Nuclear deformability and telomere dynamics are regulated by cell ...

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Dec 22, 2015 - chromatin dynamics | telomere dynamics. Physical properties of the nucleus, such as its morphology and deformability, have been associated ...
Nuclear deformability and telomere dynamics are regulated by cell geometric constraints Ekta Makhijaa, D. S. Jokhuna, and G. V. Shivashankara,b,c,1 a Mechanobiology Institute, National University of Singapore, Singapore 117411; bDepartment of Biological Sciences, National University of Singapore, Singapore 117543; and cInstitute of Molecular Oncology, Italian Foundation for Cancer Research, 20139 Milan, Italy

Edited by Dennis E. Discher, University of Pennsylvania, Philadelphia, PA, and accepted by the Editorial Board November 20, 2015 (received for review July 5, 2015)

Forces generated by the cytoskeleton can be transmitted to the nucleus and chromatin via physical links on the nuclear envelope and the lamin meshwork. Although the role of these active forces in modulating prestressed nuclear morphology has been well studied, the effect on nuclear and chromatin dynamics remains to be explored. To understand the regulation of nuclear deformability by these active forces, we created different cytoskeletal states in mouse fibroblasts using micropatterned substrates. We observed that constrained and isotropic cells, which lack long actin stress fibers, have more deformable nuclei than elongated and polarized cells. This nuclear deformability altered in response to actin, myosin, formin perturbations, or a transcriptional down-regulation of lamin A/C levels in the constrained and isotropic geometry. Furthermore, to probe the effect of active cytoskeletal forces on chromatin dynamics, we tracked the spatiotemporal dynamics of heterochromatin foci and telomeres. We observed increased dynamics and decreased correlation of the heterochromatin foci and telomere trajectories in constrained and isotropic cell geometry. The observed enhanced dynamics upon treatment with actin depolymerizing reagents in elongated and polarized geometry were regained once the reagent was washed off, suggesting an inherent structural memory in chromatin organization. We conclude that active forces from the cytoskeleton and rigidity from lamin A/C nucleoskeleton can together regulate nuclear and chromatin dynamics. Because chromatin remodeling is a necessary step in transcription control and its memory, genome integrity, and cellular deformability during migration, our results highlight the importance of cell geometric constraints as critical regulators in cell behavior.

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mechanotransduction cell geometry actomyosin contractility chromatin dynamics telomere dynamics

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(23, 24). The maintenance of nuclear physical properties is also essential in cell migration during developmental programs (25) as well as in wound healing (26, 27). Defects in nuclear morphology and its deformability have also been shown to be important in metastatic potential and cancer cell invasion (28). Further, a number of diseases have been associated with loss of the mechanical integrity of the nuclear lamina (29–31). However, the regulation of the mechanical integrity of the cell nucleus by the active cytoskeletal network is not well understood. Recent studies have revealed that cytoskeletal organization and nuclear morphology are regulated by extracellular mechanical signals, such as substrate stiffness and geometry (32–41). With the cytoskeleton physically linked to the nucleoskeleton, these extracellular mechanical signals can therefore be used to mediate changes in chromatin structure. Active cytoskeletal forces can mediate the mechanotransduction to the nucleus, to remodel 3D chromosome organization as well as permissivity to chromatin structure by regulatory molecules (42, 43). Cytoskeletal to nuclear links are also essential to the maintenance of poised euchromatin and more repressive condensed chromatin, i.e., heterochromatin assembly (44). Heterochromatin is stabilized by links between the actin cytoskeleton and the nuclear membrane (45–47). In addition, telomeres, the ends of chromosomes, are necessary for genome stability because they protect chromosomes from DNA damage response machinery (48). In this context, the dynamic control of nuclear deformability by Significance

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Physical properties of the cell nucleus are important for various cellular functions. However, the role of cell geometry and active cytoskeletal forces in regulating nuclear dynamics and chromatin dynamics is not well understood. Our results show cells with reduced matrix constraints have short actomyosin structures. These dynamic structures together with lower lamin A/C levels, resulting in softer nuclei, may provide the driving force for nuclear fluctuations. Furthermore, we observed increased dynamics of heterochromatin and telomere structures under such reduced cell–matrix interactions. We conclude that extracellular matrix signals alter cytoskeletal organization and lamin A/C expression levels, which together lead to nuclear and chromatin dynamics. These results highlight the importance of matrix constraints in regulating gene expression and maintaining genome integrity.

