimensions of the Y chromosome PAR between different mice may be attributable to variation in copy number of mo-2 and other repeats because of unequal exchange during meiosis. Nonetheless, highly similar changes in spatial organization over time in prophase were observed in all mice examined, namely progressive elongation then shortening of axes and concomitant lengthening of loops. Scale bar: 1 μm.Briefly, panels a and b show the following. At pre-leptonema, ANKRD31 blobs had a closely juxtaposed focus of the meiotic cohesin subunit REC8 (chromosome a). In leptonema and early zygonema, ANKRD31 and REC114 signals stretched along the presumptive PAR axes, with REC8 restricted to the borders (panel a, chromosomes b–e). The SYCP3-defined axis was already long as soon as it was detectable (0.73 μm) and the PARb FISH signal was compact (0.52 μm) (panel bi). At late zygonema, the PAR axis had lengthened still further (1.0 μm), while the PARb signal remained compact (panel bii). The PAR split into separate axes during this stage, each with abundant RMMAI (panel a, chromosomes f–h). The split was a REC8-poor zone bounded by REC8 foci (panel a, chromosomes f–h and Extended Data Fig. 2f). After synapsis, axes shortened and chromatin loops decompacted, with concomitant RMMAI dissociation. As cells transitioned into early pachynema and the X and Y PARs synapsed (panel a, chromosomes i–m), the PAR axes began to shorten slightly (0.85 μm) while the PARb signal expanded (0.85 μm) (panel biii). Meanwhile, the elongated ANKRD31 signals progressively decreased in intensity, collapsed along with the shortening axes, and separated from the axis while remaining nearby (panel a, chromosomes l–m). By mid-pachynema, PAR axes collapsed still further, to about half their zygotene length (0.50 μm) and the PARb chromatin expanded to more than twice the zygotene measurement (1.3 μm). ANKRD31 and REC114 enrichment largely disappeared, leaving behind a bright bolus of REC8 on the short remaining axis (panel a, chromosomes n–o and panel biv).(c) Non-homologous synapsis appears sufficient to trigger collapse of the PAR loop-axis structure. We measured REC114 signal length along the PAR axis and extension of mo-2 chromatin orthogonal to the axis in Spo11−/− spermatocytes in which the X PAR had non-homologously synapsed with an autosome while the Y PAR remained unsynapsed. Within any given cell, the unsynapsed Y PAR maintained the characteristic late zygotene configuration (long axis, short loops) whereas the synapsed X PAR adopted the configuration characteristic of mid-pachynema (short axis, long loops). Error bars are mean ± SD. Scale bar: 2μm. We do not exclude that DSB formation without synapsis may also be sufficient (Supplementary Discussion).', 'hash': 'ddb8a711ad217b0f8fbde0d30713458d3238c0e9500eca3e6dae3d4332d7717c'}, {'image_id': 'nihms-1587845-f0009', 'image_file_name': 'nihms-1587845-f0009.jpg', 'image_path': '../data/media_files/PMC7337327/nihms-1587845-f0009.jpg', 'caption': 'Mo-2 regions accumulate heterochromatin factors.(a) Costaining of ANKRD31 or mo-2 with the indicated proteins and histone marks known to localize at the pericentromeric heterochromatin (mouse major satellite), in zygotene spermatocytes (left) and pre-leptotene spermatocytes (right). Each of the heterochromatin factors shows locally enriched signal coincident with mo-2 regions (arrowheads), in addition to broader staining of other sub-nuclear regions. Scale bars: 2 μm. The CHD3/4 antibody recognizes both proteins58. The colocalization of ANKRD31 blobs with heterochromatin blobs was observed in all zygotene spermatocytes analyzed (N>20) in at least three mice for each antibody (left panel) and in one mouse for pre-leptotene spermatocytes (N>10) for each antibody (right panel). (b) CHD3/4, ATRX, HP1β, H4K20me3, H3K9me3 and macroH2A1.2 are not detectably enriched at mo-2 regions in spermatogonia (small, DMRT1-positive cells). These factors may be present at mo-2 regions in these cells, but do not appear to accumulate to elevated levels. Scale bars: 2 μm. The absence of colocalization between mo-2 FISH signals and heterochromatin factors was noted in all spermatogonia analyzed (N>30) from one mouse. (c) Heterochromatin factors can be detected in the PAR up to late pachynema. Each of the assayed proteins and histone marks showed staining on the autosomal and X-specific pericentromeric heterochromatin, the sex body, and euchromatin, albeit with variations between sites in the timing and level of accumulation. Importantly, however, they also showed enriched staining at all mo-2 regions up to early/mid-pachynema, as shown for H4K20me3 (top panel). By mid-to-late pachynema, as shown for H3K9me3 here, the signal persisted in the PAR but was usually barely detectable on chr9 or chr13 mo-2 regions. This observation indicates that, at least for the PAR, the heterochromatin factors can continue to be enriched on mo-2 chromatin after RMMAI proteins have dissociated. These results substantially extend previous observations about CHD3/4 colocalizing with PAR FISH signals; H4K20me3 being localized in the PAR and other chromosome ends; and H3K9me3, HP1β and macroH2A1.2 detection in the PAR in late pachynema58–61. Scale bars: 2 μm. The colocalization between Maj sat and H4K20me3 and H3K9me3 was observed in all spermatocytes analyzed (N>20) in one mouse. The colocalization between H4K20me3 and mo-2 FISH signals was observed in all spermatocytes analyzed (N>60), from preleptotene to mid pachytene in more than three mice. (d) Enrichment of the heterochromatin factors is independent of SPO11. Representative images of Y chromosomes from a Spo11−/− mouse are shown. Scale bar: 1 μm. The colocalization between PAR mo-2 FISH signals and heterochromatin factors was observed in all Spo11−/− spermatocytes analyzed (N>30) in more than three mice for CHD3/4 and at least one mouse each for ATRX, HP1β, HP1γ, macroH2A1.2, H3K9me3, and H4K20me3.', 'hash': 'a67e58b36d7c76eb6e6a03af1b267fbda874145ad9bd56ca9f9795a44529aca1'}]
|
{'nihms-1587845-f0001': ['X and Y usually pair late, with PARs paired in less than 20% of spermatocytes at late zygonema when most autosomes are paired2,14. At this stage, unsynapsed PAR axes (SYCP2/3) appeared thickened relative to other unsynapsed axes and had bright HORMAD1/2 staining (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1a</xref> and and <xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1a</xref>,,<xref rid="nihms-1587845-f0005" ref-type="fig">b</xref>))15. Moreover, the PAR was highly enriched for REC114, MEI4, MEI1, and IHO1—essential for genome-wide DSB formation16–19—plus ANKRD31, a REC114 partner essential for PAR DSBs20,21.', 'All five proteins (RMMAI) colocalized in several bright “blobs” for most of prophase I (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1a</xref> and and <xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1c</xref>). Two blobs were on X and Y PARs and others highlighted specific autosome ends (). Two blobs were on X and Y PARs and others highlighted specific autosome ends (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1a</xref>, , <xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1d</xref>), revisited below. Similar blobs in published micrographs were uncharacterized), revisited below. Similar blobs in published micrographs were uncharacterized16,17,19,22. The proteins also colocalized in smaller foci along unsynapsed axes16,17,19–22 (<xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1c</xref>). Enrichment on the PAR was already detectable in pre-leptonema (). Enrichment on the PAR was already detectable in pre-leptonema (<xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1e</xref>))17,22 but not in spermatogonia (<xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1f</xref>). Mass spectrometry of testis immunoprecipitates identified ZMYM3 and PTIP as new ANKRD31 interactors also enriched on the PAR (). Mass spectrometry of testis immunoprecipitates identified ZMYM3 and PTIP as new ANKRD31 interactors also enriched on the PAR (<xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1g</xref>––<xref rid="nihms-1587845-f0005" ref-type="fig">i</xref>).).', 'Structured illumination microscopy (SIM) resolved the thickened PAR as two axial cores (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1b</xref> and and <xref rid="nihms-1587845-f0006" ref-type="fig">Extended Data Fig. 2a</xref>,,<xref rid="nihms-1587845-f0006" ref-type="fig">b</xref>) decorated with RMMAI () decorated with RMMAI (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1c</xref>). PAR axes were extended and separated in late zygonema before X and Y synapsis, then collapsed during X–Y synapsis in early pachynema (). PAR axes were extended and separated in late zygonema before X and Y synapsis, then collapsed during X–Y synapsis in early pachynema (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1b</xref>). Each axial core is a sister chromatid, with a “bubble” from near the PAR boundary almost to the telomere (). Each axial core is a sister chromatid, with a “bubble” from near the PAR boundary almost to the telomere (<xref rid="nihms-1587845-f0006" ref-type="fig">Extended Data Fig. 2c</xref>––<xref rid="nihms-1587845-f0006" ref-type="fig">h</xref>). This PAR structure is distinct from what is seen at chromosome ends later in prophase I (). This PAR structure is distinct from what is seen at chromosome ends later in prophase I (Supplementary Discussion). Axis splitting and REC114 enrichment occurred independently of DSB formation (<xref rid="nihms-1587845-f0006" ref-type="fig">Extended Data Fig. 2i</xref>).).', 'We investigated temporal patterns of axis differentiation, RMMAI composition, and chromatin loop configuration on the PAR using SIM or conventional microscopy (<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1d</xref> and and <xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3a</xref>,,<xref rid="nihms-1587845-f0007" ref-type="fig">b</xref>). The SYCP3-defined axis was already long as soon as it was detectable in leptonema, and the PARb FISH signal was compact and remained so while the axis lengthened further through late zygonema, when the sister axes separated. Throughout, abundant ANKRD31 and REC114 signals stretched along the PAR axes, decorating the compact chromatin (). The SYCP3-defined axis was already long as soon as it was detectable in leptonema, and the PARb FISH signal was compact and remained so while the axis lengthened further through late zygonema, when the sister axes separated. Throughout, abundant ANKRD31 and REC114 signals stretched along the PAR axes, decorating the compact chromatin (<xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3a</xref> chromosomes a-h, and chromosomes a-h, and <xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3b</xref> i-ii). After synapsis, the axes shortened and chromatin loops decompacted, with concomitant RMMAI dissociation. A focus of the meiotic cohesin subunit REC8 was juxtaposed to ANKRD31 blobs at pre-leptonema; REC8 was mostly restricted to the borders of the PAR as its axes elongated and split, and remained highly enriched on the short axis after RMMAI proteins disappeared ( i-ii). After synapsis, the axes shortened and chromatin loops decompacted, with concomitant RMMAI dissociation. A focus of the meiotic cohesin subunit REC8 was juxtaposed to ANKRD31 blobs at pre-leptonema; REC8 was mostly restricted to the borders of the PAR as its axes elongated and split, and remained highly enriched on the short axis after RMMAI proteins disappeared (<xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3a</xref> chromosomes i-o, and chromosomes i-o, and <xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3b</xref> iii-iv). Collapse of the loop–axis structure and REC114 dissociation also occurred when the PAR underwent non-homologous synapsis in a iii-iv). Collapse of the loop–axis structure and REC114 dissociation also occurred when the PAR underwent non-homologous synapsis in a Spo11−/− mutant (<xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3c</xref>), so synapsis without recombination is sufficient for PAR reconfiguration. DSB formation without synapsis may also be sufficient (), so synapsis without recombination is sufficient for PAR reconfiguration. DSB formation without synapsis may also be sufficient (Supplementary Discussion). These findings delineate large-scale reconfiguration of loop–axis structure and establish spatial and temporal correlations between RMMAI proteins and association of a long axis with compact PAR chromatin.', '<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1a</xref>: The thickening of the PAR axis (using SYCP3 staining) and the elongation of the RMMAI signal along the PAR axis have been observed in more than three different mice in hundreds of late zygotene spermatocytes using mostly our homemade antibodies against REC114 and ANKRD31. Other antibodies such as anti-SYCP2 and anti-HORMAD2 were used to confirm the PAR axis thickening, and anti-MEI1, anti-MEI4 and anti-IHO1 were used to confirm the elongation of the REC114/ANKRD31 signal along the PAR axis, in more than 20 spermatocytes for each antibody.: The thickening of the PAR axis (using SYCP3 staining) and the elongation of the RMMAI signal along the PAR axis have been observed in more than three different mice in hundreds of late zygotene spermatocytes using mostly our homemade antibodies against REC114 and ANKRD31. Other antibodies such as anti-SYCP2 and anti-HORMAD2 were used to confirm the PAR axis thickening, and anti-MEI1, anti-MEI4 and anti-IHO1 were used to confirm the elongation of the REC114/ANKRD31 signal along the PAR axis, in more than 20 spermatocytes for each antibody.', '<xref rid="nihms-1587845-f0001" ref-type="fig">Fig. 1b</xref>: The PAR axis splitting, the extension of the RMMAI signal and the collapse of the PAR structure during X-Y synapsis have been observed by SIM in more than 60 spermatocytes in more than 3 different mice.: The PAR axis splitting, the extension of the RMMAI signal and the collapse of the PAR structure during X-Y synapsis have been observed by SIM in more than 60 spermatocytes in more than 3 different mice.'], 'nihms-1587845-f0007': ['We deduced that specific DNA sequences might recruit RMMAI proteins because autosomal blobs also hybridized to the PARb probe (<xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 1d</xref>). This repetitive probe includes a ~20-kb tandem array of a minisatellite called mo-2, with a 31-bp repeat). This repetitive probe includes a ~20-kb tandem array of a minisatellite called mo-2, with a 31-bp repeat23,24 (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2a</xref>). Clusters of mo-2 are also present at the non-centromeric ends of chr4, chr9, and chr13 (). Clusters of mo-2 are also present at the non-centromeric ends of chr4, chr9, and chr13 (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2a</xref>,,<xref rid="nihms-1587845-f0002" ref-type="fig">b</xref> and and <xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4a</xref>,,<xref rid="nihms-1587845-f0008" ref-type="fig">b</xref>))23,24. FISH with an mo-2 oligonucleotide probe showed that RMMAI blobs colocalize completely with mo-2 arrays (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2b</xref> and and <xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4c</xref>,,<xref rid="nihms-1587845-f0008" ref-type="fig">d</xref>). Mo-2 arrays become enriched at the onset of meiosis for heterochromatic histone modifications (H3K9me3, H4K20me3) and proteins (HP1β, HP1γ, and others), independent of DSB formation (). Mo-2 arrays become enriched at the onset of meiosis for heterochromatic histone modifications (H3K9me3, H4K20me3) and proteins (HP1β, HP1γ, and others), independent of DSB formation (<xref rid="nihms-1587845-f0009" ref-type="fig">Extended Data Fig. 5</xref>).).', '(a) Examples of zygotene oocytes showing the colocalization between blobs of IHO1 and REC114, MEI4 and MEI1, or ANKRD31 and mo-2 FISH signal (arrowheads). Scale bars: 2μm. RMMAI blobs colocalized with mo-2 FISH signals in all zygotene oocytes analyzed (N>30) from at least three mice. (b) PAR ultrastructure in oocytes, quantified as in <xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3b</xref>. Late zygotene cells with PAR synapsis are compiled separately from other zygotene cells. Error bars: means ± SD. Scale bar: 1 μm. . Late zygotene cells with PAR synapsis are compiled separately from other zygotene cells. Error bars: means ± SD. Scale bar: 1 μm. (c) Examples of zygotene oocytes showing colocalization of ANKRD31 blobs with enrichment for heterochromatin factors. Scale bars: 2 μm. ANKRD31 colocalized with heterochromatin factors blobs in all zygotene oocytes analyzed (N>20) from one mouse. (d) Representative SIM image of a wild-type late zygotene oocyte showing neither detectable splitting of the PAR axis nor REC8 enrichment. Scale bar: 2 μm. The absence of spermatocyte-like differentiation of the PAR axis was observed (N>30 zygotene oocytes) in more than three mice. A modest degree of differentiation was observed in a minority of oocytes (5/45) analyzed by SIM, but this did not resemble the typical PAR axis splitting found in spermatocytes. (e) Prolonged asynapsis does not allow axis splitting to occur in oocytes. Because synapsis appears sufficient to trigger collapse of PAR ultrastructure in spermatocytes (<xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3b</xref>), we asked if preventing synapsis (i.e., in a ), we asked if preventing synapsis (i.e., in a Syce1−/− mutant) could reveal a cryptic tendency toward axis splitting in oocytes. However, whereas axis splitting was clearly observed by SIM in Syce1−/− mutant spermatocytes, PAR axes were not detectably split in oocytes. Scale bars: 2 μm for main micrograph, 1 μm for insets. Axis splitting of chr9 was observed by SIM in multiple (N>20) Syce1−/− spermatocytes from three different mice. The chr13 or chr4 centromere-distal axes were also occasionally seen to be split, but we did not quantify this for these chromosomes. In males, the differentiation of the PAR or the chr9 axes becomes hardly detectable at later stages in some pachytene-like spermatocytes as cells enter apoptosis, similar to Spo11−/− or Hormad1−/− mice. However, in Syce1−/− oocytes, no significant axis differentiation or splitting was observed by conventional microscopy or by SIM in multiple spermatocytes (N>30) from three different mice, similar to what we observed in wild-type oocytes. (f,h) Delaying synapsis promotes PAR DSB formation in oocytes. Top panels: representative micrographs of pachytene XY (f) and Syce1−/− XX oocytes (h). Middle panels: RPA2 fluorescence intensity at the border of mo-2 FISH signals from PAR, chr9, and chr13. Bottom panels: Percentage of oocytes with RPA2 focus colocalizing with mo-2 regions on PAR, chr9, and chr13. Graphs show data only for pachytene oocytes in which PARs are synapsed (two mice of each genotype). Error bars: means ± SD. Scale bars: 2 μm. (g) Percentage of pachytene oocytes with one or more RPA2 foci colocalizing with mo-2 FISH signal from PAR, chr9 and chr13 in XY pachytene oocytes that had unsynapsed X and Y chromosomes. Scale bar: 2 μm, inset: 1 μm.', 'Data File S4: Excel file containing source data for <xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3</xref>', '(a) Axis thickening (SYCP2 and SYCP3) and ANKRD31 accumulation on X and Y PARs (arrowheads) in late zygonema. The asterisk shows an autosomal ANKRD31 blob. Scale bar: 2 μm. (b) Ultrastructure of the PAR before and after synapsis (montage of representative SIM images). Dashed lines indicate where chromosomes are cropped. SIM: Structured Illumination Microscopy. Scale bar: 1 μm. (c) RMMAI enrichment along split PAR axes in late zygonema. Scale bar: 1 μm. (d) Schematic showing the dynamic remodeling of the PAR loop–axis ensemble during prophase I. See measurements in <xref rid="nihms-1587845-f0007" ref-type="fig">Extended Data Fig. 3b</xref>. Scale bar: 1 μm.. Scale bar: 1 μm.'], 'nihms-1587845-f0008': ['To test if mo-2 arrays are cis-acting determinants of RMMAI recruitment, we exploited the fact that the Mus musculus molossinus subspecies has substantially lower mo-2 copy number24. The MSM/MsJ strain (MSM) showed less hybridization signal than B6 with the mo-2 FISH probe and had lower REC114 intensity in blobs (<xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4e</xref>).).', 'Data File S5: Excel file containing source data for <xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4</xref>..'], 'nihms-1587845-f0002': ['To avoid confounding strain effects, we examined spermatocytes of F1 hybrids (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2c</xref> and and <xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4f</xref>,,<xref rid="nihms-1587845-f0008" ref-type="fig">g</xref>). Less ANKRD31 accumulated on MSM PARs: the Y). Less ANKRD31 accumulated on MSM PARs: the YMSM PAR had 8-fold less ANKRD31 than the XB6 PAR in offspring from B6 mothers and MSM fathers (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2c</xref> and and <xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4g</xref>), and the X), and the XMSM PAR had 6.5-fold less than the YB6 PAR in the reciprocal cross (<xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4f</xref>,,<xref rid="nihms-1587845-f0008" ref-type="fig">g</xref>). Relative ANKRD31 levels matched mo-2 FISH. Nevertheless, MSM PARs support sex chromosome pairing efficiency and timing similar to B6 (). Relative ANKRD31 levels matched mo-2 FISH. Nevertheless, MSM PARs support sex chromosome pairing efficiency and timing similar to B6 (<xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4h</xref>), not surprisingly since MSM is fertile. Interestingly, the ssDNA binding protein RPA2 was present at lower intensity on MSM PARs (), not surprisingly since MSM is fertile. Interestingly, the ssDNA binding protein RPA2 was present at lower intensity on MSM PARs (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2c</xref> and and <xref rid="nihms-1587845-f0008" ref-type="fig">Extended Data Fig. 4f</xref>), revisited below.), revisited below.', 'These findings establish a tight correlation of RMMAI recruitment and axis remodeling with high-frequency DSB formation. Further strengthening this correlation, we noted above that MSM PARs display lower RPA2 intensity (<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2c</xref>), perhaps reflecting a lesser tendency to make multiple DSBs. Indeed, multiple PAR RPA2 foci were resolved by SIM more frequently in B6 than MSM (), perhaps reflecting a lesser tendency to make multiple DSBs. Indeed, multiple PAR RPA2 foci were resolved by SIM more frequently in B6 than MSM (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7i</xref>,,<xref rid="nihms-1587845-f0011" ref-type="fig">j</xref>).).', '<xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2b</xref>: The colocalization between REC114 blobs (or RMMAI blobs in general) and mo-2 FISH signals has been observed in all spermatocytes analyzed (N>200), from leptotene to early pachytene in more than three different mice.: The colocalization between REC114 blobs (or RMMAI blobs in general) and mo-2 FISH signals has been observed in all spermatocytes analyzed (N>200), from leptotene to early pachytene in more than three different mice.', '(a) Top panel: Self alignment of the PARb FISH probe (reproduced from <xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2a</xref>). The circled block is a 20-kb mo-2 cluster. Bottom panel: Schematic depicting the last 1.4 Mb of the non-centromeric ends of the indicated chromosomes, showing the positions of mo-2 repeats (green) adjacent to assembly gaps (mm10); mo-2 repeats were identified by BLAST search using the mo-2 consensus sequence. Mo-2 repeats also appear at the distal end of chr4 in the Celera assembly (Mm_Celera, 2009/03/04). PARb and PARd BAC clones are indicated. ). The circled block is a 20-kb mo-2 cluster. Bottom panel: Schematic depicting the last 1.4 Mb of the non-centromeric ends of the indicated chromosomes, showing the positions of mo-2 repeats (green) adjacent to assembly gaps (mm10); mo-2 repeats were identified by BLAST search using the mo-2 consensus sequence. Mo-2 repeats also appear at the distal end of chr4 in the Celera assembly (Mm_Celera, 2009/03/04). PARb and PARd BAC clones are indicated. (b) Confirmation that autosomal mo-2 FISH signals match the chromosomal locations indicated by mm10 or Celera genome assemblies. FISH was performed using an oligonucleotide probe containing the mo-2 consensus sequence in combination with BAC probes for adjacent segments of chromosomes 13, 9 and 4, as indicated. Magenta arrows point to concordant FISH signals. The chr9 BAC probe also hybridizes to the PAR. Scale bars: 2μm. The colocalization of mo-2 and the three autosomal FISH signals was observed in two mice (N>20 spermatocytes). (c) Comparison of mo-2 FISH with REC114 localization relative to the PAR boundary (PARb FISH probe) and the distal PAR (PARd probe). In mid zygonema, the mo-2 FISH signal colocalizes well with REC114 staining in between the PARb and PARd FISH signals. In late zygonema, mo-2 and REC114 are similar to one another and are elongated along the thickened SYCP3 staining of the PAR axis. From early to mid pachynema, REC114 progressively disappears, whereas the mo-2 FISH signal becomes largely extended away from the PAR axes. Note that the relative positions of the PARb and PARd probes reinforce the conclusion that the PAR does not adopt a crozier configuration. Scale bar: 1 μm. The different positioning of PARb and PARd FISH signals compared to mo-2 or REC114 signals was observed in more than 30 spermatocytes in at least three mice. (d) Illustration of the compact organization of the PAR chromatin (mo-2 FISH signal) compared to a whole-Y-chromosome paint probe. Scale bar: 2 μm. The costaining of mo-2 and full chrY probe was evaluated in one mouse (N>20 spermatocytes). (e) Lower mo-2 copy number in the M. m. molossinus subspecies correlates with lower REC114 staining in mo-2 regions. The left panels compare MSM and B6 mice for the colocalization between REC114 immunostaining and mo-2 FISH in leptotene spermatocytes. The REC114 and SYCP3 channels are shown at equivalent exposure for the two strains, whereas a longer exposure is shown for the mo-2 FISH signal in the MSM spermatocyte. Note that the mo-2-associated REC114 blobs are much brighter relative to the smaller dispersed REC114 foci in the B6 spermatocyte than in MSM. The right panel shows representative pachytene spermatocytes to confirm the locations of mo-2 clusters at autosome ends and the PAR in the MSM background. Scale bars: 2 μm. The lower intensity of REC114 blobs in MSM compared to B6 was observed in N>30 spermatocytes in three different pairs of mice. (f) PAR enrichment for ANKRD31 and RPA2 correlates with mo-2 copy number. Top panel: late zygotene spermatocytes from MSM x B6 F1 hybrid. Scale bar: 1 μm. Bottom panel: PAR-associated signals (A.U., arbitrary units) on B6-derived (YB) and MSM-derived chromosomes (XM) from the indicated number of spermatocytes (N). Red lines: means ± SD. Differences between X and Y PAR intensities are significant for both proteins and for mo-2 FISH in both F1 hybrids (p < 10−13, paired t-test; exact two-sided p values are in Data File S5). (g) Representative micrographs of late zygotene spermatocytes from reciprocal F1 hybrid males from crosses of B6 (high mo-2 copy number) and MSM (low mo-2 copy number) parents. Scale bar: 1 μm. (h) Frequency of paired X and Y at late zygonema and mid pachynema analyzed in three MSM and three B6 males. Differences between strains were not statistically significant at either stage (p = 0.241 for late zygonema and p = 0.136 for mid pachynema; two-sided Student’s t test). Note also that MSM X and Y are late-pairing chromosomes, as in the B6 background. The similar pairing kinetics indicates that the lower intensity of RMMAI staining on the MSM PAR is not attributable to earlier PAR pairing and synapsis in this strain. The number of spermatocytes analyzed is indicated (N).', 'Data File S1: Excel file containing source data for <xref rid="nihms-1587845-f0002" ref-type="fig">Fig. 2</xref>'], 'nihms-1587845-f0003': ['To identify factors important for PAR behavior, we eliminated RMMAI or axis proteins16,20,25,26. Requirements for RMMAI blobs overlap with but are distinct from those for smaller RMMAI foci, for which Hormad1 is important and Mei4 even more so, but Ankrd31 contributes only partially17,20,22 (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3a</xref>). HORMAD1 and REC8 were dispensable for RMMAI assembly on mo-2 regions, PAR axis elongation, splitting of sister axes, and formation of short loops (i.e., compact mo-2 and REC114 signals) (). HORMAD1 and REC8 were dispensable for RMMAI assembly on mo-2 regions, PAR axis elongation, splitting of sister axes, and formation of short loops (i.e., compact mo-2 and REC114 signals) (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3a</xref>,,<xref rid="nihms-1587845-f0003" ref-type="fig">b</xref>,,<xref rid="nihms-1587845-f0003" ref-type="fig">c</xref> and and <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6a</xref>,,<xref rid="nihms-1587845-f0010" ref-type="fig">b</xref>). Distal PAR axes were separated in ). Distal PAR axes were separated in Rec8−/− (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3c</xref> and and <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6c</xref>), so REC8 is essential for cohesion at the PAR end.), so REC8 is essential for cohesion at the PAR end.', 'The smaller MEI4 and REC114 foci still formed in Ankrd31−/−, but fewer and weaker (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3a</xref> and and <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6a</xref>,,<xref rid="nihms-1587845-f0010" ref-type="fig">d</xref>,,<xref rid="nihms-1587845-f0010" ref-type="fig">e</xref>))20. On mo-2 in contrast, RMMAI proteins did not accumulate detectably in Mei4−/− and Ankrd31−/− (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3a</xref> and and <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6a</xref>,,<xref rid="nihms-1587845-f0010" ref-type="fig">b</xref>). ANKRD31 was dispensable for enrichment of heterochromatin factors (). ANKRD31 was dispensable for enrichment of heterochromatin factors (<xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6f</xref>). REC114, although not IHO1, is similarly essential for RMMAI blobs). REC114, although not IHO1, is similarly essential for RMMAI blobs21. Normal PAR ultrastructure was also absent in Mei4−/− and Ankrd31−/−: axes were short with no sign of splitting and mo-2 was decompacted (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3b</xref>,,<xref rid="nihms-1587845-f0003" ref-type="fig">c</xref> and and <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6b</xref>). We conclude that PAR RMMAI blobs share genetic requirements with autosomal mo-2 blobs, and presence of blobs correlates with normal PAR structural differentiation.). We conclude that PAR RMMAI blobs share genetic requirements with autosomal mo-2 blobs, and presence of blobs correlates with normal PAR structural differentiation.', '<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3c</xref>: Axis splitting on the Y PAR has been observed by SIM in more than 100 late zygotene spermatocytes and in more than 20 zygotene-like spermatocytes from : Axis splitting on the Y PAR has been observed by SIM in more than 100 late zygotene spermatocytes and in more than 20 zygotene-like spermatocytes from Hormad1−/− mice. The fork-shaped PAR structure in Rec8−/− mice has been observed in more than 20 spermatocytes. The absence of PAR differentiation and decompaction of mo-2-containing chromatin was observed in more than 30 Ankrd31−/− spermatocytes and 20 Mei4−/− spermatocytes. This specific pattern has been confirmed in at least three different mice of each genotype using conventional microscopy. The differentiation of the PAR axis becomes hardly detectable in Hormad1−/− at later stage in some pachytene-like spermatocytes as cells enter apoptosis, similar to Spo11−/−.', '(a) Representative micrographs of ANKRD31, MEI4, IHO1 and MEI1 staining in wild type and the indicated mutants (quantification is in <xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3a</xref>). Scale bars: 2 μm. ). Scale bars: 2 μm. (b) Measurements of PAR loop–axis organization, as in <xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3b</xref>, on two additional males. Data from mouse 1 are reproduced from , on two additional males. Data from mouse 1 are reproduced from <xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3b</xref> to facilitate comparison. Means of each measurement for each mouse at each stage are given below, along with the means across all three mice. Means are rounded to two significant figures; the grand means were calculated using unrounded values from individual mice. The number of cells of each stage from each mouse is given (N). to facilitate comparison. Means of each measurement for each mouse at each stage are given below, along with the means across all three mice. Means are rounded to two significant figures; the grand means were calculated using unrounded values from individual mice. The number of cells of each stage from each mouse is given (N). (c) REC8 is dispensable for splitting apart of PAR sister chromatid axes, but is required to maintain the connection between sisters at the distal tip of the chromosome. A representative SIM image is shown of a Y chromosome from a late zygotene Rec8−/− spermatocyte. The SYCP3-labeled axes adopt an open-fork configuration. Note that the distal FISH probe (PARd) shows that there are clearly disjoined sisters whereas the PAR boundary (PARb) shows only a single compact signal comparable to wild type. The disposition of the probes and SYCP3 further rules out the crozier configuration as an explanation for split PAR axes. Scale bar: 1 μm. The Y or X PAR structure was resolved by SIM as “fork-shaped” in all spermatocytes analyzed (N>20) from three mice. (d) Quantification of REC114 and MEI4 foci in two additional pairs of wild-type and Ankrd31−/− mice. Horizontal lines indicate means. Fewer foci were observed in the Ankrd31−/− mutant (two-sided Student’s t tests for each comparison of mutant to wild type: p = 5.6 × 10−6 (2nd set, REC114); p = 1.1 × 10−5 (2nd set, MEI4); p = 2.1 × 10−6 (3rd set, REC114); p = 0.017 (3rd, MEI4)). (e) Reduced REC114-staining intensity of axis-associated foci in Ankrd31−/− mutants. To rigorously control for slide-to-slide and within-slide variation in immunostaining, we mixed together wild-type and Ankrd31−/− testis cell suspensions before preparing chromosome spreads. A representative image is shown of a region from a single microscopic field containing two wild-type zygotene spermatocytes (left) and two Ankrd31−/− spermatocytes of equivalent stage (right). Note the diminished intensity of REC114 foci in the Ankrd31−/− spermatocytes. Scale