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. 2020 May 5;118(9):2319-2332.
doi: 10.1016/j.bpj.2020.04.001. Epub 2020 Apr 14.

Chromatin and Cytoskeletal Tethering Determine Nuclear Morphology in Progerin-Expressing Cells

Maria Chiara Lionetti  1 , Silvia Bonfanti  2 , Maria Rita Fumagalli  3 , Zoe Budrikis  2 , Francesc Font-Clos  2 , Giulio Costantini  2 , Oleksandr Chepizhko  4 , Stefano Zapperi  5 , Caterina A M La Porta  6
Affiliations

Chromatin and Cytoskeletal Tethering Determine Nuclear Morphology in Progerin-Expressing Cells

Maria Chiara Lionetti et al. Biophys J. .

Abstract

The nuclear morphology of eukaryotic cells is determined by the interplay between the lamina forming the nuclear skeleton, the chromatin inside the nucleus, and the coupling with the cytoskeleton. Nuclear alterations are often associated with pathological conditions as in Hutchinson-Gilford progeria syndrome, in which a mutation in the lamin A gene yields an altered form of the protein, named progerin, and an aberrant nuclear shape. Here, we introduce an inducible cellular model of Hutchinson-Gilford progeria syndrome in HeLa cells in which increased progerin expression leads to alterations in the coupling of the lamin shell with cytoskeletal or chromatin tethers as well as with polycomb group proteins. Furthermore, our experiments show that progerin expression leads to enhanced nuclear shape fluctuations in response to cytoskeletal activity. To interpret the experimental results, we introduce a computational model of the cell nucleus that explicitly includes chromatin fibers, the nuclear shell, and coupling with the cytoskeleton. The model allows us to investigate how the geometrical organization of the chromatin-lamin tether affects nuclear morphology and shape fluctuations. In sum, our findings highlight the crucial role played by lamin-chromatin and lamin-cytoskeletal alterations in determining nuclear shape morphology and in affecting cellular functions and gene regulation.