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hysical properties of the nucleus, such as its morphology and deformability, have been associated with important cellular functions like gene expression, genome integrity, and cell behavior (1–3). The major cellular components that regulate these physical properties are the cytoskeleton to nuclear links and the nuclear lamina (4–8). Lineage-specific physical properties of the nucleus emerge during cellular differentiation; although stem cell nuclei are highly deformable (9, 10) and have a dynamic chromatin with hyperdynamic chromatin proteins (11), with differentiation, nuclei lose their deformability and become less deformable (12). The nucleus in a differentiated cell is physically coupled to the cytoskeleton via lamins and the linker of nucleoskeleton and cytoskeleton (LINC) complex, which comprises transmembrane Sad1p, UNC-84 (SUN) and Klarsicht, ANC-1, Syne Homology (KASH) domain proteins (13–18). Any perturbation to these components is linked to changes in nuclear morphology and deformability (19). Therefore, the meshwork of actin stress fibers and lamin A/C serves as a critical physical intermediate in the maintenance of nuclear functional homeostasis. The physical properties of the nucleus govern the spatiotemporal packaging of chromatin, which regulates lineage-specific gene expression programs (20–22). In addition, modulations in cytoskeletal to nuclear links have been implicated in DNA damage and genome integrity E32–E40 | PNAS | Published online December 22, 2015

Author contributions: E.M., D.S.J., and G.V.S. designed research; E.M. and D.S.J. performed research; E.M., D.S.J., and G.V.S. analyzed data; and E.M., D.S.J., and G.V.S. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. D.E.D. is a guest editor invited by the Editorial Board. Freely available online through the PNAS open access option. 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1513189113/-/DCSupplemental.

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Results Reduced Matrix Constraints Enhance Nuclear Deformability. Various studies have shown that the geometry of the cell regulates cytoskeletal organization. Hence, to probe the effect of cytoskeletal organization on nuclear dynamics, NIH 3T3 fibroblast cells were cultured on fibronectin micropatterns of two extreme geometries; namely, large polarized (1,800 μm2 1:5 rectangle) or constrained isotropic (500 μm2 circle). Cells cultured on these geometries exhibited distinct cytoskeletal organization and nuclear morphologies. Consistent with previous studies (32), large polarized (LP) cells were flat, their actin was organized as long apical stress fibers, and the nucleus was also flat and elongated. On the other hand, constrained isotropic (CI) cells were taller, their actin was

organized as short filaments or patches, and the nucleus was rounded (Fig. 1A and Fig. S1 A and B). Despite the nuclear height being greater in CI cells, the projected nuclear area, surface area, and volume were less compared with LP cells (Fig. S1C). Next, to study the dynamics of nuclear morphology as a function of the two extreme cytoskeletal organizations, time lapse imaging was performed using fibroblasts stably expressing H2B-EGFP and cultured on LP or CI fibronectin micropatterns (Movie S1). The time lapse images were thresholded to obtain the nuclear periphery before the time series was converted to a z stack (Fig. S1D) and reconstructed in Imaris to form a surface (Fig. 1B). These kymographs revealed increased nuclear periphery fluctuations in CI cells. Also, superimposition of nuclear peripheries at different time points (Fig. 1C) revealed that in LP cells, the nuclear periphery does not undergo a significant alteration with time as opposed to CI cells, which show significant fluctuations of the nuclear periphery within 10 min. To quantify the fluctuations of the nuclear envelope, projected nuclear area was plotted as a function of time. This was obtained by thresholding either the widefield images of the nucleus or the maximum intensity projection of all confocal z-slices of the nucleus. Such projected nuclear area vs. time curves were then fitted with a third-order polynomial, and the residuals were normalized by the value of the polynomial fit at each time point. These normalized residual fluctuations were then plotted as a function of time. Typical time traces of projected nuclear area fluctuations (PNAFs) revealed a relatively constant projected nuclear area in LP cells over a period of 20 min and up to 10% fluctuations in CI cells (Fig. 1D). This increased nuclear deformability of CI cells was consistently different from LP cells, as observed over multiple cells (Fig. 1E). Typical time traces of absolute residual area (in μm2) also showed higher fluctuations in CI cells compared with LP cells (Fig. S1E). However, the nuclear surface area and volume in CI cells did not show such large fluctuations (Fig. S1 F and G). To quantify the amplitude of fluctuations, PNAF data from all cells and time points were combined to obtain a distribution. SD (σ) of this distribution (or the full width at half maximum of its Gaussian fit, which equals 2.355σ) was used to compare the amplitude of area fluctuations (Fig. 1F). The σ of PNAF was 5.3% in CI cells, compared with only 1.7% in LP cells (Fig. 1F, Inset). To understand this cell geometry-mediated threefold change in nuclear deformability, we next probed the role of cytoskeletal forces and nuclear stiffness.