bar: 2 μm. REC114 (non-blob) foci showed lower fluorescence intensity in Ankrd31−/− compared to wild type in all pairs of spermatocytes captured in the same imaging field (N=8 pairs), from one pair of mice. (f) PAR enrichment of heterochromatin-associated factors is independent of ANKRD31. Representative images of the Y chromosome at late zygonema/early pachynema showing colocalization between the decompacted mo-2 chromatin and the indicated proteins. Note that both the FISH and immunofluorescence signals are localized mostly off the axis. Compare with the same signals in absence of SPO11 (<xref rid="nihms-1587845-f0009" ref-type="fig">Extended Data Fig. 5d</xref>). Scale bar: 1 μm. Mo-2 FISH signal colocalized off the axis with the heterochromatin factors in ). Scale bar: 1 μm. Mo-2 FISH signal colocalized off the axis with the heterochromatin factors in Ankrd31−/− mice in all spermatocytes analyzed (N>30) in more than three mice for CHD3/4 and at least one mouse for ATRX, HP1β, HP1γ, macroH2A1.2, H3K9me3, and H4K20me3.', '(a) Loop-axis organization of the mo-2 region of chr9 in late zygonema. Compare with the PAR (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3b</xref>). Scale bars: 1 μm. Error bars: means ± SD. ). Scale bars: 1 μm. Error bars: means ± SD. (b) Low mo-2 copy number correlates with less loop–axis reorganization (SIM images of late-zygotene F1-hybrid spermatocytes). Scale bars: 1 μm. The differentiation of the B6 PAR was observed in both hybrids B6 × MSM and MSM × B6 in 3 and 4 spermatocytes, respectively by SIM (1 mouse for each) and in more than 20 spermatocytes by conventional microscopy in two mice of each genotype. (c,d,e) Immuno-FISH for RPA2 and mo-2 was used to detect DSBs cytologically in wild type and the indicated mutants. To analyze Rec8 and Hormad1 mutations, we compared to mutants lacking SYCE1 (a synaptonemal complex central element component62) because Syce1−/− mutants show similar meiotic progression defects without defective RMMAI recruitment. Panel c shows representative images. Scale bars: 2 μm, inset 1 μm. Panel d shows the global counts of RPA2 foci for zygotene (zyg) or zygotene-like cells and for pachytene (pach) or pachytene-like cells. Panel e shows, for each cell, the fraction of mo-2 regions that had a colocalized RPA2 focus. Red lines: means ± SD. Statistical significance is indicated in panels c and d for comparisons (two-sided Student’s t tests) of wild type to Ankrd31−/− or of Syce1−/− to either Rec8−/− or Hormad1−/− for matched stages. Exact p values are in Data File S7. Note that the number of discretely scorable mo-2 regions in panel e varied from cell to cell depending on pairing status. (f) Frequent DSB formation at mo-2 regions in the PARs and on autosomes does not require HORMAD1. Micrograph at left shows two adjacent spermatocytes (boundary indicated by dashed line). Scale bar: 2 μm. Insets at right show higher magnification views of the numbered mo-2 regions, all of which are associated with RPA2 immunostaining of varying intensity. This picture illustrates the preferential RPA2 focus formation in mo-2 regions in a Hormad1−/− mouse; quantification is in panel e. (g) Autosomal mo-2 regions often form DSBs late. Immuno-FISH for RPA2, mo-2, and PARb was used to detect DSBs cytologically in wild type from leptonema to mid-pachynema, and to distinguish the X or Y PAR from chromosomes 9 and 13. Chr4 was not assayed because the mo-2 FISH signal was often barely detectable. The top panel shows the global number of RPA2 foci per cell. Black lines are means ± SD. The bottom panel shows the percentage of spermatocytes with an RPA2 focus overlapping the PAR (X, Y, or both) or overlapping chr9 or chr13. A representative image of an early pachytene spermatocyte is shown. Note that, as previously shown for the PAR2, autosomal mo-2 regions continue to accumulate RPA2 foci beyond the time when global RPA2 foci have largely or completely ceased accumulating. Scale bar: 2 μm. (h) X–Y pairing status, quantified by immuno-FISH for SYCP3 and the PARd probe. (i) Montage of SIM images from a B6 male showing that multiple, distinct RPA2 foci can be detected from late zygonema to mid pachynema, suggesting that multiple PAR DSBs can be formed during one meiosis (see also ref. 2 for further discussion). Scale bar: 1μm. The presence of multiple RPA2 foci in the PAR was observed by SIM in more than 20 spermatocytes from late zygonema to mid pachynema in one mouse. (j) Percentage of spermatocytes at the zygotene-pachytene transition with no (0), 1, 2 or 3 distinguishable RPA2 foci on the unsynapsed Y chromosome PAR of MSM and B6 mice. The difference between the strains is statistically significant (negative binomial regression, p = 7.2 × 10−5). N indicates the number of spermatocytes analyzed. A representative picture is shown for each genotype, with one RPA2 focus on the MSM PAR and two apparent sites of RPA2 accumulation on the B6 PAR. The detection of multiple foci is consistent with reported double crossovers6. Scale bar: 1 μm.', 'Data File S2: Excel file containing source data for <xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3</xref>'], 'nihms-1587845-f0004': ['If mo-2 arrays are cis-acting determinants of high-level RMMAI recruitment that in turn governs PAR structural dynamics, then autosomal mo-2 should also form PAR-like structures. Indeed, the distal end of chr9 underwent splitting in spermatocytes where this region was late to synapse (<xref rid="nihms-1587845-f0004" ref-type="fig">Fig. 4a</xref>) and showed a PAR-like pattern of extended axes and compact chromatin dependent on ) and showed a PAR-like pattern of extended axes and compact chromatin dependent on Ankrd31 (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7a</xref>). Thus, mo-2 (and/or linked elements) may be sufficient for both RMMAI recruitment and axis remodeling. Less axis remodeling for MSM PARs (). Thus, mo-2 (and/or linked elements) may be sufficient for both RMMAI recruitment and axis remodeling. Less axis remodeling for MSM PARs (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7b</xref>) reinforced the correlation between mo-2 copy number, RMMAI levels, and PAR ultrastructure.) reinforced the correlation between mo-2 copy number, RMMAI levels, and PAR ultrastructure.', 'We hypothesized that RMMAI recruitment and axis remodeling create an environment conducive to high-level DSB formation. This idea predicts that mutations should affect all of these processes coordinately and that autosomal mo-2 regions should experience PAR-like DSB formation. We counted axial RPA2 foci as a proxy for global DSB numbers and assessed mo-2 overlap with RPA2 (<xref rid="nihms-1587845-f0004" ref-type="fig">Fig. 4b</xref> and and <xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7c</xref>––<xref rid="nihms-1587845-f0011" ref-type="fig">f</xref>).).', 'We used maps of ssDNA bound by the strand-exchange protein DMC1 (ssDNA sequencing, or SSDS)7,29,30 to test more directly whether autosomal mo-2 regions experience PAR-like DSB formation, i.e., dependent on ANKRD31 but largely independent of the histone methyltransferase PRDM9 (<xref rid="nihms-1587845-f0004" ref-type="fig">Fig. 4c</xref> and and <xref rid="nihms-1587845-f0012" ref-type="fig">Extended Data Fig. 8a</xref>))7,20,21. Indeed, the region encompassing the chr9 mo-2 cluster displayed accumulation of SSDS reads that was substantially reduced in Ankrd31−/− but not in Prdm9−/−. A modest ANKRD31-dependent, PRDM9-independent peak was also observed near the mo-2 cluster on chr13 (<xref rid="nihms-1587845-f0012" ref-type="fig">Extended Data Fig. 8a</xref>). Thus, autosomal mo-2 regions not only accumulate PAR-like levels of RMMAI proteins and undergo PAR-like axis remodeling in spermatocytes, they frequently form DSBs in a PAR-like manner.). Thus, autosomal mo-2 regions not only accumulate PAR-like levels of RMMAI proteins and undergo PAR-like axis remodeling in spermatocytes, they frequently form DSBs in a PAR-like manner.', 'XY oocytes pair and synapse their PARs relatively late: only 28% of late zygotene cells had X and Y paired and/or synapsed (25 of 90 cells from two mice), increasing to 66% at pachynema (115 of 174 cells). This late pairing and synapsis is reminiscent of spermatocytes, but appears less efficient. Most pachytene XY oocytes that synapsed their PARs had a PAR-associated RPA2 focus, at twice the frequency and with higher immunofluorescence intensity than in XX oocytes (<xref rid="nihms-1587845-f0004" ref-type="fig">Fig. 4d</xref>, , <xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9f</xref>). RPA2 foci were also seen on most PARs that failed to synapse (). RPA2 foci were also seen on most PARs that failed to synapse (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9g</xref>). In contrast, chr9 and chr13 had lower RPA2 frequency and intensity that was comparable to XX PARs and that did not differ between XY and XX (). In contrast, chr9 and chr13 had lower RPA2 frequency and intensity that was comparable to XX PARs and that did not differ between XY and XX (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9f</xref>).).', '<xref rid="nihms-1587845-f0004" ref-type="fig">Fig. 4a</xref>: The differentiation of the non-centromeric end of the chr9 was observed in 6 spermatocytes by SIM and was observed in more than 20 late zygotene spermatocytes by conventional microscopy in three different mice.: The differentiation of the non-centromeric end of the chr9 was observed in 6 spermatocytes by SIM and was observed in more than 20 late zygotene spermatocytes by conventional microscopy in three different mice.'], 'nihms-1587845-f0011': ['In wild-type zygotene spermatocytes, RPA2 foci overlapped on average 35% of each cell’s mo-2 regions, increasing to 70% at pachynema (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7e</xref>). Similar to the PAR). Similar to the PAR2, autosomal mo-2 often acquired DSBs late (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7g</xref>). In contrast, ). In contrast, Ankrd31−/− mutants had starkly reduced overlap of RPA2 foci with mo-2, so X and Y paired in only 6% of mid-pachytene spermatocytes (<xref rid="nihms-1587845-f0004" ref-type="fig">Fig. 4b</xref> and and <xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7e</xref>,,<xref rid="nihms-1587845-f0011" ref-type="fig">h</xref>). This is distinct from autosomes: global RPA2 foci were only modestly reduced (). This is distinct from autosomes: global RPA2 foci were only modestly reduced (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7d</xref>) and most ) and most Ankrd31−/− cells pair and synapse all autosomes20,21. (Ankrd31−/− mutants form fewer RPA2 foci at leptonema and early zygonema, but normal numbers thereafter20,21.)', 'Rec8 deficiency did not reduce RPA2 focus formation on mo-2 or more globally relative to a synapsis-deficient control (Syce1−/−) (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7c</xref>––<xref rid="nihms-1587845-f0011" ref-type="fig">e</xref>). However, X–Y pairing was reduced (). However, X–Y pairing was reduced (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7h</xref>), presumably because REC8 promotes interhomolog recombination), presumably because REC8 promotes interhomolog recombination27. Hormad1−/− spermatocytes had comparable or higher frequencies of mo-2-overlapping RPA2 foci and X–Y pairing as the Syce1−/− control (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7e</xref>,,<xref rid="nihms-1587845-f0011" ref-type="fig">h</xref>). The high frequency of mo-2 RPA2 foci was striking given the global reduction in RPA2 foci (). The high frequency of mo-2 RPA2 foci was striking given the global reduction in RPA2 foci (<xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7d</xref>,,<xref rid="nihms-1587845-f0011" ref-type="fig">f</xref>) and DSBs) and DSBs28, but consistent with HORMAD1 dispensability both for RMMAI recruitment to mo-2 and for PAR ultrastructure (<xref rid="nihms-1587845-f0003" ref-type="fig">Fig. 3a</xref>––<xref rid="nihms-1587845-f0003" ref-type="fig">c</xref>).).', 'Data File S7: Excel file containing source data for <xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7</xref>..', '(a) The mo-2 region of chr9 undergoes axis elongation and splitting similar to PARs (SIM image of a wild-type zygotene spermatocyte). Scale bar: 1 μm. (b) ANKRD31 is required for high-level DSB formation in mo-2 regions and XY pairing. Immuno-FISH for RPA2 and mo-2 was used to detect DSBs. Illustration from <xref rid="nihms-1587845-f0011" ref-type="fig">Extended Data Fig. 7c</xref>. . (c) PAR-like DSB formation near autosomal mo-2 regions. Excerpt from <xref rid="nihms-1587845-f0012" ref-type="fig">Extended Data Fig. 8a</xref>. SSDS coverage. SSDS coverage7,20 is shown for the Y PAR (left) and the mo-2-adjacent region of chr9 (right). Positions of mo-2 repeats are shown below. (d) Early pachytene XY oocyte showing bright RPA2 focus in the PAR. Scale bar: 2 μm.'], 'nihms-1587845-f0013': ['In females, recombination between the two X chromosomes is not restricted to the PAR, so oocytes do not require PAR DSBs like spermatocytes31. We therefore asked whether the PAR undergoes spermatocyte-like structural changes in oocytes. RMMAI proteins robustly accumulated on PAR and autosomal mo-2 regions from leptonema to pachynema (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9a</xref>), consistent with studies of MEI4 and ANKRD31), consistent with studies of MEI4 and ANKRD3116,21. Oocytes also displayed an extended PAR axis and compact PARb FISH signal from leptonema to zygonema and transitioned to a shorter axis and more extended PARb signal in pachynema, with loss of REC114 signal upon synapsis (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9b</xref>). Heterochromatin factors were also enriched (). Heterochromatin factors were also enriched (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9c</xref>). However, we did not detect spermatocyte-like thickening or splitting of the PAR axis or REC8 accumulation (). However, we did not detect spermatocyte-like thickening or splitting of the PAR axis or REC8 accumulation (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9d</xref>), even in the absence of synapsis in ), even in the absence of synapsis in Syce1−/− mutants (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9e</xref>). Moreover, similar to the PAR). Moreover, similar to the PAR31, autosomal mo-2 regions showed little enrichment for SSDS signal in wild-type ovaries (<xref rid="nihms-1587845-f0012" ref-type="fig">Extended Data Fig. 8b</xref>,,<xref rid="nihms-1587845-f0012" ref-type="fig">c</xref>).).', 'These findings suggest that delayed PAR synapsis allows oocytes to more efficiently form DSBs. Supporting this conclusion, absence of synapsis in Syce1−/− oocytes was accompanied by an increase in both the frequency and intensity of RPA2 on PARs and autosomal mo-2 regions alike (<xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9h</xref>). Our results do not exclude the possibility of spermatocyte-oocyte differences in trans-acting factors, but we infer that the ability to manifest high-level DSB formation depends substantially on the result of a race between DSB formation and completion of synapsis (). Our results do not exclude the possibility of spermatocyte-oocyte differences in trans-acting factors, but we infer that the ability to manifest high-level DSB formation depends substantially on the result of a race between DSB formation and completion of synapsis (Supplementary Discussion).', 'Data File S8: Excel file containing source data for <xref rid="nihms-1587845-f0013" ref-type="fig">Extended Data Fig. 9</xref>..'], 'nihms-1587845-f0014': ['We demonstrate that the PAR in male mice undergoes a striking rearrangement of loop–axis structure prior to DSB formation involving recruitment of RMMAI proteins, dynamic axis elongation, and splitting of sister chromatid axes (<xref rid="nihms-1587845-f0014" ref-type="fig">Extended Data Fig. 10</xref>). Most of these behaviors also occur in oocytes and can support high-level DSB formation if synapsis is delayed. The mo-2 array may be a key cis-acting determinant and RMMAI proteins are crucial trans-acting determinants. Although the function of sister axis splitting is unclear (). Most of these behaviors also occur in oocytes and can support high-level DSB formation if synapsis is delayed. The mo-2 array may be a key cis-acting determinant and RMMAI proteins are crucial trans-acting determinants. Although the function of sister axis splitting is unclear (Supplementary Discussion), the full suite of PAR behaviors appears essential for pairing, recombination, and segregation of heteromorphic sex chromosomes.'], 'nihms-1587845-f0012': ['SSDS sequencing data were from previously described studies7,20,31 and are all available at the Gene Expression Omnibus (GEO) repository under accession numbers GSE35498, GSE99921, GSE118913. To define enrichment values presented in <xref rid="nihms-1587845-f0012" ref-type="fig">Extended Data Fig. 8b</xref>, the SSDS coverage was summed across the indicated coordinates adjacent to the mo-2 repeats. A chromosomal mean and standard deviation for chr9 was estimated by dividing the chromosome into 4-kb bins, summing the SSDS coverage in each bin, and calculating the mean and standard deviation after excluding those bins that overlapped a DSB hotspot. The enrichment score was then defined as the difference between the coverage in the mo-2-adjacent region and the chr9 mean coverage, divided by