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Figures

Figure 4
Figure 4
Progerin induction leads to larger shape fluctuations due to cytoskeletal activity. (A) Illustration of local displacements method is shown: given meshes for two time points T0, T1, the algorithm finds the vectors vi used to compute σ(d); see Materials and Methods for details. (B) Boxplot of σ(d) over time for untreated cells is shown, comparing cells overexpressing WT lamin A (dark gray) or Δ50 lamin A (light gray). (C) and (D) show boxplots of σ(d) over time, comparing control cells (gray) with blebbistatin-treated cells (green) for (C) WT lamin A and (D) progerin cases. Additional panels show cells treated with (E and F) cytochalasin (blue boxplots), (G and H) jasplakinolide (orange boxplots), and (I and J) SMIFH2 (violet boxplots). For each time point, local displacements are computed with respect to the previous time point. Statistical significance is measured with Kolmogorov-Smirnov tests, with ∗∗ (∗) marking p < 0.01 (0.05). p-Values are corrected for multiple testing; see Materials and Methods for details. Data have been collected over at least three independent experiments. Error bars in boxplots indicate minimum and maximum quartiles, boxes are first and third quartiles. To see this figure in color, go online.
Figure 1
Figure 1
Progerin expression affects nuclear morphology. Typical nuclei obtained from (A) subconfluent HGPS cells (HGPS), healthy mother of HGPS patient (Healthy), or (D) HeLa Tet-On progerin-expressing cells without Doxy treatment (No Doxy) or after induction with 10 ng/mL with doxycyclin (Doxy) are shown. Subconfluent cells were fixed with ice-cold methanol, then incubated with anti-PanLamin (1:50, ab20740; Abcam) at 4°C overnight and with AlexaFluo488 (1:250, ab15113; Abcam) for 1 h at RT. Nuclei were stained with DAPI. Images were acquired with a Leica SP2 laser scanning confocal microscope. Quantification of morphological alterations of (B and C) HGPS cells and mother of HGPS cells and (E and F) HeLa Tet-On progerin-expressing cells (Doxy) and HeLa Tet-On cells without Doxy treatment (No Doxy) by computing the number of blebs (B and E) and the curvature fluctuations (C and F) as described in the Materials and Methods. Statistics was performed over 297 No Doxy nuclei, 330 Doxy nuclei, 87 healthy nuclei, and 87 HGPS nuclei. The p-values reported were obtained via a Kolmogorov-Smirnov test comparing the distribution of the relevant magnitude among the two groups (Healthy versus HGPS or Doxy versus No Doxy). The computation is implemented via the “ks_2samp” function from the SciPy library (35). Data have been collected over at least three independent experiments. Error bars in boxplots indicate minimum and maximum quartiles, boxes are first and third quartiles. To see this figure in color, go online.
Figure 2
Figure 2
Progerin induction leads to fascin phosphorylation in S39 without affecting nesprin-2 expression. (A) Typical experiment of Western blot of giant nesprin in HeLa Tet-On 3G progerin-expressing cells treated with increasing concentration of Doxy or without (No Doxy) is shown. 20 μg total protein was loaded on 10% polyacrylamide gel, transferred on PVDF, and incubated with nesprin-2 (1:1000, MABC86; Merck Millipore) overnight at 4°C. Anti-β-tubulin antibody (1:5000, T8328; Sigma-Aldrich) for 1 h at RT was used as housekeeping. (B) Densitometric analysis of two independent Western blot experiments of giant nesprin Western blot as shown in (A) is given. Densitometric analysis was carried out with ImageJ software. The y axis shows the ratio between the mean of the densitometric value of giant nesprin with respect to the housekeeping β-tubulin. Statistical significance was established by t-test. (C) Typical Western blot of S39-fascin in fascin-immunoprecipitated samples obtained from HeLa Tet-On progerin-expressing cells treated or untreated with Doxy is shown. Subconfluent cells were processed for immunoprecipitation as described in the Materials and Methods. Briefly, 500 μg of total proteins was incubated with anti-fascin antibody (1:100, ab126772; AbCam) overnight at 4°C under stirring. 50% bead slurry of Protein A-Agarose (P9269; Sigma-Aldrich) was added to the lysate and reincubated with gentle rocking for 2 h at 4°C. After three washes with 500 μL of lysis buffer, the sample was resuspended in 30 μL 2× Laemmli sample buffer and heated to 90°C for 5 min, and 15 μL of immunoprecipitated sample was loaded on 10% SDS-PAGE for Western blot. (D) Densitometric analysis of two independent experiments as shown in (C) is given. The y axis shows the ratio between the mean of the densitometric value of S39-fascin and fascin.
Figure 3
Figure 3
Effect of progerin induction on nuclear tethering factors by proximity ligation assay. The tethering is quantified by the proximity ligation assay measuring the interaction between all lamins detected using PanLamin antibody and (A and B) SUN1 or (D and E) emerin. Briefly, subconfluent cells were fixed on slides with ice-cold 100% methanol for 5 min. Slides were then incubated in a humidity chamber overnight at 4°C with PanLamin (1:50, ab20740; Abcam) antibody and with SUN1 (1:200, ab103021; Abcam) or emerin (1:200, ab40688; Abcam). After washing, the samples were incubated in a preheated humidity chamber for 1 h at 37°C with anti-rabbit PLUS and anti-mouse MINUS PLA probes diluted 1:5. Ligation and amplification steps were performed according to the manufacturer’s instructions. Slides were mounted with Duolink In Situ Medium. The number of aggregates linking (C) SUN1 or (F) emerin and lamins are quantified as described in the Materials and Methods. The analysis has been carried out on 72 nuclei for SUN1 (36 without Doxy and 36 with Doxy) and 76 nuclei for emerin (53 without Doxy and 23 with Doxy). Statistical significance is established by the Kolmogorov-Smirnov method. Data have been collected over at least three independent experiments. Error bars in boxplots indicate minumum and maximum quartiles, boxes are first and third quartiles. To see this figure in color, go online.
Figure 5
Figure 5
Effect of progerin induction on the interaction of polycomb protein SUZ12 and lamins by proximity ligation assay. The tethering is quantified by the proximity ligation assay measuring the interaction between (A and B) all lamins (panLMN) or (D and E) lamin A and PcG SUZ12. Briefly, subconfluent cells were fixed on slides with ice-cold 100% methanol for 5 min. Slides were then incubated in a humidity chamber overnight at 4°C with PanLamin (1:50, ab207404; Abcam) or anti-lamin (1:100, ab8980; Abcam) antibody with SUZ12 (1:800, mAb 3737; Cell Signaling). After washing, samples were incubated in a preheated humidity chamber for 1 h at 37°C with anti-rabbit PLUS and anti-mouse MINUS PLA probes diluted 1:5. Ligation and amplification steps were performed according to manufacturer’s instructions. Slides were mounted with Duolink In Situ Medium. (A), (B), (D), and (E) show typical experiments for each experimental condition. The number of aggregates linking SUZ12 and lamins (C for PanLamin and F for lamin A) are quantified as described in the Materials and Methods. The analysis was carried out on 79 nuclei for PanLamin (33 without Doxy and 46 with Doxy) and 81 for lamin A (41 without Doxy and 40 with Doxy). Statistical significance is established by the Kolmogorov-Smirnov method. Data have been collected over at least three independent experiments. Error bars in boxplots indicate minimum and maximum quartiles, boxes are first and third quartiles. To see this figure in color, go online.
Figure 6
Figure 6
Formation of nuclear blebs is induced by a strong chromatin tethers localized on lamin domain boundaries. (A–C) Simulations without lamin domains and uniform distribution of lamin-chromatin tether are shown. (D–F) Simulations with lamin domains and uniform distribution of lamin-chromatin tether are shown. (G–I) Simulations with lamin domains and lamin-chromatin tethers localized along the domain boundaries are shown. Only in the last case do we observe the formation of blebs. In all cases, the link density is p = 0.4. To see this figure in color, go online.
Figure 7
Figure 7
Nuclear shape fluctuations depend on cytoskeletal tethering stiffness. Simulations of shape fluctuations induced by cytoskeletal contraction for different values of the stiffness of the tether kcyto are shown. (A) kcyto = 10−3 N/m, (B) kcyto = 5 × 10−3 N/m, and (C) kcyto = 10−2 N/m. (D) The standard deviation of the radial displacements is shown as a function of kcyto. All the comparisons are statistically significant (p < 10−10 according to the Kolmogorov-Smirnov test). Errror bars in the boxplot indicate minimum and maximum quartiles, boxes are first and third quartiles. To see this figure in color, go online.
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