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actomyosin contractility and its effect on heterochromatin and telomere dynamics are still unclear. To understand the mechanism underlying the cytoskeleton mediated alterations in nuclear and chromatin dynamics, we modulated cytoskeletal organization using cell geometric constraints and measured nuclear deformability and heterochromatin and telomere dynamics. We found that in cells with constrained and isotropic geometry, the nucleus is more deformable than cells with elongated and polarized geometry. We further showed that this can be attributed to differential force generating actomyosin structures and differential lamin A/C expression levels in the two geometries. These active cytoskeletal forces were also found to regulate the dynamics of subnuclear heterochromatin and telomere structures. Our observations suggest that active forces from the cytoskeleton regulate nuclear and chromatin dynamics, which could in turn affect the spatiotemporal regulation of genomic processes and thus cell behavior.

Fig. 1. Reduced matrix constraints enhance nuclear deformability. (A) Maximum intensity projection for confocal images of typical large polarized (LP) and constrained isotropic (CI) cell stained with Phalloidin (green) and Hoechst (blue). (Scale bar, 10 μm.) See also Fig. S1. (B) Surface rendering of nuclear periphery kymographs for a typical LP and CI cell time series. (Scale bar, 10 μm.) (C) Widefield epifluorescence images of nuclei in LP and CI cells expressing H2B-EGFP. Colored outlines mark the periphery of these nuclei at various time points. (D) Typical PNAFs of LP and CI cells as a function of time. (E) PNAF vs. time plot for multiple cells. CI, 27 cells; LP, 83 cells. (F) Gray and light red curves represent a normalized histogram of combined PNAFs for all cells and all time points in LP and CI patterns, respectively. Black and red curves represent the Gaussian fittings. Inset shows SDs of the two distributions. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nLP = 3,204 from 83 cells and nCI = 1,803 from 27 cells.

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To study the role of cytoskeletal forces in PNAF, actin organization was perturbed in both LP and CI cells by treating them with actin depolymerizing and actin stabilizing agents cytochalasin-D and jasplakinolide, respectively. In each case, time lapse imaging was first performed on control cells (n > 15). These same cells were then treated with pharmacological agents and reimaged. Periphery kymographs for all treatments are shown in Fig. 2A. Here the PNAF showed a nonmonotonic dependence on the state of actin polymerization. Depolymerization of F-actin in LP cells using cytochalasin-D decreased the projected nuclear area exponentially with time. In this case, the PNAF was calculated by normalizing the residuals of an exponential fit with the value of the fit at each time point. Cytochalasin-D treatment in LP cells enhanced PNAF from 1.7% to 5.3% (Fig. 2 A and B and Movie S2), and actin stabilization (using jasplakinolide) in CI cells reduced PNAF from 5.3% to 1.6% (Fig. 2 A and B and Movie S3). Surprisingly, further actin depolymerization in CI cells using cytochalasin-D also reduced the PNAF from 5.3% to 2.2% (Fig. 2 A and B and Movie S4). Consistent PNAF were obtained upon actin perturbation in multiple cells (Fig. S2 A–C). Such nonmonotonic dependence of PNAF on actin polymerization (Fig. 2B and Fig. S3) suggests that only cells with intermediate state of actin polymerization exhibit fluctuations in the projected nuclear area. PNAS | Published online December 22, 2015 | E33

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Actin, Myosin, and Formin Regulate Matrix-Assisted Nuclear Deformability.