the chr9 standard deviation., the SSDS coverage was summed across the indicated coordinates adjacent to the mo-2 repeats. A chromosomal mean and standard deviation for chr9 was estimated by dividing the chromosome into 4-kb bins, summing the SSDS coverage in each bin, and calculating the mean and standard deviation after excluding those bins that overlapped a DSB hotspot. The enrichment score was then defined as the difference between the coverage in the mo-2-adjacent region and the chr9 mean coverage, divided by the chr9 standard deviation.'], 'nihms-1587845-f0006': ['Briefly, panels a and b show the following. At pre-leptonema, ANKRD31 blobs had a closely juxtaposed focus of the meiotic cohesin subunit REC8 (chromosome a). In leptonema and early zygonema, ANKRD31 and REC114 signals stretched along the presumptive PAR axes, with REC8 restricted to the borders (panel a, chromosomes b–e). The SYCP3-defined axis was already long as soon as it was detectable (0.73 μm) and the PARb FISH signal was compact (0.52 μm) (panel bi). At late zygonema, the PAR axis had lengthened still further (1.0 μm), while the PARb signal remained compact (panel bii). The PAR split into separate axes during this stage, each with abundant RMMAI (panel a, chromosomes f–h). The split was a REC8-poor zone bounded by REC8 foci (panel a, chromosomes f–h and <xref rid="nihms-1587845-f0006" ref-type="fig">Extended Data Fig. 2f</xref>). After synapsis, axes shortened and chromatin loops decompacted, with concomitant RMMAI dissociation. As cells transitioned into early pachynema and the X and Y PARs synapsed (panel a, chromosomes i–m), the PAR axes began to shorten slightly (0.85 μm) while the PARb signal expanded (0.85 μm) (panel biii). Meanwhile, the elongated ANKRD31 signals progressively decreased in intensity, collapsed along with the shortening axes, and separated from the axis while remaining nearby (panel a, chromosomes l–m). By mid-pachynema, PAR axes collapsed still further, to about half their zygotene length (0.50 μm) and the PARb chromatin expanded to more than twice the zygotene measurement (1.3 μm). ANKRD31 and REC114 enrichment largely disappeared, leaving behind a bright bolus of REC8 on the short remaining axis (panel a, chromosomes n–o and panel biv).). After synapsis, axes shortened and chromatin loops decompacted, with concomitant RMMAI dissociation. As cells transitioned into early pachynema and the X and Y PARs synapsed (panel a, chromosomes i–m), the PAR axes began to shorten slightly (0.85 μm) while the PARb signal expanded (0.85 μm) (panel biii). Meanwhile, the elongated ANKRD31 signals progressively decreased in intensity, collapsed along with the shortening axes, and separated from the axis while remaining nearby (panel a, chromosomes l–m). By mid-pachynema, PAR axes collapsed still further, to about half their zygotene length (0.50 μm) and the PARb chromatin expanded to more than twice the zygotene measurement (1.3 μm). ANKRD31 and REC114 enrichment largely disappeared, leaving behind a bright bolus of REC8 on the short remaining axis (panel a, chromosomes n–o and panel biv).'], 'nihms-1587845-f0010': ['Data File S6: Excel file containing source data for <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6</xref>..', '(a) Quantification of REC114, ANKRD31, MEI4, and IHO1 foci along unsynapsed axes in leptotene/early zygotene spermatocytes. Error bars: means ± SD. Comparisons to wild type are indicated (two-sided Student’s t test): * = p<0.02, ** = p≤10−7, ns = not significant (p>0.05); exact p values are in Data File S2. Representative micrographs of REC114 staining are shown; other proteins are in <xref rid="nihms-1587845-f0010" ref-type="fig">Extended Data Fig. 6a</xref>. Presence of mo-2 associated blobs (arrowheads) is indicated in the bottom panel. Scale bars: 2 μm. . Presence of mo-2 associated blobs (arrowheads) is indicated in the bottom panel. Scale bars: 2 μm. (b) Genetic requirements for PAR loop–axis organization (length of REC114 and mo-2 FISH signals along the PAR axis and axis-orthogonal extension of mo-2). Error bars: means ± SD. (c) Representative SIM images of Y-PAR loop–axis structure in each mutant at late zygonema. Scale bar: 1 μm.'], 'nihms-1587845-f0005': ['Data File S3: Excel file containing source data for <xref rid="nihms-1587845-f0005" ref-type="fig">Extended Data Fig. 1</xref> (including mass spectrometry data) (including mass spectrometry data)']}
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Ensuring meiotic DNA break formation in the mouse pseudoautosomal region
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Nature
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1593241200
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None
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other
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PMC7337327
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[
""
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Nature. 2020 Jun 27; 582(7812):426-431
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NO-CC CODE
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XBP1s interacts with T-BET and binds to the proximal GZMB promoter.a, Cytoplasmic and nuclear proteins were fractionated from primary human NK cells, followed by immunoblot analysis. b, FLAG-XBP1s and T-BET were co-transfected into 293T cells, followed by immunoprecipitation (IP) and immunoblot (IB) analyses. c, T-BET-expressing 293T cells were transfected with FLAG-XBP1u, FLAG-XBP1s or the control (pCDH) vector, followed by IP and immunoblot analyses. d, NK cells were treated with IL-15 (100 units/ml) for 24 h, followed by immunofluorescent staining with an anti-XBP1 antibody recognizing both XBP1u and XBP1s as well as anti-T-BET and anti-β-Actin antibodies. The nuclei were stained by DAPI (blue). e,f, NK cells were treated with IL-15 for 16 h, followed by ChIP assays performed with specific antibody or control IgG. Precipitated DNA was analyzed by PCR (e) or qPCR (f). Bar graphs display mean ± s.d. of n = 3 donors. *P < 0.05 by Student’s two-tailed unpaired t test. g, XBP1s or control vector was co-transduced with a pGL3 plasmid containing the GZMB promoter and a pRL-TK plasmid (as a control for data normalization) into 293T cells for 48 h. Cells were lysed to determine the promoter activity of GZMB by luciferase reporter assays. Bar graphs display mean ± s.e.m. of n = 4 independently experiments. ***P < 0.001 by Student’s two-tailed unpaired t test. The experiment was repeated independently two times (a,b) or three times (c,d,e) with similar results. Blot (a,b,c) and gel (e) images were cropped, and the full scans are shown in Supplementary figures.
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nihms-1510059-f0003
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nihms-1510059-f0003.jpg
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[{'image_id': 'nihms-1510059-f0001', 'image_file_name': 'nihms-1510059-f0001.jpg', 'image_path': '../data/media_files/PMC6293989/nihms-1510059-f0001.jpg', 'caption': 'XBP1s is induced by IL-15 at the protein level and controls degranulation in NK cells.(a,b) NK cells were treated with IL-2 (100 units/ml), IL-12 (10 ng/ml), or IL-15 (100 units/ml) for 24 h for flow cytometric analysis (a, n = 9 donors) and/or immunoblotting (b, n = 4 donors). ***P\u2009 < \u20090.001 by two-tailed paired t test. The experiment in (b) was repeated 3 times with similar results; images were cropped, and the full scans are shown in the supplementary figures. c, The expression of XBP1s target genes was assessed by qPCR after NK cells were treated as in (a,b). Bar graphs display mean\u2009± s.e.m. of n\u2009= 3 donors. *P < 0.05 by linear mixed model. d, NK cells were transduced with an XBP1s lentiviral construct or empty vector (EV) and 48h later were FACS-sorted for transduced GFP+ cells. Sorted cells were co-cultured with indicated leukemia cells for 4 h, followed by quantifying CD107a+ cells by flow cytometry. n = 4 donors. *P\u2009< 0.05, **P\u2009< 0.01 by two-tailed paired t test. e, NK cells were transduced with a XBP1 or a scramble shRNA lentiviral construct (pLKO.1) and FACS-sorted for GFP+ cells after 48 h, then co-cultured with the MOML13 leukemia cell line for 4 h, followed by quantification of CD107a+ cells. Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 6 donors. **P\u2009<\u20090.01 by linear mixed model. f, NK cells were treated with IL-15 for 24 h and co-cultured with the MM1.S cell line for 4 h, followed by evaluation of XBP1s and CD107a expression by flow cytometry. n = 8 donors. ***P\u2009< 0.001 by two-tailed paired t test.', 'hash': 'b8ae7483a54610b37b0d4168b44f7cf1fa41e7716c9e06b41e758b38c33a4e92'}, {'image_id': 'nihms-1510059-f0004', 'image_file_name': 'nihms-1510059-f0004.jpg', 'image_path': '../data/media_files/PMC6293989/nihms-1510059-f0004.jpg', 'caption': 'XBP1s contributes to IL-15-mediated NK cell survival.a,b, NK cells were transiently transfected or lentivirally transduced with XBP1 esiRNA, XBP1u, XBP1s, or control [Scramble in (a) and empty vector (EV) in (b), followed by culture for 48 h with IL-15 (100 units/ml). Densitometric quantification shows the ratio of C-CASP3 protein to β-Actin protein (right panel). Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 3 donors (a,b). *P\u2009< 0.05, **P < 0.01 by Student’s two-tailed unpaired t test (a) or linear mixed model (b). c, NK cells were pretreated with 4µ8C for 1 h and then with or without IL-15 for 4 h. Densitometric quantification shows the ratio of C-CASP3 protein to β-Actin protein (right panel). Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 3 donors. *P\u2009< 0.05, **P\u2009< 0.01 by linear mixed model. d, NK cells were pre-treated with or without 4µ8C (50 µM) for 1 h, followed by an incubation with IL-15 for 96 h. Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 3 donors. **P\u2009< 0.01 by linear mixed model. e, NK cells were pre-treated with or without 4µ8C (50 µM) for 4 h, then washed and labeled with CFSE or Far Red live staining dye, respectively. Cells were mixed 1:1 and cultured with IL-15. Live cell ratios (CFSE+ vs Far Red+) were analyzed by flow cytometry. The experiment was repeated independently for four donors with similar results. f,g, The percentages and absolute quantities of live cells depicted in (e) are shown in (f) and (g), respectively. Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 4 donors. ***P\u2009<\u20090.001 by Student’s two-tailed unpaired t test. N.S., no significance. Blot images (a,b,c) were cropped, and the full scans are shown in Supplementary figures.', 'hash': '72217804c97d15c7cbb88e466596210208594d34b7c6036d34b8244c8c994b41'}, {'image_id': 'nihms-1510059-f0003', 'image_file_name': 'nihms-1510059-f0003.jpg', 'image_path': '../data/media_files/PMC6293989/nihms-1510059-f0003.jpg', 'caption': 'XBP1s interacts with T-BET and binds to the proximal GZMB promoter.a, Cytoplasmic and nuclear proteins were fractionated from primary human NK cells, followed by immunoblot analysis. b, FLAG-XBP1s and T-BET were co-transfected into 293T cells, followed by immunoprecipitation (IP) and immunoblot (IB) analyses. c, T-BET-expressing 293T cells were transfected with FLAG-XBP1u, FLAG-XBP1s or the control (pCDH) vector, followed by IP and immunoblot analyses. d, NK cells were treated with IL-15 (100 units/ml) for 24 h, followed by immunofluorescent staining with an anti-XBP1 antibody recognizing both XBP1u and XBP1s as well as anti-T-BET and anti-β-Actin antibodies. The nuclei were stained by DAPI (blue). e,f, NK cells were treated with IL-15 for 16 h, followed by ChIP assays performed with specific antibody or control IgG. Precipitated DNA was analyzed by PCR (e) or qPCR (f). Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 3 donors. *P\u2009< 0.05 by Student’s two-tailed unpaired t test. g, XBP1s or control vector was co-transduced with a pGL3 plasmid containing the GZMB promoter and a pRL-TK plasmid (as a control for data normalization) into 293T cells for 48 h. Cells were lysed to determine the promoter activity of GZMB by luciferase reporter assays. Bar graphs display mean\u2009±\u2009s.e.m. of n\u2009= 4 independently experiments. ***P\u2009< 0.001 by Student’s two-tailed unpaired t test. The experiment was repeated independently two times (a,b) or three times (c,d,e) with similar results. Blot (a,b,c) and gel (e) images were cropped, and the full scans are shown in Supplementary figures.', 'hash': '0b75bfa27c412bf4c48e4f3fa19fbac1fcee53e9bb3cf0dcf5c64ed382354f7f'}, {'image_id': 'nihms-1510059-f0002', 'image_file_name': 'nihms-1510059-f0002.jpg', 'image_path': '../data/media_files/PMC6293989/nihms-1510059-f0002.jpg', 'caption': 'XBP1s regulates GZMB and IFNγ expression in NK cells.a, NK cells were transduced with an XBP1s lentiviral construct or empty vector (EV) and 48 h later were FACS-sorted for transduced GFP+ cells, which were co-cultured with K562 cells for 4 h, and then fractionated with anti-CD56 magnetic beads, followed by qPCR analysis. Bar graphs display mean\u2009±\u2009s.e.m. of n\u2009= 3 donors. **P\u2009< 0.01, ***P\u2009<\u20090.001 by Student’s two-tailed unpaired t test. b, NK cells were transduced with XBP1u, XBP1s, or pCDH-EV by lentiviral infection and cultured with IL-15 (100 units/ml) for 48 h and then were FACS-sorted for GFP+ cells to determine XBP1s and GZMB protein expression by immunoblotting. The experiment was repeated 3 times with similar results. c, Densitometric quantification of the ratio of the level of XBP1s or GZMB protein to the level of β-Actin protein for (b). Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 3 donors. *P\u2009<\u20090.05 by linear mixed model. d, NK cells were transduced with a XBP1 or scramble shRNA lentiviral construct (pLKO.1) and 48 h later were FACS-sorted as in (a). FACS-purified cells were co-cultured with K562 cells for 4 h, and then fractionated with anti-CD56 magnetic beads, followed by qPCR analysis. Bar graphs display mean\u2009±\u2009s.e.m. of n\u2009= 3 donors. *P < 0.05 by linear mixed model. e, NK cells were treated with IL-15, followed by immunoblotting or qPCR analysis. The experiment was repeated independently three times with similar results (b,e). N.S., no significance. Blot images (b,e) were cropped, and the full scans are shown in the Supplementary figures.', 'hash': 'd2d76ff6c87c034f4bab6d0050002848d1fb219a1136e95558986474ef4ffce7'}, {'image_id': 'nihms-1510059-f0005', 'image_file_name': 'nihms-1510059-f0005.jpg', 'image_path': '../data/media_files/PMC6293989/nihms-1510059-f0005.jpg', 'caption': 'AKT mediates stability of XBP1s.a, XBP1 splicing assays by PCR (upper panel) and quantification of XBP1s by qPCR in NK cells (lower panel). Bar graphs display mean\u2009±\u2009s.e.m. of n = 3 donors. b, Immunoblotting of cytoplasmic and nuclear protein fractions of NK cells treated with IL-15 (100 units/ml) for indicated time period. c, NK cells were pre-treated with or without AKTi-1/2 (10 µM) for 30 min, followed by stimulation with IL-15 for 6 h prior to immunoblotting. n = 3 donors. d, NK cells were pre-treated with IL-15 alone or plus AKTi-1/2 for 1 h, then cultured with or without CHX (10 µg/ml) for 30 min prior to immunoblotting. Data are summarized in Supplementary Fig. 6a. n = 3 donors. e, 293T cells were transfected with indicated amount of pECE-myrAKTΔ4−129 vector and/or pCDH-FLAG-tagged XBP1s (FLAG-XBP1s) vector or respective control vector (dash) (1 µg each) and cultured for 24 h. XBP1s protein was analyzed by immunoprecipitation (IP) with anti-FLAG antibody combined with immunoblotting. Data are summarized in Supplementary Fig. 6b. n = 3 independent experiments. f, XBP1s mRNA expression was analyzed by qPCR in the pECE-myrAKTΔ4–129- or pECE control-transfected 293T cells co-transfected with pCDH-FLAG-XBP1s for 24 h (endogenous XBP1s is very low). Bar graphs display mean\u2009±\u2009s.d. of n\u2009= 3 independent experiments. N.S., not significant, by Student’s two-tailed unpaired t test. g, 293T cells were co-transfected with pCDH-FLAG-XBP1s and pECE-myrAKTΔ4−129 or pECE empty vector (EV) for 24 h and then treated with CHX for the indicated time periods with or without MG132 (10 µM). n = 3 independent experiments. h, myrAKTΔ4−129 or control (dash) was co-transfected with FLAG-XBP1s into 293T cells for 24 h with or without MG132 for 4 h. Protein ubiquitination of XBP1s was analyzed by IP with anti-FLAG antibodies, followed by immunoblotting with densitometric quantification. i, NK cells were pre-treated with or without AKTi-1/2 for 1 h followed by MG132 treatment for 3 h in the presence of IL-15. Ubiquitination of XBP1s was analyzed by IP with anti-ubiquitin antibodies combined with immunoblotting using XBP1s antibodies. The experiment was repeated independently for three times (b,h,i) with similar results. Gel (a) and blot images (b-e, g-i) were cropped, and the full scans are shown in Supplementary figures.', 'hash': 'f4590bb06c7f921980704f6bf5ec614ff61e88c922273222329eb64e406cab06'}]
