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First published online 26 September 2006
doi: 10.1242/jcs.03180

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Changes in lamina structure are followed by spatial reorganization of heterochromatic regions in caspase-8-activated human mesenchymal stem cells

¹ Department of Molecular Cell Biology, Leiden University Medical Center, Einsteinweg 20, 2300RC Leiden, The Netherlands
² Quantitative Imaging group, Department of Applied Sciences Delft University of Technology, Delft, The Netherlands
³ Department of Physics, Bar-Ilan University, Ramat-Gan 52900, Israel

View larger version (61K): [in a new window]   Fig. 1. Induction of apoptosis in hMSCs using lentivirus vector expressing a constitutive (CC8) or an inducible caspase-8 (FKC8). (A) Human MSCs at passage four were transduced with lentivirus vector containing FKC8. Graphs show the cultures before or after treatment with the FKC8 activator AP20187. Bar, 20 µm. (B) Quantification of apoptotic cells after caspase-8 activation. Human MSCs were transduced with CC8 or FKC8. FKC8 was activated with 100 nM AP20187 (+AP) and caspase activity was inhibited with 50 nM Z-VAD.fmk (+Z). Treatments were carried out for 6 hours. Nuclei were stained with DAPI. Histograms show the fraction of cells with condensed DNA as calculated from a population of 250 nuclei. (C) Detection of FKC8-activation pathway using immunostaining. Human MSCs at passage six were transduced with FKC8 followed by AP20187 treatment for 6 hours (+FKC8). Control cells were left non-transduced. Subsequently, cells were fixed and immunostained with anti-caspase-8 (green) or anti-cleaved caspase-3 (red) antibodies. DAPI staining was used to monitor changes in DNA morphology. Nuclear morphology is indicated on the left side. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 2. Changes in lamina morphology during FKC8-induced apoptosis. (A) Human MSCs at passage four were transduced with lentivirus vectors encoding lamin A-DsRed, histone H4-CFP or lamin B-GFP. Cells were finally transduced with FKC8. Control cells were left without AP20187 treatment (mock-treated), and FKC8 was activated by incubating the cells with 100 nM AP20187 for 6 hours. Confocal images were taken from representative nuclei. Images were processed with TeloView and show views of the xy-axis (square) and xz-axis (rectangle) of cells. Bar, 5 µm. (B) Visualization of fragmented DNA and the lamina after FKC8 activation. Cells expressing FKC8 were treated with 100 nM AP20187 for 6 hours or were left without AP20187 treatment. Fragmented DNA (green) was visualized with the Cell Death Detection kit, whereas the lamina was detected with anti lamin B1 antibody (red). Short arrows indicate convoluted nuclei and long arrows indicate nuclei with degraded lamin B. Bar, 10 µm. (C) The effect of Z-VAD.fmk treatment on lamina organization. Human MSCs at passage four were transduced with lamin B-GFP and FKC8 lentiviral vectors. Cells were treated with 50 nM Z-VAD.fmk for two days and 10 nM AP20187 was added during the last 5 hours (+AP+Z). Control cells were treated with 10 nM AP20187 only (+AP). Confocal images were taken from living cells. Images obtained after TeloView processing show maximum projections in xy-axis and xz-axis in green, and a single optical section in the xy-axis in gray. Bar, 5 µm. (D) Statistical analyses of changes in nuclear depth upon caspase-8 activation. Human MSCs at passage four were subsequently transduced with vectors coding for lamin B-GFP and CC8 or FKC8. Control cells were transduced with lamin B-GFP vector only. CC8-transduced cells were examined 16 hours post-transduction. FKC8-transduced cells were treated with either 10 nM or 100 nM AP20187 for 5 hours. Nuclear depth was measured from confocal images. 120 nuclei were evaluated.

View larger version (40K): [in a new window]   Fig. 3. Centromere spatial localization in FKC8-activated hMSCs. Human MSCs at passage four were transduced with CenpA-GFP followed by FKC8 lentiviral vectors. Confocal images were taken from living cells before (mocked-treated) or 6 hours after 100 nM AP20187 treatment (+AP20187). Images were processed with TeloView. Bar, 5 µm. (A) 3D representation of a nucleus exhibiting a disk or a convoluted morphology. CenpA-GFP is shown in green. DIC image in the xy-axis reveals the nuclear rim. Underneath each image, a graph shows the spatial distance of centromeres from the center of mass (CM) after analyses in the algorithm centromeres. (B) Statistical analysis of centromere distribution in FKC8-transduced cells. The spatial localization of CenpA-GFP was evaluated using the centromeres algorithm. The average of CenpA-GFP dots close to center of mass (CM) (0-0.3 normalized distance) or close to the periphery (0.7-1 normalized distance) was calculated. FKC8-transduced cells were treated with 100 nM AP20187 (+AP) only or together with 50 nM Z-VAD.fmk (+AP+Z) for 5 hours. Control cells were mock-treated. AP20187-treated cells were sorted according to nuclear shape (round or convoluted). 50 nuclei were evaluated.