units of actin, myosin is also necessary to induce the observed nuclear fluctuations. The blebbistatin mediated decrease in PNAF was highly reproducible (Fig. S2E). Consistent with this, ATP depletion also significantly decreased the PNAF (Fig. S4). To assess if nucleators of actin polymerization, such as formin, could be generating these active forces, we inhibited formin activity using SMIFH2 in cells with enhanced PNAF, i.e., CI cells and cytochalasin-D–treated LP cells. The PNAF in both cases were reduced by half in all cells (Fig. 2 G and H, Fig. S2F, and Movie S7), confirming that formin indeed plays a role in nuclear fluctuations. Next, we assessed if the physical links between the cytoskeleton and the cell nucleus are required to generate the observed projected area fluctuations. LINC Complex and Microtubules Affect Amplitude of Nuclear Area Fluctuations. Actin is physically linked to the nucleus via nesprin,

which is a component of the LINC complex (49). To understand whether this physical link is necessary for nuclear deformability, cells were transfected with dominant negative (DN)-KASH fused with GFP, which displaces the endogenous nesprin from the nuclear envelope to the endoplasmic reticulum, thereby disrupting the LINC complex (50). These cells were then cultured on CI fibronectin micropatterns, and time lapse imaging of H2B-mRFP labeled nuclei was performed in cells expressing DN-KASH GFP (Fig. 3A and Movie S8). Periphery kymographs of these nuclei (Fig. 3B) showed similar fluctuations as control CI cells (Fig. 1B). Typical time traces of the normalized PNAF (Fig. 3C) and the σ of their distribution (Fig. 3C, Inset) revealed that nesprin perturbed cells still exhibit nuclear fluctuations and the fluctuation amplitude reduced only 1.3-fold compared with control CI cells. Because this fluctuation amplitude is still higher than control LP cells (which show threefold reduction compared with control CI cells), LINC Fig. 2. Actin, myosin, and formin regulate matrix assisted nuclear deformability. (A) Surface rendering of nuclear periphery kymographs for typical control and treated LP and CI cells. (Scale bar, 10 μm.) (B) SDs of PNAF distributions of multiple control and treated LP and CI cells. Left to right represents increasing actin polymerization. nCI+CytoD = 625 from 16 cells, nCI = 1,803 from 27 cells, nLP+CytoD = 1,177 from 43 cells, nLP+CytoD+Washoff = 1,288 from 22 cells, nLP = 3,204 from 83 cells, and nCI+Jas = 1,067 from 13 cells. Performing twosample F-test for variance on the various distributions yields **P < 0.001 for all conditions. (C) A typical PNAF trace for a LP cell sequentially treated with cytochalasin-D and blebbistatin. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points. Performing twosample F-test for variance on the two distributions yields **P < 0.001 for nLP+CytoD = 1,177 from 43 cells and nLP+CytoD+Blebb = 1,196 from 20 cells. (D) Merge of nuclear periphery outlines at 15-min interval for a typical LP cell in untreated, cytochalasin-D, and cytochalasin-D + blebbistatin conditions. (E) PNAF vs. time plot for multiple control and blebbistatin-treated CI cells. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nCI = 800 and nCI+Blebb = 805 from 25 cells. (F) Merge of nuclear periphery outlines at 15-min interval for a typical CI cell in untreated and blebbistatin conditions. (G) Typical PNAF trace for a LP cell sequentially treated with cytochalasin-D and SMIFH2. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nLP+CytoD = nLP+CytoD+SMIFH2 = 144 from four cells. (H) PNAF vs. time plot for multiple control and SMIFH2-treated CI cells. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nCI = 1,222 and nCI+SMIFH2 = 1,194 from 26 cells. See also Fig. S2.

To further explore the origin of such nuclear fluctuations mediated by intermediate state of actin polymerization, the myosin activity was perturbed using blebbistatin in cells with enhanced PNAF, i.e., LP cells treated with cytochalasin-D and CI cells. In each case the PNAF decreased to half (Fig. 2 C–F, Fig. S2 D and E, and Movies S5 and S6), suggesting that along with small polymerized E34 | www.pnas.org/cgi/doi/10.1073/pnas.1513189113

Fig. 3. Role of LINC complex and microtubules in nuclear deformability. (A) Widefield epifluorescence image of a typical CI cell coexpressing H2BmRFP (green) and Nesprin DN-KASH EGFP (red). H2B-mRFP is shown in green to maintain consistency across the figures. (B) Surface rendering of nuclear periphery kymograph for a typical Nesprin mutant CI cell. (C) PNAF vs. time plot for multiple Nesprin mutant CI cells. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points for control and DNKASH CI conditions. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nCI = 1,803 from 27 cells and nCI+DNKASH = 530 from 5 cells. (D) Typical PNAF trace for a CI cell treated with nocodazole. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nCI = 820 and nCI+Noco = 640 from 26 cells.

