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First published online December 3, 2008
doi: 10.1242/10.1242/jcs.034876

Journal of Cell Science 121, 4018-4028 (2008)
Published by The Company of Biologists 2008
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The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells

Vered Raz1,2,*, Bart J. Vermolen3,4, Yuval Garini4,5, Jos J. M. Onderwater1, Mieke A. Mommaas-Kienhuis1, Abraham J. Koster1, Ian T. Young4, Hans Tanke1 and Roeland W. Dirks1

1 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
2 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
3 Biophysical Engineering Group, Faculty of Sciences and Technology, Twente University, 7500 AE Enschede, The Netherlands
4 Quantitative Imaging Group, Department of Applied Sciences Delft University of Technology, Delft, The Netherlands
5 Physics Department and Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel


Figure 1
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  		Fig. 1. Spatial changes in lamina organization during cellular senescence in hMSCs. Changes in the spatial organization of lamin A during senescence of hMSCs. Cells at passage 3 were transduced with the lamin A-EGFP lentiviral vector and cultured for 9 passages. (A) Confocal images were taken from living cells at passage 4, 6, and 12 (PS 4, PS 6 and PS 12, respectively). Maximum projections of x-y and y-z axes from representative nuclei (left). Cross-sections 55 nm deep were made from the y-z axis. Shown are five serial sections at equal intervals (right). (B) Electron microscope images of horizontal (i,ii) or vertical (iii,iv) sections of hMSCs at passage 9, taken from normal (i,iii) or senescent cells (ii,iv). N, nucleus.

 

Figure 2
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  		Fig. 2. Centromeres shift to peripheral localization in cellular senescent hMSCs. Cells at passage 3 or 9 were transduced with the CenpA-EGFP (green) and the Lamin A-DsRed (red) lentiviral vectors, and imaging was carried out on cells at passages 6 and 12. (A) Maximum projections of cells at passage 6 or 12 on the x-y and y-z axis. The spatial localization of centromeres was obtained from cells at passage 6 and 12. CDF plots of centromere spatial localization are shown (right). CDF plots show the normalized frequency as a function of a normalized nucleus radius. Statistics show pooled data taken from 12 cells per passage. The Kolmogorov-Smirnov Test (KS Test) P value is indicated and suggests that the underlying distributions differ significantly. (B) Quantification of spatial overlap between CenpA and lamin A. Images of CenpA-EGFP and lamina-DsRed were taken from hMSCs as described in A. Quantitative overlap fluorescence intensities between CenpA and lamina A were spatially analyzed in cross-sections. Maximum projections (x-y axis) of lamin A-DsRed (red) and CenpA-EGFP (green) in a representative cell at passage 6 or passage 12. Fluorescence intensities of both probes are plotted in the y-z or x-z axes (graphs below and to right of confocal images). The spatial overlap between lamin A and CenpA is indicated by arrows. Histograms show the percentage of CenpA overlapping with lamin A, in cells at passage 6 and 12. Results are mean ± s.d. of ten cells.

 

Figure 3
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  		Fig. 3. Telomeres form aggregates during cellular senescence in hMSCs. (A) Telomeres form aggregates in cells at passage 12. Cells at passage 3 and 12 were labeled with PNA probes to identify telomeres. 3D image processing and quantitative image analysis of PNA fluorescence was carried out using TeloView. (Ai) Maximum projections of PNA hybridization from representative nuclei at passage 3 (a) and 12 (b and c). A nucleus with mainly telomere aggregates (c) represents a class of cells found at passage 12, which were excluded from statistical analyses. (Aii) Telomere fluorescence intensity in cells at passage 3 and 12. The PNA fluorescent dots were sorted and plotted according to their intensity. Shown are the intensity plots obtained from a typical cell in passage 3 (open squares) and 12 (closed triangles). The thresholds for definition of small and big telomere aggregates are indicated with straight or dashed red lines, respectively. (Aiii) B-box plots of the ratio of big telomere intensities (i>T) or small telomere intensities (t<i<T) to all telomeres in cells at passage 3 and passage 12. (Aiv) B-box plots of the ratio in the fluorescent intensity between telomere aggregates (i>t) (agg.) to normal telomeres, and the ratio between the number of spots found in aggregates to total PNA spots. Statistical analyses represent 1400 PNA fluorescent dots obtained from 20 cells per passage. Based on fluorescence intensity, the PNA fluorescent dots are calculated as equivalent to 90-100% of expected telomeres. Cells with less telomeres (Fig. 2Aic) were excluded from statistical analyses. (B) Telomere organization correlates with changes in nuclear shape. hMSCs at PS 9 were labeled with PNA followed by immunofluorescence with anti lamin B antibody (visualized in green). 3D reconstructions of confocal images were carried out using Teloview. Based on lamin B organization and the nuclear depth, cells were grouped into normal and senescent groups. Maximum projections of the x-y and y-z axis show representative normal or senescent cells B-box plots on the right show the ratio in the fluorescent intensity between telomere aggregates (i>t) (agg.) to normal telomeres, and the ratio between the number of spots found in aggregates and total PNA spots. Statistical analysis involved 1050 PNA fluorescent dots obtained from 15 cells per group.