View larger version (56K): [in a new window]   Fig. 4. Spatial organization of telomeres during FKC8-induced apoptosis in hMSCs. (A) Human MSCs at passage four were transduced with lentivirus vectors encoding either Trf2-citrine (shown in green) or Trf1-DsRed (shown in red), followed by a transduction with the lamin A-DsRed (red) or the lamin B-GFP (green) encoding vectors. The cells were finally transduced with FKC8. Confocal images were taken from living cells before (mock-treated) or 4-6 hours after 100 nM AP20187 treatment (+AP20187). Images were processed with TeloView to quantify the fluorescence dots. Images show representative nuclei. Bar, 5 µm. (B) Quantification of telomere fluorescence intensity. The fluorescent dots obtained in A were sorted and plotted according to their intensity. The intensity graph obtained from the control cells is shown in yellow, and that obtained from FKC8-activated cells, showing a convoluted nuclear shape, is shown in green. (C) Statistical analyses of telomere organization. Images of mock-treated or AP20187-treated cells, as described in A, were processed in TeloView. Statistical analyses of Trf1-DsRed spatial organization included the percentage of telomeres in close spatial distance, the percentage of fluorescent particles in aggregates, and the ratio between the two intensity slopes. AP20187-treated cells (+AP) were sorted according to the lamina shape. Forty nuclei were evaluated. (D) DIPimage image analysis shows one optical section along the xz-axis of lamin B-GFP-expressing (green) and Trf1-DsRed-expressing (red) cells. Images of representative nuclei within the subclasses, yellow, red, blue and green of C (a, b, c and d, respectively) were processed in DIPimage. Yellow shows overlap between lamin B-GFP and Trf1-DsRed (indicated by arrows). Bar, 5 µm.

View larger version (29K): [in a new window]   Fig. 5. Mutations in lamin A or lamin B genes that make them caspase-3-sensitive affect lamina and telomere spatial organization. (A) Western blot analysis of lamin degradation in 293T cells. 293T cells were transduced with lentiviral vectors expressing wild-type lamin B-GFP, lamin B-L158D-GFP, wild-type lamin A-DsRed or lamin A V221D-GFP. Subsequently, half of the transformed cells were transduced with LV-CC8 (+). Proteins were extracted 18 hours after transduction and subjected to Western blot analysis. Lamin B-GFP was detected with an anti-GFP antibody and lamin A-DsRed was detected with an anti-lamin A/C antibody. Long arrows indicate the fused protein and small arrows the cleaved products. (B) Quantification of lamina morphologies in hMSCs. Human MSCs at passage four were transduced with lentivirus vectors coding for wild-type lamin B-GFP, lamin B L158D-GFP, wild type lamin A-DsRed, or the lamin A V221D-DsRed. Lamina morphology was determined by fluorescence microscopy. Histograms show the percentage of cells exhibiting intranuclear structures (purple) or degraded (red) lamina structures (as shown in Fig. 2). Maximum projections of confocal images show telomere organization in cells expressing lamin mutants. (C) hMSCs at PS6 were transduced with Trf1-DsRed (red) and lamin B L158D-GFP (green). In cells expressing lamin A V221D-DsRed (red), TRF2 was detected by immunostaining (blue). After TeloView processing, maximum projections in the xy-axis and xz-axis (in green), and a single section in the xy-axis (gray) are shown. Bar, 5 µm.

View larger version (73K): [in a new window]   Fig. 6. Spatial organization of PML bodies in caspase-8 activated hMSCs. Human MSCs at passage four were transduced with CenpA-GFP (A) or lamin B-GFP (B), and with FKC8 lentiviral vectors. FKC8 was activated with 100 nM AP20187 for 6 hours (+AP20187). Mock-treated cells were left without AP20187 treatment. Subsequently, cells were subjected to immunostaining using an anti-hPML antibody. Confocal images were processed with TeloView. CenpA-GFP and lamin B-GFP are shown in green and PML is shown in red. Bars, 5 µm.