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complex may only partially be responsible for actomyosin mediated nuclear fluctuations. We next explored whether microtubules serve as additional force transducers between the actomyosin structures and the nuclear periphery. Microtubules are physically coupled to the nucleus via LINC complex (51) and to actin via microtubule– actin crosslinking factors (MACFs) (52). To probe whether the microtubule network that surrounds the nucleus could transmit force for nuclear fluctuations, microtubules were depolymerized using nocodazole in cells on CI patterns. Typical time traces of the normalized PNAF (Fig. 3D) and the σ of their distribution (Fig. 3D, Inset) revealed 1.5-fold increase compared with control CI cells. This can be explained by either loss of perinuclear microtubule cage and/or enhanced actomyosin contractile forces following microtubule depolymerization (53). Taken together, these results show that perturbations of the physical links only partially affect the amplitude of PNAF, suggesting that there are additional mechanisms that drive nuclear fluctuations.

Deformable Nuclei Have Increased Heterochromatin Dynamics. To understand whether PNAF have an effect on chromatin dynamics, we followed the trajectories of heterochromatin foci, visible as bright spots in H2B-EGFP labeled nuclei. XY trajectories (Fig. 5 A and B) and line kymographs across the nucleus (Fig. S5A) were obtained from maximum intensity projected time series of cells on CI and LP micropatterns. These trajectories and kymographs showed an increase in heterochromatin dynamics in CI compared with LP cells. Such dynamics in CI cells were abolished by blebbistatin treatment. In contrast, heterochromatin Makhija et al.

Fig. 4. Role of lamin A/C in nuclear deformability. (A) Widefield epifluorescence image of a typical CI cell coexpressing H2B-EGFP (green) and lamin A/C-mRFP (red). (B) PNAF vs. time plot for multiple lamin A/C overexpressing CI cells. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points for control and lamin A/C overexpression CI conditions. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nCI = 1,803 from 27 cells and nCI+laminA=C = 210 from 4 cells. (C) mRNA levels obtained by qRT-PCR for CI cells normalized with respect to LP cells (n = 3 samples). Error bars represent SE. **P < 0.001. (D) Surface rendering of a nuclear periphery kymograph for typical lamin A/C overexpressing CI cell, control MEF cell, and lamin−/− MEF cell. (E) Widefield epifluorescence image of a typical MEF cell expressing H2B-EGFP. (F) PNAF vs. time plot for multiple MEF cells cultured on LP patterns (10 cells). (G) Widefield epifluorescence image of a typical lamin−/− MEF cell expressing H2B-EGFP. (H) PNAF vs. time plot for multiple lamin−/− MEF cells cultured on LP patterns. Inset represents normalized SDs of PNAF distributions obtained by combining all cells and time points for MEFs and lamin−/− MEFs. Performing two-sample F-test for variance on the two distributions yields **P < 0.001 for nMEF = 340 from 10 cells and nlamin=  MEF = 495 from 15 cells.

foci became more dynamic in LP cells following cytochalasin-D treatment. Mean squared displacement (MSD) vs. time curves show that although the foci are usually confined in LP cells, they become more diffusive in CI cells (Fig. 5C). Further, the MSD vs. time curves for cytochalasin-D and blebbistatin treatments suggest that such confined and diffusive dynamics of the heterochromatin foci in LP and CI patterns, respectively, are regulated by actomyosin contractility. We then sought to answer whether the correlation between heterochromatin foci trajectories changes as a function of PNAF. For this, 3D trajectories of individual foci were obtained using Imaris (Movie S11), and their pairwise vector Pearson correlation coefficient was calculated (Materials and Methods and Fig. S5 B and C). In a typical CI cell, most pairs of foci trajectories were uncorrelated (Fig. 5 D and F). On the other hand, in a typical LP cell, the foci trajectories were highly correlated (Fig. 5 E and G). Additionally, the foci pairs in LP cells could be distinguished into two groups, based on their z-position. The foci in the apical region were correlated with other foci in apical region, whereas those in the basal region were correlated with other foci in basal region, but apical and basal foci were uncorrelated with each other (Fig. 5 E and G and Fig. S5 D and E). The foci trajectory correlations increased drastically in CI cells following blebbistatin PNAS | Published online December 22, 2015 | E35