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{'nihms-1510059-f0001': ['Unspliced XBP1 mRNA, known as XBP1u, encodes an unstable cytoplasmic protein with no transactivation domains. As a result of unconventional splicing mediated by the serine/threonine-protein kinase/endoribonuclease, IRE1α, mature XBP1 mRNA is converted to XBP1s4. The protein encoded by XBP1s can act as a transcription factor2,3. XBP1s has multiple roles in regulating the immune response. It regulates major histocompatibility complex class II (MHC II) gene transcription in HeLa and COS cells5, as well as the differentiation of plasma cells, eosinophils and CD8+ T cells6–8. XBP1s also modulates anti-tumor immunity by disrupting dendritic cell homeostasis9. We investigated the expression of XBP1s in primary human NK cells purified from the blood of healthy donors in response to interleukin 2 (IL-2), IL-12 or IL-15 for 24 h prior to analysis by flow cytometry or immunoblot. IL-15 induced the expression of XBP1s protein, whereas IL-2 and IL-12 showed reduced effects compared to IL-15 (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1a,b</xref>). Although IL-2 and IL-15 share the cognate receptors IL-2Rβ and IL-2Rγc on NK cells, induction of XBP1s by IL-15 was significantly higher than that triggered by similar concentrations of IL-2 (). Although IL-2 and IL-15 share the cognate receptors IL-2Rβ and IL-2Rγc on NK cells, induction of XBP1s by IL-15 was significantly higher than that triggered by similar concentrations of IL-2 (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1b</xref> and and Supplementary Fig. 1a). This suggests that the IL-15Rα chain expressed on NK cells may play a critical role in inducing XBP1s. In addition, the expression of transcripts for XBP1s target genes, including ERDJ4 and SEC61A19, was significantly increased in IL-15-treated primary human NK cells compared to non-treated, IL-2- or IL-12-treated cells (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1c</xref>).).', 'We next investigated the effects of XBP1s overexpression on NK cell function. Primary human NK cells transfected with pCDH lentivirus carrying a wild-type XBP1s gene (pCDH-XBP1s) and co-cultured with K562, MOLM-13 or U937 leukemia cell lines had a higher percentage of CD107a+ NK cells compared to NK cells transfected with the lentivirus carrying an empty PCDH vector (pCDH-EV) (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1d</xref>). Upon co-culture with MOML-13 target cells, the percentage of CD107a). Upon co-culture with MOML-13 target cells, the percentage of CD107a+ cells in primary human NK cells transduced with pLKO.1 lentivirus carrying XBP1 shRNAs (XBP1-knockdown, KD) was significantly decreased (an approximately 35% reduction) compared to cells transduced with pLKO.1 lentivirus carrying scramble shRNAs (scramble-KD) (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1e</xref>). In addition, primary human NK cell degranulation against multiple myeloma MM.1S cells was observed in IL-15-treated, but not in non-treated primary human NK cells (). In addition, primary human NK cell degranulation against multiple myeloma MM.1S cells was observed in IL-15-treated, but not in non-treated primary human NK cells (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1f</xref>). When co-cultured with MM.1S multiple myeloma cells, the percentage of CD107a). When co-cultured with MM.1S multiple myeloma cells, the percentage of CD107a+ NK cells expressing XBP1s was approximately 4-fold greater than that of CD107a+ NK cells lacking XBP1s (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1f</xref>). Moreover, the expression of XBP1s protein was significantly higher in CD107a). Moreover, the expression of XBP1s protein was significantly higher in CD107a+ compared to CD107a─ primary human NK cells co-cultured with MM.1S cells (Supplementary Fig. 1b), indicating that expression of XBP1s correlates with NK cell cytotoxicity against tumor cells. Collectively, our results suggest that IL-15 induces XBP1s protein expression and the expression level of the transcriptional factor directly correlates with cytotoxic activity in human NK cells.', 'To investigate how XBP1s regulates NK cell function, we analyzed the expression of genes related to NK cell effector functions, including GZMB (granzyme B), IFNG (interferon-γ), and PRF1 (perforin). Expression of GZMB and IFNG but not PRF1 mRNA was higher in pCDH-XBP1s-transduced primary human NK cells compared to pCDH-EV control NK cells (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1a</xref>), along with increased expression of GZMB protein (), along with increased expression of GZMB protein (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2b,c</xref>). Overexpression of the unspliced form of XBP1, XBP1u, which can be processed into XBP1s through IRE1α-mediated mRNA splicing, in primary human NK cells by transduction with pCDH lentivirus carrying a wild-type ). Overexpression of the unspliced form of XBP1, XBP1u, which can be processed into XBP1s through IRE1α-mediated mRNA splicing, in primary human NK cells by transduction with pCDH lentivirus carrying a wild-type XBP1u gene (pCDH-XBP1u) also increased the expression of GZMB compared to pCDH-EV NK cells (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2b,c</xref>). Moreover, primary human NK cells treated with thapsigargin (Thap), a chemical drug that induces ER stress and IRE1α catalytic activity). Moreover, primary human NK cells treated with thapsigargin (Thap), a chemical drug that induces ER stress and IRE1α catalytic activity10, increased XBP1s protein and GZMB mRNA and protein when compared to NK cells without Thap treatment, in the absence or presence of IL-15 (Supplementary Fig. 2a-c). In addition, downregulation of GZMB protein expression was observed in primary human NK cells transfected with XBP1 siRNAs compared to cells transfected with scramble control siRNAs (Supplementary Fig. 2d,e). We also observed decreased expression of both GZMB and IFNG genes, but not PRF1, in primary human NK cells with XBP1-KD using shRNA, compared to scramble-KD control NK cells (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1d</xref>). Inhibition of ). Inhibition of XBP1 mRNA splicing in primary human NK cells with 4µ8C, an inhibitor of IRE1α-mediated mRNA splicing11, resulted in decreased expression of XBP1s protein and suppression of IL-15-induced GZMB protein and mRNA compared to the cells without 4µ8C treatment (Supplementary Fig. 2f-h). The expression of XBP1s protein was positively correlated with the mRNA expression of GZMB and IFNG, all of which were induced by treatment with IL-15 at multiple time points in primary human NK cells (<xref rid="nihms-1510059-f0001" ref-type="fig">Fig. 1e</xref>). Thus, XBP1s positively regulates the expression of GZMB and IFN-γ in NK cells.). Thus, XBP1s positively regulates the expression of GZMB and IFN-γ in NK cells.'], 'nihms-1510059-f0002': ['We next investigated the cellular localization of XBP1s. Immunoblot analysis of the cytoplasmic and nuclear protein fractions in primary human NK cells treated with IL-15 for 24 h indicated that XBP1s exists almost exclusively in the nucleus following induction by IL-15 (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2a</xref>), consistent with its role in regulating transcription), consistent with its role in regulating transcription12. Next, we overexpressed T-BET and FLAG-XBP1u or FLAG-XBP1s in 293T cells. Co-immunoprecipitation followed by immunoblot using antibodies against FLAG or T-BET indicated that overexpressed FLAG-XBP1s interacted with T-BET (<xref rid="nihms-1510059-f0003" ref-type="fig">Fig. 3b,c</xref> and and Supplementary Fig. 3a), a transcriptional regulator important for NK cell function. T-BET was previously assumed to associate with the GZMB promoter, although no specific binding sites have been identified13,14. Of note, FLAG-XBP1s did not interact with endogenous STAT5 in 293T cells, an important transcriptional factor downstream of IL-15 signaling (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2c</xref>). Using confocal imaging with antibodies identifying both XBP1u and XBP1s, and a T-BET antibody, we observed the co-localization of T-BET and XBP1 in the nuclei of primary human NK cells (). Using confocal imaging with antibodies identifying both XBP1u and XBP1s, and a T-BET antibody, we observed the co-localization of T-BET and XBP1 in the nuclei of primary human NK cells (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2d</xref>) and in the human NK cell lymphoma cell line NK-92 () and in the human NK cell lymphoma cell line NK-92 (Supplementary Fig. 3b). Confocal microscopy indicated that T-BET has an almost exclusively nuclear distribution in NK cells (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2d</xref>), which was validated by staining with an alternative antibody against T-BET in both primary NK cells and NK-92 cells (), which was validated by staining with an alternative antibody against T-BET in both primary NK cells and NK-92 cells (Supplementary Fig. 3c).', 'Next, we tested whether XBP1s interacted with its canonical binding motifs (G/C)ACGT15,16 located within the GZMB proximal promoter (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2e</xref>). Chromatin immunoprecipitation (ChIP) indicated that XBP1s and T-BET bound to the same proximal region of the ). Chromatin immunoprecipitation (ChIP) indicated that XBP1s and T-BET bound to the same proximal region of the GZMB promoter in primary human NK cells treated with IL-15, but with little or no binding in untreated cells (<xref rid="nihms-1510059-f0003" ref-type="fig">Fig. 3e,f</xref>). In contrast, T-BET did not bind to the ). In contrast, T-BET did not bind to the GZMB promoter in primary human NK cells treated with 4µ8C (which do not express XBP1s; Supplementary Fig. 2g) in the presence of IL-15, compared to control cells only treated with IL-15 (Supplementary Fig. 3d). Using a luciferase assay to evaluate GZMB promoter activity, we observed that 293T cells transfected with XBP1s had much higher GZMB promoter activity compared to that of the empty vector-transfected cells (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2g</xref>). Of note, we also observed STAT5 binding to the ). Of note, we also observed STAT5 binding to the GZMB promoter in primary human NK cells by ChIP assays (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2e</xref>), consistent with previous reports), consistent with previous reports17. STAT5 is known to positively regulate GZMB expression17–19; however, STAT5 did not interact with FLAG-XBP1s in 293T cells by co-immunoprecipitation assays (<xref rid="nihms-1510059-f0002" ref-type="fig">Fig. 2c</xref>). To test whether STAT5 was required for ). To test whether STAT5 was required for GZMB induction by XBP1s we knocked down the expression of STAT5A or STAT5B using shRNAs in 293T cells that were co-transfected with a pCDH-XBP1s or pCDH-EV and a PGL3 vector carrying the GZMB promoter reporter (PGL3-GZMB) (Supplementary Fig. 4a). The induction of the GZMB promoter reporter by XBP1s overexpression was not inhibited by either STAT5A-KD (279%) or STAT5B-KD (184%) in 293T cells compared to control 293T cells (175%) (Supplementary Fig. 4b), indicating that induction of GZMB promoter activity by XBP1s and T-BET in 293T cells does not require STAT5. Together, our data suggest that XBP1s interacts with T-BET but not STAT5 and regulates the transcriptional activity of GZMB via promoter binding.'], 'nihms-1510059-f0003': ['XBP1 is a survival gene that protects cells from stress-induced death20,21, and IL-15 is a critical cytokine for NK cell survival1,22. Knockdown of XBP1 by transfection with XBP1 siRNAs resulted in increased expression of cleaved caspase-3 in primary human NK cells compared to cells transfected with scramble siRNAs (<xref rid="nihms-1510059-f0003" ref-type="fig">Fig. 3a</xref>), indicating increased apoptosis in XBP1-KD NK cells. In contrast, pCDH-XBP1u- or pCDH-XBP1s-transduced primary human NK cells showed decreased expression of cleaved caspase-3 compared to pCDH-EV control NK cells (), indicating increased apoptosis in XBP1-KD NK cells. In contrast, pCDH-XBP1u- or pCDH-XBP1s-transduced primary human NK cells showed decreased expression of cleaved caspase-3 compared to pCDH-EV control NK cells (<xref rid="nihms-1510059-f0003" ref-type="fig">Fig. 3b</xref>). Moreover, in IL-15-activated primary human NK cells in which ). Moreover, in IL-15-activated primary human NK cells in which XBP1 splicing was inhibited by treatment with 4µ8C, cleaved caspase-3 was higher and survival was lower compared to similarly activated cells without 4µ8C treatment (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4c-f</xref>). Taken together, these data indicate that XBP1s modulates IL-15-induced survival in human NK cells.). Taken together, these data indicate that XBP1s modulates IL-15-induced survival in human NK cells.'], 'nihms-1510059-f0005': ['Next, we investigated the molecular mechanism by which IL-15 regulated the expression of XBP1s protein. IL-15 stimulation of primary human NK cells did not increase the amount of XBP1s mRNA compared to unstimulated NK cells, as evaluated by XBP1 splicing assays and qPCR; however, XBP1s protein accumulated in the nucleus of IL-15-treated primary human NK cells within 2 h of stimulation (<xref rid="nihms-1510059-f0005" ref-type="fig">Fig. 5a,b</xref>). IL-15 stimulation also induced the phosphorylation of the serine-threonine kinase AKT in NK cells primarily in the cytoplasm (). IL-15 stimulation also induced the phosphorylation of the serine-threonine kinase AKT in NK cells primarily in the cytoplasm (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4b</xref>), as previously reported), as previously reported23. Blockade of AKT phosphorylation with the AKT inhibitor AKTi-1/224 in IL-15-stimulated primary human NK cells resulted in decreased expression of XBP1s protein compared to cells treated with IL-15 in the absence AKTi-1/2 (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4c</xref> and and Supplementary Fig. 5a). Moreover, the expression of GZMB mRNA and GZMB protein was significantly downregulated in primary NK cells transduced with AKT1-shRNA compared to scramble shRNA (Supplementary Fig. 5b,c). On the other hand, the expression of GZMB mRNA was significantly upregulated and GZMB protein was moderately upregulated in primary NK cells transduced with a constitutively active form of AKT (pCDH-myrAKTΔ4−129, encoding a 14 amino acid src myristoylation signal sequence fused to the N-terminus of AKT delta4–12925), compared to cells transduced with control pCDH-EV (Supplementary Fig. 5d,e). Treatment with AKTi-1/2 also reduced the level of XBP1s protein in IL-15-stimulated NK cells in the presence of cycloheximide (CHX), which blocks de novo protein synthesis and thus prevents any confounding effects of increased protein translation26 (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4d</xref> and and Supplementary Fig.6a). These data indicate that AKT plays a role in increasing the IL-15-induced protein levels, but not mRNA expression of XBP1s in primary human NK cells.'], 'nihms-1510059-f0004': ['To further test whether AKT is required for maintaining the level of XBP1s protein, we co-transfected various concentrations of pECE-myrAKTΔ4−129 or pECE-EV with pCDH-FLAG-XBP1s or pCDH-EV into 293T cells. Immunoprecipitation experiments indicated that the level of FLAG-XBP1s protein was markedly increased in a dose-dependent manner by myrAKTΔ4−129 overexpression (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4e</xref> and and Supplementary Fig. 6b), while XBP1s mRNA was not induced by overexpression of myrAKTΔ4−129 (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4f</xref>), consistent with the idea that IL-15-induced XBP1s upregulation is independent of ), consistent with the idea that IL-15-induced XBP1s upregulation is independent of XBP1s transcription. In addition, co-transduction of pCDH-XBP1s and pECE-myrAKTΔ4−129 enhanced the activity of the GZMB promoter in 293T cells compared to transfection of pCDH-XBP1s alone (Supplementary Fig. 6c). These data indicate that AKT activation is required for maintaining the level of XBP1s protein. As time advanced during a 4-h incubation, a decrease was observed in FLAG-XBP1s protein in 293T cells transfected with pECE-EV, but not with pECE-myrAKTΔ4−129 following CHX treatment (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4g</xref> and and Supplementary Fig. 6d), suggesting that signaling downstream of AKT protects XBP1s from degradation. Block of proteosomal degradation with the cell-permeable proteasome and calpain inhibitor MG13227 recovered FLAG-XBP1s protein in pECE-EV- but not pECE-myrAKTΔ4−129-transfected 293T cells treated with CHX (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4g</xref> and and Supplementary Fig. 6d), indicating that overexpression of myrAKTΔ4−129 enhanced the protein stability of XBP1s in 293T cells. Ubiquitination of FLAG-XBP1s was reduced following transfection of pECE-myrAKTΔ4−129 compared to pECE-EV in 293T cells (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4h</xref>), while ubiquitination of XBP1s was substantially increased in IL-15-stimulated primary human NK cells treated with the AKT inhibitor AKTi-1/2 compared to untreated cells (), while ubiquitination of XBP1s was substantially increased in IL-15-stimulated primary human NK cells treated with the AKT inhibitor AKTi-1/2 compared to untreated cells (<xref rid="nihms-1510059-f0004" ref-type="fig">Fig. 4i</xref>). Our data suggest that AKT controls XBP1s stability, possibly through a mechanism involving the ubiquitination of XBP1s.). Our data suggest that AKT controls XBP1s stability, possibly through a mechanism involving the ubiquitination of XBP1s.']}
|
The IL-15-AKT-XBP1s signaling pathway contributes to effector functions and survival in human NK cells
| null |
Nat Immunol
|
1547107200
|
Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.