 

Figure 4
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  		Fig. 4. Comparison of telomere and centromere spatial organization in 35y and 81y hMSCs. The 35y and 81y cells were isolated from two individuals at age 35 (35y) and 81 (81y). (A) Formation of telomere aggregates in 81y cells at passage 6. Cells at passage 5 were transduced with TRF1-DsRed lentiviral vectors. 3D B-box plots show the ratio in the fluorescent intensity between small telomere aggregates and normal telomeres [(t<i<T)/(i<t)], the ratio in fluorescent intensity between large telomere aggregates and normal telomeres [(i>T)/(i<t)] and the ratio between the number of dots found in aggregates to total TRF1 fluorescent dots. CDF plots (bottom left) show the normalized frequency as a function of a normalized nucleus radius. The P value (KS Test) is indicated and suggests that the underlying distributions differ significantly. Statistical analyses represent 1750 TRF1-DsRed fluorescent dots obtained from 25 cells per cell class. (B) CDF plots of CenpA spatial localization in 35y and 81y cells. Cells at passage 5 were transduced with CenpA-EGFP lentivirus vectors. Confocal images were taken from living cells at passage 6. The plots show the normalized frequency as a function of a normalized nucleus radius. The P value (KS Test) is indicated and suggests that the underlying distributions differ significantly. Statistical analyses represent 525 CenpA-fluorescent dots obtained from 15 cells per cell class.

 

Figure 5
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  		Fig. 5. Telomere aggregates are associated with {gamma}-H2AX foci but not with TERT. (A) Telomere aggregates do not overlap with TERT in senescent cells. The 81y cells were transduced at passage 6 with Trf2-citrine lentiviral vector, and immunodetection with anti-TERT and anti-lamin-A antibodies was carried out on cells at passage 7. Left panels show maximum projections of TERT (red) and lamin A (blue) in normal (i) or senescent (ii,iii) cells. Right panels show maximum projections of TRF2 and TERT in the x-y axis. Fluorescence intensity graphs on the y-z or x-z axis show overlap in distribution (indicated with white arrows). Gray arrows indicate large TRF2 aggregates. (B) TERT colocalizes with intranuclear structures of the lamina in senescent hMSCs. Immunofluorescence with anti-TERT (green) and anti-lamin A (red) antibodies was carried out on hMSCs at passage 9. (i) Wide-field fluorescence microscope images show a correlation between lamina deformed shape and reduced expression level of TERT in senescent cells. Scale bars are equal 15 µm. (ii,iii) Cross-sections (x-y axis) of confocal images show a cell with a normal ellipse lamina structure (ii) or a distorted lamina shape (iii). The overlap in spatial localization between lamin A (red) and TERT (green) is indicated in the intensity plots in the merged images. (C) Large telomere aggregates overlap with {gamma}-H2AX in cells with a folded lamina structure. The 81y cells were transduced at passage 6 with TRF1-citrine followed by transduction with lamin A. At passage 7, cells were immunolabeled with anti {gamma}-H2AX antibody. Left panel shows merged images of lamin A-EGFP (blue) and TRF1-citrine (yellow), middle panel shows merged images of lamin A-EGFP (blue) and {gamma}-H2AX (red), and right panel shows merged images of TRF1-citrine (yellow) and {gamma}-H2AX (red) in normal (i) or senescent (ii,iii) cells.

 

Figure 6
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  		Fig. 6. Increase in telomere binding to lamina intranuclear structures. Telomeres bind to the nuclear lamina. (A) U2OS cells were transduced with lentiviral vectors that express the lamin B (LB)-WT or lamin A (LA)-WT fused to GFP. As controls, cells were transduced with CMV-GFP or TRF1-citrine lentiviral vectors, or were left nontransduced (NT). Cells were crosslinked with formaldehyde 2 days after transduction, and isolated chromatin was used for chromatin immunoprecipitation with antibodies against GFP. Purified DNA from the immunoprecipitations and input fractions were used for QPCR using telomere primers. Histograms show % recovery after normalization to data obtained from NT cells. Data represent the average of two independent experiments. (B) Enrichment of telomere binding to the nuclear lamina in cells expressing mutation in lamina genes. Human MSCs at passage 6 were transduced with the lentiviral vectors that express the lamin B(LB)-WT, lamin A (LA)-WT, LA-R133L, LA-R220Q or LB-L158D fused to EGFP. Five days after transduction, cells were crosslinked with formaldehyde, and isolated chromatin was used for chromatin immunoprecipitation (ChIP) with antibodies against GFP. Purified DNA from the immunoprecipitations and input fractions were used for QPCR using telomere-specific primers. Percentage recovery was normalized to data obtained from NT cells. Histograms show fold enrichment in % input LA-R133L or LA-R220Q over LA-WT, and LB-L158D over LB-WT. Averages represent two independent experiments. (C) Maximum projections of x-y axis taken from representative hMSCs at passage 5 expressing TRF1-DsRed together with LA-WT-EGFP, LA-R133L-EGFP, LA-R220Q-EGFP and LB-L158D-EGFP. Overlap between lamina intranuclear structures and telomere aggregates is shown in representative single cross-sections at the x-z and y-z axis. Quantification of telomere aggregates in hMSCs expressing the LA-R133L, LA-R220Q and LB-L158D mutants. (D) TRF1 fluorescent intensities were measured from confocal images taken from cells. Plots show the fluorescent intensities of TRF1-DsRed dots from representative cells expressing LA-WT (blue dots), LA-R133L (pink dots), LA-R220Q (red dots) and LB-L158D (green dots) fused to EGFP. The thresholds for calculation of small and large telomere aggregates are indicated with straight or dotted lines, respectively. (E) hMSCs at passage 6 were transduced with Lamin-B-EGFP (green) and the TRF1-DsRed (red) lentiviral vectors. Confocal images were taken from normal or senescent living cells at passage 8. 3D reconstructions were processed in TeloView, single sections on the x-z axis were produced in DipImage at equal intervals. The boxed images are a x1.65 magnification of the x-z sections.

 

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© The Company of Biologists Ltd 2008