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Lamin A/C Levels Inversely Regulate Nuclear Deformability. Structural lamin proteins in the nuclear envelope regulate nuclear stiffness: whereas lamin A/C confers rigidity to the nucleus, its absence increases nuclear deformability (54, 55). To understand the relation between lamin-mediated nuclear stiffness and cytoskeletal-mediated nuclear fluctuations we overexpressed lamin A/C using transient transfection in fibroblasts before culturing them on CI patterns. Time lapse imaging was then performed for cells expressing both lamin A/C-RFP and H2B-EGFP (Fig. 4A and Movie S9). Typical time traces of normalized PNAF in these cells (Fig. 4B) showed significantly lower amplitude than in control CI cells (Fig. 1E). The σ of the distribution of the area fluctuations was reduced 1.5-fold compared with control CI cells (Fig. 4B, Inset). The nuclear periphery kymograph (Fig. 4D) also showed a significant decrease in periphery fluctuations compared with control CI cells (Fig. 1B). This result suggested that geometric constraints placed on a cell might regulate lamin A/C expression and thus alter PNAF. We therefore checked whether the endogenous lamin A/C levels were altered in wild-type cells on CI compared with LP patterns. To achieve this, lamin A/C mRNA levels were measured using quantitative (q)RT-PCR in both geometries. CI cells showed 80% reduction in lamin A/C mRNA levels compared with LP cells (Fig. 4C). Decreases in lamin A/C protein in poorly spread cells on soft substrates have been reported (56). To further investigate the role of lamin A/C in the inhibition of PNAF, lamin A/C knockout and control (stably transfected with empty vector) MEFs (57) transfected with H2B-EGFP were cultured on LP micropatterns (Fig. 4 E and G). Time lapse imaging of the nuclei in these cells (Fig. 4 G and H and Movie S10) revealed increased PNAF in the knockout cells (Fig. 4 F and H). The σ of distribution of area fluctuations showed 3.4-fold increase in the knockout cells compared with control MEFs (Fig. 4H, Inset). The nuclear periphery kymograph (Fig. 4D) also showed increased fluctuations in knockout MEFs. Taken together, these results suggest that lamin A/C-mediated nuclear stiffness inversely regulates the cytoskeletal-mediated nuclear deformability.

results indicate the reversible nature of cytoskeletal-mediated chromatin dynamics. Telomere Dynamics Are Regulated by Active Cytoskeletal Forces.

Fig. 5. Actomyosin contractility regulates matrix-associated heterochromatin dynamics. (A and B) XY trajectories of multiple heterochromatin foci before and after blebbistatin and cytochalasin-D perturbations in CI and LP cells, respectively. (C) Mean squared displacement vs. time plots for heterochromatin foci before and after blebbistatin and cytochalasin-D perturbations in CI and LP cells, respectively. Error bars represent SE. (D and E) Maximum intensity z-projected images of H2B-EGFP nucleus in typical CI and LP cells. Heterochromatin foci have been numbered and color-coded based on their z-position. Blue represents basal plane, and yellow represents apical plane. (F and G) Pearson correlation coefficient calculated between 3D trajectories of all heterochromatin foci pairs labeled in D and E. See also Fig. S5. (H and I) Pearson correlation coefficient calculated between 3D trajectories of the same heterochromatin foci pairs upon blebbistatin and cytochalasin-D treatments in CI and LP cells, respectively. (J) Widefield epifluorescence images of H2B-EGFP nucleus in typical LP cell in untreated, cytochalasin-D, and washoff conditions. See also Fig. S6. (K) Pearson correlation coefficient for H2B-EGFP intensity histograms in control, cytochalasin-Dtreated, and washoff conditions. Error bars represent SE. *P = 0.05, n = 7 cells. (L) Pearson correlation coefficient calculated between 3D trajectories of same heterochromatin foci pairs (same as in E, G, and I) upon washoff of cytochalasIn-D.