|
[
"Animals",
"B-Lymphocytes",
"CRISPR-Cas Systems",
"Carcinogenesis",
"DNA, Intergenic",
"Germinal Center",
"Histone Demethylases",
"Hyperplasia",
"Immunological Synapses",
"Introns",
"Lymphoma",
"Mice",
"Mice, Inbred C57BL",
"Mice, Knockout",
"Proto-Oncogene Proteins c-bcl-6"
] |
other
|
PMC6293989
| null |
53
|
[
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"{'Citation': 'Geng H et al. Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia. Cancer Discov 2, 1004–1023 (2012).', 'ArticleIdList': {'ArticleId': [{'@IdType': 'pmc', '#text': 'PMC3516186'}, {'@IdType': 'pubmed', '#text': '23107779'}]}}",
"{'Citation': 'Yoav Benjamini YH Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society 57, 289–300 (1995).'}",
"{'Citation': 'Shaffer AL et al. A library of gene expression signatures to illuminate normal and pathological lymphoid biology. Immunol Rev 210, 67–85 (2006).', 'ArticleIdList': {'ArticleId': {'@IdType': 'pubmed', '#text': '16623765'}}}"
] |
Nat Immunol. 2019 Jan 10; 20(1):10-17
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NO-CC CODE
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Plaque assays reveal that some individual schizonts generate mixed sexual and asexual plaques.a, Schematic of the possible types of plaques originated from schizonts expressing or not expressing PfAP2-G (expression indicated by red nuclei). Early gametocytes expressing the Pfs16 marker are indicated in green. Mixed plaques can arise from asexual schizonts if some rings activate PfAP2-G expression and convert within the same cycle (SCC route). b, Representative IFA images of pure asexual, mixed, and pure sexual plaques. Asexual parasites are multinucleated schizonts, Pfs16-negative and contain a large hemozoin pigment, whereas gametocytes are mono-nucleated, Pfs16-positive and have small hemozoin pigment granules. Arrowheads indicate free hemozoin pigment (residual body) from the parental schizont that originated the plaque. Single nucleated parasites without hemozoin pigment or Pfs16 signal are merozoites that failed to develop. Images are representative of four independent experiments, each including at least three different samples. Scale bar, 5 µm. c, Distribution of plaque types in the wild type line E5, E5-HA-DD treated with Shld at 0-5 hpi (favoring the SCC route), and E5-HA-DD treated at ~30 hpi of the previous cycle (favoring the NCC route). At least 100 plaques of each culture were counted in each experiment. d, Distribution of pure and mixed plaques only among plaques containing ≥1 Pfs16-positive parasite. At least 100 plaques of each culture containing ≥1 sexual parasite were counted in each experiment. In panels c and d, values are the average of three independent biological replicates (red dots), with S.E.M. The distribution of plaque types in panels c and d was significantly different between E5, E5-HA-DD-SCC and E5-HA-DD-NCC (p=0.000 using a two-tailed Fischer’s exact test).
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nihms-1509554-f0002
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f9bd0ee605eca374a7ef270169d50081b202625d1c0d0ab461ebbedd6d38f188
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multiple
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multiple panels: images & plots
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[
"Microscopy",
"Plots and Charts",
"Immuno Assays"
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[
"fluorescence microscopy",
"bar plot",
"immunoblot"
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[
800,
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[{'image_id': 'nihms-1509554-f0001', 'image_file_name': 'nihms-1509554-f0001.jpg', 'image_path': '../data/media_files/PMC6294672/nihms-1509554-f0001.jpg', 'caption': 'Gametocytes can form at the same cycle of PfAP2-G activation.a, Proposed nomenclature for the initial steps and stages of sexual differentiation. Sexual conversion is marked by the onset of gametocyte-specific expression of proteins absent from any replicating blood stages (asexual or sexually-committed). Sexual commitment is defined as a cell state that deterministically results in sexual conversion at a later point. Currently, sexually-committed forms are morphologically undistinguishable from their asexual counterparts. Sexual rings, which have also been named as ring gametocytes or gametorings, are still morphologically indistinguishable from asexual rings, but they mature into stage I gametocytes. Expression of known protein markers for the different stages is shown. Vertical lines indicate reinvasion. b, E5-HA-DD cultures with PfAP2-G stabilized at the ring stage (+Shld) form stage I gametocytes before reinvasion. Gametocytes were detected as mononucleated parasites positive for the early gametocyte marker Pfg27. Similar results were obtained in four independent experiments. Scale bar, 5 µm. c, Gametocytes develop from E5-HA-DD cultures treated simultaneously with Shld and GlcNAc at the ring stage. Similar results were obtained in five independent experiments. Scale bar, 5 µm. d-e, Sexual conversion (proportion of parasites that differentiate into gametocytes, see Methods) in E5-HA-DD cultures with Shld added at different h post-invasion (hpi). GlcNAc was added at the ring stage of the next cycle to determine total sexual conversion (d) or simultaneously with Shld to measure SCC (e). Individual data points, mean and SEM from three independent biological replicates (except for 10-15 hpi, n=2) are shown. p values were calculated using one-way ANOVA. f, FACS-sorted PfAP2-G-eYFP-negative schizonts of the E5-eYFP line produce gametocytes within the first cycle after reinvasion. Bar charts show the proportion of PfAP2-G-eYFP-positive schizonts in unsorted controls and sorted samples, and the proportion of Pfs16-positives among pigmented parasites (within 3 h of sorting and 48 h later), as determined by IFA. In each experiment, >1,000 parasites of each sample were counted. Values are the average of four independent experiments, with S.E.M (except for Pfs16 “sorted”, n=3). p values were calculated using a two-tailed unpaired t-test with equal variance. Scale bar, 5 µm.', 'hash': '96e5ca1426fd4a5fe426a15fe033845d4fd9f45156855006a81e5c4ca68fe551'}, {'image_id': 'nihms-1509554-f0003', 'image_file_name': 'nihms-1509554-f0003.jpg', 'image_path': '../data/media_files/PMC6294672/nihms-1509554-f0003.jpg', 'caption': 'Single-cell RNA-seq identification and characterization of same cycle conversion (SCC) gametocytes.a, Cluster analysis of previously published single-cell transcriptomics data from parasites of the E5-HA-DD line treated with Shld at ~4-16 h post-invasion (hpi) and isolated at ~30, ~36 or ~42 hpi of the same cycle (cycle 1) or at ~42 hpi of the next cycle (cycle 2, stage I gametocytes). See Supplementary Figure 6 legend for a detailed definition of tSNE axes. The plot at the right shows the proportion of parasites from cycle 1 and cycle 2 in selected clusters (normalized per number of cells in each sample) from the analysis of 7,472 cells (5,736 from cycle 1 and 1,736 from cycle 2). The cycle 1 versus cycle 2 composition of cluster 13, cluster 14, and the combined remaining clusters were all non-randomly distributed (all p<1×10−15, two-sided Exact Poisson test). b, Distribution of cells from cycle 1 (Shld-treated and untreated) and cycle 2 between the different clusters from the analysis of 10,509 cells. Cycle 1 values are normalized to account for the number of cells analyzed at each time point. c, Relative abundance of cycle 1 Shld-treated (bright shading) or untreated (pale shading) cells within each cluster. Abundance is normalized to account for the number of cells collected at each treatment condition. Numbers at the right show the relative enrichment for treated cells within each cluster (RE) and the number of cells in each cluster (n). Error bars indicate 95% confidence interval based on two-sided Exact Poisson test. d, Average expression of cluster 13 cycle 2 gametocyte markers (also see Supplementary Table 3 and Supplementary Fig. 6–7) shown for Shld-treated cycle 1 cells outside of cluster 13 (5,552 cells) or within cluster 13 (184 cells), and for Shld-treated cycle 2 cluster 13 cells (1,003 cells). Expression is normalized to 10,000 transcripts per cell. Mean and estimated 95% confidence intervals are shown. e, Same analysis for additional gametocyte markers.', 'hash': '76140dca950fd737c41b0fd876f46c68e27850e72616037df8199e8596a0fe2b'}, {'image_id': 'nihms-1509554-f0004', 'image_file_name': 'nihms-1509554-f0004.jpg', 'image_path': '../data/media_files/PMC6294672/nihms-1509554-f0004.jpg', 'caption': 'Temporal dynamics of pfap2-g transcript levels.a, Reverse transcription-quantitative PCR (RT-qPCR) time-course analysis of pfap2-g expression in tightly synchronized cultures of the non-transgenic parasites lines F12 and E5. Parasite age is expressed in h post-invasion (hpi). b, Expression of pfap2-g in cultures in which 80 nM ML10 was added at 25-30 hpi, and in control cultures without the inhibitor. At 45-50 hpi ML10-treated cultures contained only mature schizonts, whereas control cultures already contained abundant rings. ML10 was washed out of treated cultures and RNA collected 2 h later, when substantial re-invasion had occurred (“wash +2 h”). Images of Giemsa-stained smears of the different preparations (representative of three independent experiments) are shown. Scale bar, 5 µm. c, Time-course analysis of pfap2-g expression in cultures of the E5-HA-DD line maintained in the absence of Shld (-Shld) or with Shld added at 0-5 hpi. Re-synchronization to a 5 h age window was performed between cycles 1 and 2. In cycle 1, pfap2-g expression was significantly higher in the presence of Shld compared to the -Shld condition at all time points analyzed (p=0.002, 0.002 and 0.027 at 10-15, 20-25 and 30-35 hpi, respectively, using a two-sided t-test with equal variance). In all panels, transcript levels were normalized against ubiquitin-conjugating enzyme (uce). Values are the average of two (c) or three (a-b) independent biological replicates (red dots). Error bars in panels a-b are S.E.M.', 'hash': 'c938826a9a2a250266a21415bcd13ffdf1e43d9451a62efaf32277316e3e9781'}, {'image_id': 'nihms-1509554-f0005', 'image_file_name': 'nihms-1509554-f0005.jpg', 'image_path': '../data/media_files/PMC6294672/nihms-1509554-f0005.jpg', 'caption': 'PfAP2-G expression dynamics.a, IFA analysis of PfAP2-G-HA and the early gametocyte marker Pfs16 in E5-HA cultures. Images are representative of six independent experiments. Scale bar, 2 µm. b, IFA analysis of PfAP2-G-HA and the gametocyte marker Pfg27 in E5-HA GlcNAc-treated gametocyte cultures. Images are representative of three independent experiments. Scale bar, 2 µm. c, IFA analysis of PfAP2-G-HA and the gametocyte marker Pfs16 at different h post-invasion (hpi) in Shld-treated E5-HA-DD cultures. d, Proportion of stage I gametocytes (Pfs16-positive) positive for PfAP2-G-HA nuclear signal. In panels c and d, values are the average of two independent biological replicates (red dots). e, IFA analysis of PfAP2-G-eYFP expression in rings arising from FACS-sorted eYFP-negative schizonts of the E5-eYFP line. IFA was performed 3 h (“sorted”) and 20 h (“sorted +20h”, ≤20 hpi rings) after sorting. Pfs16-expression and p values were determined as in Fig. 1f. Values are the average of four independent experiments (red dots), with S.E.M. f, Sexual conversion in E5-HA-DD cultures treated with Shld at 20-25 hpi and with GlcNAc at 5-10 hpi of the following cycle. Shld was removed at the times indicated by the horizontal bars. Gametocytemia was determined at day 7 after GlcNAc addition by analysis of Giemsa-stained smears. “Relative sexual conversion” is the level of sexual conversion (%) relative to the condition in which Shld is present all the time. g, Same as in panel f, but GlcNAc was added simultaneously with Shld at 0-5 hpi to obtain only gametocytes formed by the SCC route. For consistency with panel f, age is expressed as if parasites continued the replicative cycle. In panels f and g, values are the average of three independent biological replicates, with S.E.M. h, A new model of the P. falciparum life cycle in the human blood. Red circles indicate nuclear PfAP2-G expression. When PfAP2-G expression is activated early during the ring stage, gametocyte differentiation proceeds without additional replication (same cycle conversion, SCC). In contrast, when PfAP2-G is activated later, parasites go through one additional round of replication as committed forms before differentiating into gametocytes (next cycle conversion, NCC).', 'hash': '055c92f50c094f4ceaead3f3bf84b7008d20e34ff0f1de991c7c4b3d257c0170'}, {'image_id': 'nihms-1509554-f0002', 'image_file_name': 'nihms-1509554-f0002.jpg', 'image_path': '../data/media_files/PMC6294672/nihms-1509554-f0002.jpg', 'caption': 'Plaque assays reveal that some individual schizonts generate mixed sexual and asexual plaques.a, Schematic of the possible types of plaques originated from schizonts expressing or not expressing PfAP2-G (expression indicated by red nuclei). Early gametocytes expressing the Pfs16 marker are indicated in green. Mixed plaques can arise from asexual schizonts if some rings activate PfAP2-G expression and convert within the same cycle (SCC route). b, Representative IFA images of pure asexual, mixed, and pure sexual plaques. Asexual parasites are multinucleated schizonts, Pfs16-negative and contain a large hemozoin pigment, whereas gametocytes are mono-nucleated, Pfs16-positive and have small hemozoin pigment granules. Arrowheads indicate free hemozoin pigment (residual body) from the parental schizont that originated the plaque. Single nucleated parasites without hemozoin pigment or Pfs16 signal are merozoites that failed to develop. Images are representative of four independent experiments, each including at least three different samples. Scale bar, 5 µm. c, Distribution of plaque types in the wild type line E5, E5-HA-DD treated with Shld at 0-5 hpi (favoring the SCC route), and E5-HA-DD treated at ~30 hpi of the previous cycle (favoring the NCC route). At least 100 plaques of each culture were counted in each experiment. d, Distribution of pure and mixed plaques only among plaques containing ≥1 Pfs16-positive parasite. At least 100 plaques of each culture containing ≥1 sexual parasite were counted in each experiment. In panels c and d, values are the average of three independent biological replicates (red dots), with S.E.M. The distribution of plaque types in panels c and d was significantly different between E5, E5-HA-DD-SCC and E5-HA-DD-NCC (p=0.000 using a two-tailed Fischer’s exact test).', 'hash': 'f9bd0ee605eca374a7ef270169d50081b202625d1c0d0ab461ebbedd6d38f188'}]
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{'nihms-1509554-f0001': ['The process by which parasites commit to sexual development and subsequently convert into gametocytes is not completely understood. Two models for gametocytogenesis were put forward almost forty years ago, proposing that gametocyte formation may take place either within the same cycle of commitment, or after one additional round of multiplication7. Plaque assays, in which the progeny of individual schizonts is visualized in immobilized erythrocytes, demonstrated a non-random distribution of sexual and asexual parasites, such that some schizonts predominantly produced sexual forms whereas others only produced asexual forms8. Later studies combining plaque assays with immunofluorescence assay (IFA) analysis of sexual and asexual markers concluded that the progeny of a single schizont produces only sexual or only asexual parasites, implying commitment at the cycle before differentiation9. Based on these results, the currently accepted model for gametocytogenesis holds that parasites that commit to sexual development go through an additional cycle of replication as committed forms before sexual conversion/differentiation1,3–6. Since there are discrepancies in the terminology used for the initial steps and stages of sexual development, we propose the nomenclature shown in <xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1a</xref>, in which sexual commitment and conversion are defined as distinct, sequential steps of the process., in which sexual commitment and conversion are defined as distinct, sequential steps of the process.', 'In the E5 PfAP2-G-DD parasite line10 (E5-HA-DD hereafter), in which endogenous PfAP2-G is fused to the FKBP destabilization domain, PfAP2-G is stable only in the presence of the Shield-1 (Shld) ligand, whereas in its absence PfAP2-G is degraded and gametocytes never form10. When adding Shld to synchronized E5-HA-DD cultures at the ring stage, we observed single-nucleated Pfg27-positive parasites within as little as ~30 h after Shld addition, when the majority of parasites were still at the schizont stage (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1b</xref>). As Pfg27 is a well-established marker of early gametocytes from stage I onwards). As Pfg27 is a well-established marker of early gametocytes from stage I onwards1,2,4,23, this result suggests that asexual parasites can convert directly into gametocytes without going through an additional round of multiplication after PfAP2-G stabilization. To test this idea, we treated E5-HA-DD cultures at the ring stage simultaneously with Shld and N-acetyl-D-glucosamine (GlcNAc), which at the concentration used (50 mM) inhibits schizont maturation24,25. Indeed, mature gametocytes emerged in these cultures (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1c</xref>), demonstrating that gametocytes can form without going through the PfAP2-G-positive committed schizont stage. This suggests that sexual conversion can occur through a same cycle conversion (SCC) route, in addition to the canonical next cycle conversion (NCC) route involving an additional round of replication after commitment. We define the new SCC route as conversion to sexual gametocyte in the progeny of PfAP2-G-negative schizonts, without additional replication. This implies that commitment, as marked by PfAP2-G expression, and conversion to gametocyte can occur within the same cycle.), demonstrating that gametocytes can form without going through the PfAP2-G-positive committed schizont stage. This suggests that sexual conversion can occur through a same cycle conversion (SCC) route, in addition to the canonical next cycle conversion (NCC) route involving an additional round of replication after commitment. We define the new SCC route as conversion to sexual gametocyte in the progeny of PfAP2-G-negative schizonts, without additional replication. This implies that commitment, as marked by PfAP2-G expression, and conversion to gametocyte can occur within the same cycle.', 'Next, we investigated whether the timing of PfAP2-G stabilization during the intraerythrocytic cycle affects the frequency of sexual conversion via the NCC or SCC routes. E5-HA-DD cultures were tightly synchronized to a 5 h window and Shld was added at 0-5, 10-15, 20-25 or 30-35 h post-invasion (hpi). When we added GlcNAc at the ring stage (~5-10 hpi) of the next cycle to measure sexual conversion by either route, we observed high conversion rates (≥25%) regardless of the timing of PfAP2-G stabilization (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1d</xref>). In sharp contrast, when we measured conversion within the same cycle of PfAP2-G stabilization by adding GlcNAc simultaneously with Shld, conversion was maximal when Shld was added shortly after invasion (0-5 hpi) and nearly negligible when PfAP2-G was stabilized at 20-25 hpi or later (). In sharp contrast, when we measured conversion within the same cycle of PfAP2-G stabilization by adding GlcNAc simultaneously with Shld, conversion was maximal when Shld was added shortly after invasion (0-5 hpi) and nearly negligible when PfAP2-G was stabilized at 20-25 hpi or later (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1e</xref>). These results indicate that when PfAP2-G is stabilized at the early ring stage, sexual conversion frequently occurs within the same growth cycle through the SCC route, whereas PfAP2-G stabilization at later stages results in conversion predominantly at the next cycle via the NCC route. Gametocyte activation and egress assays didn’t reveal differences between gametocytes generated by the SCC or the NCC routes (). These results indicate that when PfAP2-G is stabilized at the early ring stage, sexual conversion frequently occurs within the same growth cycle through the SCC route, whereas PfAP2-G stabilization at later stages results in conversion predominantly at the next cycle via the NCC route. Gametocyte activation and egress assays didn’t reveal differences between gametocytes generated by the SCC or the NCC routes (Supplementary Fig. 1).', 'The E5-HA-DD line shows an unusually high sexual conversion rate when Shld is added (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1d-e</xref>). In other parasite lines, high rates of ). In other parasite lines, high rates of pfap2-g activation would result in high sexual conversion and lower multiplication rates, posing a fitness cost, but this doesn’t occur in the E5-HA-DD line maintained in the absence of Shld. This raises the possibility that the regulation of pfap2-g expression may be altered in this line. To determine whether parasites in which pfap2-g is regulated normally can convert via the SCC route, we tagged endogenous PfAP2-G with the fluorescent marker eYFP using the CRISPR-Cas9 system (E5-eYFP line, Supplementary Fig. 2) and used tightly synchronized E5-eYFP late schizont cultures to FACS-sort PfAP2-G-eYFP-negative parasites (Supplementary Fig. 3). The almost complete absence of PfAP2-G-eYFP-positive schizonts after sorting was validated by IFA (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1f</xref>). IFA analysis with antibodies against the very early gametocyte marker Pfs16). IFA analysis with antibodies against the very early gametocyte marker Pfs161–4,26 conducted 48 h after sorting, when most parasites that continued asexual growth were at the schizont stage, revealed the presence of new Pfs16-positive gametocytes (<xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1f</xref>). For these experiments, we used Pfs16 instead of Pfg27 as a gametocyte marker because we found that it is an absolutely specific marker that is never expressed in PfAP2-G-deficient parasites and its expression starts earlier during gametocyte development (). For these experiments, we used Pfs16 instead of Pfg27 as a gametocyte marker because we found that it is an absolutely specific marker that is never expressed in PfAP2-G-deficient parasites and its expression starts earlier during gametocyte development (Supplementary Fig. 4), as previously reported26. These results show that gametocytes can arise from PfAP2-G-negative asexual schizonts in the first cycle after reinvasion, confirming conversion via the SCC route.', 'a, IFA analysis of PfAP2-G-HA and the early gametocyte marker Pfs16 in E5-HA cultures. Images are representative of six independent experiments. Scale bar, 2 µm. b, IFA analysis of PfAP2-G-HA and the gametocyte marker Pfg27 in E5-HA GlcNAc-treated gametocyte cultures. Images are representative of three independent experiments. Scale bar, 2 µm. c, IFA analysis of PfAP2-G-HA and the gametocyte marker Pfs16 at different h post-invasion (hpi) in Shld-treated E5-HA-DD cultures. d, Proportion of stage I gametocytes (Pfs16-positive) positive for PfAP2-G-HA nuclear signal. In panels c and d, values are the average of two independent biological replicates (red dots). e, IFA analysis of PfAP2-G-eYFP expression in rings arising from FACS-sorted eYFP-negative schizonts of the E5-eYFP line. IFA was performed 3 h (“sorted”) and 20 h (“sorted +20h”, ≤20 hpi rings) after sorting. Pfs16-expression and p values were determined as in <xref rid="nihms-1509554-f0001" ref-type="fig">Fig. 1f</xref>. Values are the average of four independent experiments (red dots), with S.E.M. . Values are the average of four independent experiments (red dots), with S.E.M. f, Sexual conversion in E5-HA-DD cultures treated with Shld at 20-25 hpi and with GlcNAc at 5-10 hpi of the following cycle. Shld was removed at the times indicated by the horizontal bars. Gametocytemia was determined at day 7 after GlcNAc addition by analysis of Giemsa-stained smears. “Relative sexual conversion” is the level of sexual conversion (%) relative to the condition in which Shld is present all the time. g, Same as in panel f, but GlcNAc was added simultaneously with Shld at 0-5 hpi to obtain only gametocytes formed by the SCC route. For consistency with panel f, age is expressed as if parasites continued the replicative cycle. In panels f and g, values are the average of three independent biological replicates, with S.E.M. h, A new model of the P. falciparum life cycle in the human blood. Red circles indicate nuclear PfAP2-G expression. When PfAP2-G expression is activated early during the ring stage, gametocyte differentiation proceeds without additional replication (same cycle conversion, SCC). In contrast, when PfAP2-G is activated later, parasites go through one additional round of replication as committed forms before differentiating into gametocytes (next cycle conversion, NCC).'], 'nihms-1509554-f0002': ['Previous reports described that in immobilized cultures individual schizonts produce plaques comprised of either only gametocytes or only asexual forms9, which led to the generally accepted view that sexual commitment always occurs in the cycle before conversion1,3,4,6. In the light of our identification of the SCC route we wanted to re-examine this observation: if commitment and conversion can occur within the same cycle, parasites arising from an asexual schizont could become gametocytes independently of the choice made by their sibling progeny, resulting in mixed plaques (<xref rid="nihms-1509554-f0002" ref-type="fig">Fig. 2a</xref>). One-cycle plaque assays (). One-cycle plaque assays (<xref rid="nihms-1509554-f0002" ref-type="fig">Fig. 2</xref>) performed with tightly synchronized cultures of the wild type 3D7 subclone E5 indeed revealed that almost 40% of gametocyte-containing plaques consisted of a mixture of Pfs16-positive gametocytes and multinucleated asexual parasites () performed with tightly synchronized cultures of the wild type 3D7 subclone E5 indeed revealed that almost 40% of gametocyte-containing plaques consisted of a mixture of Pfs16-positive gametocytes and multinucleated asexual parasites (<xref rid="nihms-1509554-f0002" ref-type="fig">Fig. 2d</xref>). This proportion of mixed plaques cannot be explained by multiply-infected erythrocytes in the overlaid schizonts preparation (). This proportion of mixed plaques cannot be explained by multiply-infected erythrocytes in the overlaid schizonts preparation (Supplementary Table 1 and Supplementary Fig. 5). To confirm that the levels of mixed plaques reflect the use of the SCC route, in these experiments we included E5-HA-DD cultures treated with Shld at times that result in conversion predominantly via the SCC or the NCC routes, which revealed a much higher proportion of mixed plaques in SCC cultures (<xref rid="nihms-1509554-f0002" ref-type="fig">Fig. 2c-d</xref>). Additional characterization of the plaques is provided in ). Additional characterization of the plaques is provided in Supplementary Fig. 5b-c. Of note, IFA analysis of PfAP2-G in >200 PfAP2-G-positive mature schizonts of the E5-HA-DD and E5-HA lines10 (the latter expressing HA-tagged endogenous PfAP2-G) did not identify any schizont containing a mixture of HA-positive and HA-negative merozoites, supporting the view that mixed plaques arise from direct conversion via the SCC route and not from single schizonts containing sexual and asexual merozoites (<xref rid="nihms-1509554-f0002" ref-type="fig">Fig. 2a</xref>). Abundant mixed plaques were also observed in assays performed with a different 3D7 stock (). Abundant mixed plaques were also observed in assays performed with a different 3D7 stock (Supplementary Table 2 and Supplementary Fig. 5d). Altogether, these results confirm that sexual conversion can occur at the same cycle of commitment in wild type parasites.'], 'nihms-1509554-f0003': ['In a previous study, E5-HA-DD cultures with Shld added at the ring stage (~4-16 hpi) were used for single-cell RNA-seq analysis either during the commitment cycle (~30, ~36 and ~42 hpi of the same cycle of Shld treatment, cycle 1) or at the next cycle (stage I gametocytes, cycle 2)13. Cluster analysis of transcriptional patterns of individual parasites revealed that the majority of cycle 2 gametocytes fall within clusters 13 and 14. Notably, some parasites from the commitment cycle (cycle 1) also fall within these two clusters (<xref rid="nihms-1509554-f0003" ref-type="fig">Fig. 3a-b</xref>). Comparing the relative abundance of Shld-treated versus untreated cells from cycle 1 shows an especially strong enrichment of treated cells for cluster 13 (). Comparing the relative abundance of Shld-treated versus untreated cells from cycle 1 shows an especially strong enrichment of treated cells for cluster 13 (<xref rid="nihms-1509554-f0003" ref-type="fig">Fig. 3c</xref> and and Supplementary Fig. 6). These results suggest that cycle 1 parasites that fall within cluster 13 (4.8% of the total cycle 1 cells, <xref rid="nihms-1509554-f0003" ref-type="fig">Fig. 3b</xref>) converted into stage I gametocytes at the same cycle of Shld treatment using the SCC route.) converted into stage I gametocytes at the same cycle of Shld treatment using the SCC route.', 'To confirm that parasites from the commitment cycle (cycle 1) that fall in cluster 13 are actually stage I gametocytes, we identified the most up-regulated genes expressed by cycle 2 stage I gametocytes in cluster 13 as compared to expression in cells from other clusters. For all of these genes, which include known early gametocyte markers such as pfg27 or pfgexp0223,27, and also for the well-established early gametocyte markers pfs16 and pfg14.748, transcript levels in cycle 1 cluster 13 cells were higher than in cycle 1 cells from other clusters (<xref rid="nihms-1509554-f0003" ref-type="fig">Fig. 3d-e</xref>, , Supplementary Table 3 and Supplementary Fig. 6–7). However, average pfap2-g expression was similar between cluster 13 and other clusters. This is because in contrast to gametocyte-specific markers, pfap2-g is abundantly expressed in sexually-committed cells13, which account for pfap2-g expression within other clusters (<xref rid="nihms-1509554-f0003" ref-type="fig">Fig. 3e</xref> and and Supplementary Fig. 6–7).', 'In spite of high overall transcriptional similarity between cycle 1 and cycle 2 cluster 13 cells (<xref rid="nihms-1509554-f0003" ref-type="fig">Fig. 3a,d</xref>), we identified a small number of transcriptional differences that are likely associated with the route of conversion (), we identified a small number of transcriptional differences that are likely associated with the route of conversion (Supplementary Fig. 8). The early gametocyte marker pfg14.74828 and two other genes with maximal expression in gametocytes29 showed >3-fold higher transcript levels in parasites that converted via the NCC route (cycle 2).'], 'nihms-1509554-f0004': ['The discovery of the SCC route predicts that initial pfap2-g expression can be activated in rings. To test this idea, we employed the gametocyte non-producer F12 line, which contains a non-sense mutation that results in a non-functional PfAP2-G protein10,30. This makes it ideally suited to determine the temporal dynamics of pfap2-g transcription in the absence of gametocytes or the PfAP2-G transcriptional feedback-loop10,13. Time-course analysis of pfap2-g relative transcript levels in F12 revealed a clear peak of expression in 10-15 hpi rings. In contrast, in E5 cultures transcript levels were already high at 0-5 hpi and decreased until 30-35 hpi before increasing again at 40-45 hpi (<xref rid="nihms-1509554-f0004" ref-type="fig">Fig. 4a</xref>).).', 'In addition to late schizonts, 40-45 hpi samples already contain early rings of the new growth cycle. To determine whether mature schizonts actually express pfap2-g, we collected RNA at 45-50 hpi from cultures treated with the cyclic GMP-dependent protein kinase (PfPKG) inhibitor ML10, which blocks merozoite egress and reinvasion31. These experiments revealed expression of pfap2-g in mature schizonts (<xref rid="nihms-1509554-f0004" ref-type="fig">Fig. 4b</xref>), which was confirmed by analysis of magnet-purified schizonts (), which was confirmed by analysis of magnet-purified schizonts (Supplementary Fig. 9a). As a cautionary note, we observed that measuring pfap2-g transcripts from RNA extracted with Trizol directly from Percoll-purified schizonts yielded artifactual results (Supplementary Fig. 9a).', 'To directly assess the effect of PfAP2-G protein on pfap2-g expression, we used cultures of the E5-HA-DD line with or without Shld. As expected, the pfap2-g temporal expression profile in E5-HA-DD without Shld was similar to F12, with peak expression at 10-15 hpi, whereas in Shld-treated cultures expression was high at all the time points analyzed except at 30-35 hpi (<xref rid="nihms-1509554-f0004" ref-type="fig">Fig. 4c</xref>), similar to E5. The higher expression in Shld-treated versus untreated cultures is consistent with PfAP2-G auto-regulating the expression of its own gene), similar to E5. The higher expression in Shld-treated versus untreated cultures is consistent with PfAP2-G auto-regulating the expression of its own gene10,13. Characterization of the expression of other early gametocyte markers4 in the same time-course experiments showed that pfg14.744 transcripts28 are absolutely specific for gametocytes, whereas pfgexp5 transcripts32 and low levels of pfs16 and pfg27 transcripts23,26 can be detected in the absence of functional PfAP2-G and gametocytes (Supplementary Fig. 10), consistent with previous reports17,32,33.'], 'nihms-1509554-f0005': ['We previously described that PfAP2-G-HA is localized in the nucleus of some parasites in cultures synchronized to the ring, trophozoite, or schizont stage10. However, if sexually-committed rings and trophozoites exist, in IFA experiments they would be morphologically indistinguishable from sexual rings and stage I gametocytes, respectively. Using Pfs16 protein as a gametocyte marker26 that is absent from sexually-committed parasites, we found that PfAP2-G-HA is expressed in the nucleus from presumably committed trophozoites to stage I gametocytes (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5a</xref>), although some stages could not be unambiguously identified by this approach (see below). The distribution of PfAP2-G within the nucleus shows very limited overlap with heterochromatin, as expected from the euchromatic location of the majority of its predicted targets), although some stages could not be unambiguously identified by this approach (see below). The distribution of PfAP2-G within the nucleus shows very limited overlap with heterochromatin, as expected from the euchromatic location of the majority of its predicted targets10,13, but in many parasites the PfAP2-G signal appeared to concentrate in the nuclear periphery (Supplementary Fig. 11a-b). At later stages of sexual development (stage II-V gametocytes) PfAP2-G-HA signal is near background levels over the whole parasite and does not show nuclear localization (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5b</xref>). Western blot analysis confirmed a decrease of PfAP2-G-HA protein levels with gametocyte development (). Western blot analysis confirmed a decrease of PfAP2-G-HA protein levels with gametocyte development (Supplementary Fig. 11c).', 'We observed Pfs16 signal in some parasites without detectable pigment (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5a</xref>, sexual ring), consistent with the observation that expression of this marker starts before the previously reported 30-40 hpi, sexual ring), consistent with the observation that expression of this marker starts before the previously reported 30-40 hpi26 (Supplementary Fig. 4). These results suggest that single-nucleated, hemozoin-containing parasites that are positive for PfAP2-G but negative for Pfs16 are sexually-committed trophozoites rather than very early stage I gametocytes that don’t express Pfs16 at detectable levels yet. To confirm that committed forms preceding the schizont stage occur, we analyzed tightly synchronized E5-HA-DD cultures by IFA with Shld added at times that result in different use of the SCC route (10-15 or 20-25 hpi) (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5c</xref>). IFA performed at 40-45 hpi of the same cycle of Shld addition unambiguously distinguished sexually-committed parasites (all multinucleated schizonts, PfAP2-G-positive/Pfs16-negative) from stage I gametocytes (single-nucleated, Pfs16-positive). At 30-35 hpi, the majority of PfAP2-G-positive/Pfs16-negative parasites were mononucleated, suggesting that they are sexually-committed trophozoites. If they were stage I gametocytes in which Pfs16 is not detectable yet, many of them would become Pfs16-positive at 40-45 hpi, but this was not observed: the distribution of parasite types between the two IFA time points indicates that the majority of PfAP2-G-positive/Pfs16-negative parasites at 30-35 hpi actually are committed trophozoites that later develop to committed schizonts. Many Pfs16-positive stage I gametocytes did not express nuclear PfAP2-G-HA and the proportion of nuclear PfAP2-G-HA-positive stage I gametocytes decreased between 30-35 and 40-45 hpi (). IFA performed at 40-45 hpi of the same cycle of Shld addition unambiguously distinguished sexually-committed parasites (all multinucleated schizonts, PfAP2-G-positive/Pfs16-negative) from stage I gametocytes (single-nucleated, Pfs16-positive). At 30-35 hpi, the majority of PfAP2-G-positive/Pfs16-negative parasites were mononucleated, suggesting that they are sexually-committed trophozoites. If they were stage I gametocytes in which Pfs16 is not detectable yet, many of them would become Pfs16-positive at 40-45 hpi, but this was not observed: the distribution of parasite types between the two IFA time points indicates that the majority of PfAP2-G-positive/Pfs16-negative parasites at 30-35 hpi actually are committed trophozoites that later develop to committed schizonts. Many Pfs16-positive stage I gametocytes did not express nuclear PfAP2-G-HA and the proportion of nuclear PfAP2-G-HA-positive stage I gametocytes decreased between 30-35 and 40-45 hpi (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5d</xref>), indicating that PfAP2-G-HA is present in the nucleus of early but not older stage I gametocytes.), indicating that PfAP2-G-HA is present in the nucleus of early but not older stage I gametocytes.', 'The SCC route implies that de novo expression of PfAP2-G can start before nuclear replication. Indeed, we observed PfAP2-G-eYFP-positive rings among the progeny of FACS-sorted PfAP2-G-eYFP-negative schizonts of the E5-eYFP line analyzed by IFA 20 h after sorting (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5e</xref>). In this set of experiments we again determined the proportion of new Pfs16-positive parasites (gametocytes) formed in the first cycle after reinvasion, which roughly corresponded to the proportion of PfAP2-G-positive rings (). In this set of experiments we again determined the proportion of new Pfs16-positive parasites (gametocytes) formed in the first cycle after reinvasion, which roughly corresponded to the proportion of PfAP2-G-positive rings (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5e</xref>). This result suggests that activation of PfAP2-G in rings typically results in SCC, although it remains possible that some PfAP2-G-positive rings develop as replicating sexually-committed forms (those would be sexually-committed rings). The idea that PfAP2-G expression can be activated before the committed schizont stage was also supported by IFA analysis over consecutive stages, which revealed a higher proportion of PfAP2-G-positive parasites in cultures at the ring or trophozoite stages than in schizonts of the previous cycle (). This result suggests that activation of PfAP2-G in rings typically results in SCC, although it remains possible that some PfAP2-G-positive rings develop as replicating sexually-committed forms (those would be sexually-committed rings). The idea that PfAP2-G expression can be activated before the committed schizont stage was also supported by IFA analysis over consecutive stages, which revealed a higher proportion of PfAP2-G-positive parasites in cultures at the ring or trophozoite stages than in schizonts of the previous cycle (Supplementary Fig. 12).', 'To identify at which stages PfAP2-G is needed for productive sexual conversion via the NCC and the SCC routes, Shld was removed from tightly synchronized E5-HA-DD cultures at different times. Under conditions promoting the NCC route, PfAP2-G stabilization was required until at least the ring stage of the second cycle (sexual ring stage) for high gametocyte production (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5f</xref>). In contrast, when conversion was restricted to the SCC route, more than half of the gametocytes could still form when Shld was removed as early as 41-46 hpi of the first cycle (). In contrast, when conversion was restricted to the SCC route, more than half of the gametocytes could still form when Shld was removed as early as 41-46 hpi of the first cycle (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5g</xref>). These results indicate that for both conversion routes PfAP2-G is no longer essential at the gametocyte stage I or even earlier.). These results indicate that for both conversion routes PfAP2-G is no longer essential at the gametocyte stage I or even earlier.', 'Based on the results described here and published data, we propose an extended model for the early steps of sexual differentiation in P. falciparum. After the chromatin at the pfap2-g locus adopts a permissive state10,19,20, either spontaneously18,35 or induced by external cues1–6,36,37, pfap2-g is transcribed mainly at the ring and late schizont stages. High expression of the gene requires a positive feedback loop involving the PfAP2-G protein. In some parasites, pfap2-g expression starts at the early ring stage and PfAP2-G levels reach a threshold sufficient to trigger the sexual transcriptional program before the onset of nuclear division, resulting in expression of early gametocyte markers and inhibition of the replicative asexual program. In such parasites, sexual conversion initiates within the same cycle of commitment as marked by PfAP2-G expression (SCC route) (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5h</xref>). In contrast, in other parasites in which PfAP2-G expression is activated, the protein levels necessary to trigger conversion are not reached early enough for SCC. We predict the existence of a checkpoint after which conversion in the same cycle is no longer possible. These parasites complete the replicative cycle as sexually-committed forms, which can include at least sexually-committed trophozoites and schizonts. After reinvasion, the new rings (sexual rings) have the ). In contrast, in other parasites in which PfAP2-G expression is activated, the protein levels necessary to trigger conversion are not reached early enough for SCC. We predict the existence of a checkpoint after which conversion in the same cycle is no longer possible. These parasites complete the replicative cycle as sexually-committed forms, which can include at least sexually-committed trophozoites and schizonts. After reinvasion, the new rings (sexual rings) have the pfap2-g locus in an active chromatin conformation inherited from the previous cycle by epigenetic mechanisms18, and already carry nuclear PfAP2-G protein ready to drive its own expression10,13. Together, these conditions guarantee that high PfAP2-G levels are reached very early during the ring stage, resulting in sexual conversion one cycle after commitment (NCC route) (<xref rid="nihms-1509554-f0005" ref-type="fig">Fig. 5h</xref>). The observation that PfAP2-G is absent from the nucleus and dispensable from early stages of gametocyte development is consistent with a role for this protein largely restricted to triggering the sexual transcriptional program, which later involves additional ApiAP2 transcriptional regulators). The observation that PfAP2-G is absent from the nucleus and dispensable from early stages of gametocyte development is consistent with a role for this protein largely restricted to triggering the sexual transcriptional program, which later involves additional ApiAP2 transcriptional regulators13,38,39.']}
|
Plasmodium falciparum Revisiting the initial steps of sexual development in the malaria parasite
| null |
Nat Microbiol
|
1548489600
|
[{'@Label': 'BACKGROUND', '#text': 'Very preterm (VPT) infants are at-risk for altered growth, slower speed of processing (SOP), and hypertension. This study assesses the relationship between postnatal body composition (BC), neurodevelopment (indexed by SOP), and blood pressure (BP) in VPT infants.'}, {'@Label': 'METHODS', '#text': 'Thirty-four VPT infants underwent weekly measurements and BC testing until discharge and post-discharge at 4\u2009mos CGA and 4\u2009yrs. At post-discharge visits, SOP was assessed using visual evoked potentials and the NIH Toolbox; BP was also measured.'}, {'@Label': 'RESULTS', '#text': 'In-hospital rate of weight, length and fat-free mass (FFM) gains were associated with faster SOP at 4 yrs. Higher rate of gains in weight and FFM from discharge to 4\u2009mos CGA were associated with faster SOP at 4\u2009mos CGA, while higher fat mass (FM) gains during the same time were positively associated with BP at 4\u2009yrs. BC at 4\u2009yrs nor gains beyond 4\u2009mos CGA were associated with outcomes.'}, {'@Label': 'CONCLUSIONS', '#text': 'In VPT infants, early FFM gains are associated with faster SOP, whereas post-discharge FM gains are associated with higher BPs at 4\u2009yrs. This shows birth to 4\u2009mos CGA is a sensitive period for growth and its relation to neurodevelopmental and metabolic outcomes. Close monitoring and early nutritional adjustments to optimize quality of gains may improve outcomes.'}]
|
[
"Anthropometry",
"Blood Pressure",
"Body Composition",
"Central Nervous System",
"Child, Preschool",
"Evoked Potentials, Visual",
"Female",
"Humans",
"Infant, Extremely Premature",
"Male",
"Prospective Studies"
] |
other
|
PMC6294672
| null |
41
|
[
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] |
Nat Microbiol. 2019 Jan 26; 4(1):144-154
|
NO-CC CODE
|
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