treatment (Fig. 5H), whereas they were reduced for most foci pairs in LP cells upon cytochalasin-D treatment (Fig. 5I, Fig. S5F, and Movie S12). These results suggest that active cytoskeletal forces modulate correlated dynamics of chromatin domains. To understand whether such cytoskeletal mediated chromatin perturbation was reversible, LP cells treated with cytochalasin-D for 30 min were washed, and time lapse imaging was performed using the same cells. Surprisingly, the spatial map of the chromatin images, which was altered upon cytochalasin-D treatment, was fully restored after the agent was washed off (Fig. 5J). Photobleached regions in the nucleus were also restored after the drug was washed off in LP cells, whereas in CI cells the bleach patterns were lost even before the drug treatment (Fig. S6 A and B). The correlation between H2B-EGFP intensity histograms (Fig. 5K) and polarization anisotropy histograms (Fig. S6C) of drug-perturbed and washed nuclei with control nuclei revealed that an hour after the drug was washed off, the nuclei had almost returned to their initial configuration. Additionally, the pairwise foci trajectory correlations were completely restored an hour after the drug was washed off, and apical and basal groupings similar to control nuclei were established (Fig. 5L). The XYZ trajectories of some heterochromatin foci during the nuclear deformation phase immediately upon cytochalasin-D treatment also overlapped with their trajectory during the restoration phase immediately following the washout (Fig. S6D). These E36 | www.pnas.org/cgi/doi/10.1073/pnas.1513189113

Next, we assessed the effect of PNAF on the dynamics of specific functional units inside the nucleus: the telomeres. To probe the effect of PNAF on telomeres, H2B-EGFP fibroblasts were transfected with telomeric repeat binding factor 1 (TRF1) tagged with dsRed. These cells were cultured on LP and on CI micropatterns, and time lapse confocal imaging was performed. Telomeres were visible as distinct bright punctae of TRF1 dsRed in the nucleus. An overlap of z-projected TRF1 dsRed images with a time difference of 3 min showed almost perfect merge in case of LP cells, whereas there was a significant shift in telomere positions in case of CI cells (Fig. 6 A and B and Movies S13 and S14). To probe long-timescale dynamics of telomeres, line kymographs across the nucleus in the z-projected TRF1 dsRed time series were plotted for 1 h, which showed higher dynamics of telomeres in CI cells compared with LP cells (Fig. S7 A and B). Typical 3D trajectories of telomeres revealed that in LP cells, telomeres were more confined compared with CI cells, where they explored a volume of ∼0.15 μm3 compared with 0.01 μm3 in LP cells (Fig. 6C). FWHM of the histogram plotted for distance from mean position by combining data from all time points and all telomeres showed almost double radial spread of the telomere trajectory in CI cells compared with LP cells (Fig. 6D). Because CI cells showed a reduction in lamin A/C levels and the associated enhanced telomere dynamics, we next assessed its role in regulating the observed dynamics. For this, lamin A/C−/− cells were plated on LP patterns, and telomere dynamics were measured (Movie S15). The radial spread in lamin A/C-deficient cells on LP patterns was doubled compared with WT LP cells, suggesting that lamin A/C could be an essential intermediate in stabilizing telomere dynamics. Because actomyosin contractility was a critical determinant of nuclear and chromatin dynamics, we measured telomere dynamics in blebbistatin-treated CI cells (Movie S16) and cytochalasinD–treated LP cells (Movie S17). The radial spread of the trajectory decreased from 0.66 μm to 0.22 μm upon blebbistatin treatment in CI cells, whereas it did not show drastic change in LP cells upon cytochalasin-D treatment (Fig. 6E). In addition, telomere speeds showed a similar trend to their radial spreads. Telomeres in CI cells and lamin-deficient cells on LP patterns moved significantly faster than control LP cells. Further, blebbistatin treatment significantly slowed down the telomeres in CI cells, whereas cytochalasin-D treatment did not alter the speed of telomeres in LP cells (Fig. S7 C and D). We characterized the telomere motion further by quantifying the MSD of individual telomeres in each case. MSD vs. time plots showed much more diffusive telomeres in CI cells compared with LP cells (Fig. 6F) with the exponent α, which defines the type of motion, varying from 0.75 in CI to 0.55 in LP cells (Fig. 6G). The exponent α was also higher in lamin-deficient cells on LP patterns and cytochalasin-D–treated LP cells compared with control LP cells. Blebbistatin treatment of CI cells reduced α significantly compared with control CI cells (Fig. 6H). Next, to probe whether the correlation between telomere trajectories change as a function of PNAF, pairwise vector Pearson correlation coefficient was calculated for each pair of telomeres in different conditions. In a typical LP cell, most telomere trajectories were correlated (mean correlation ∼0.65), whereas in CI cells, much fewer pairs were correlated (mean correlation ∼0.15) (Fig. 6 I and J). Cytochalasin-D treatment altered the correlation map of telomere trajectories in LP cells making them more uncorrelated (mean correlation ∼0.1), whereas blebbistatin treatment in CI cells increased the trajectory correlations (mean correlation ∼0.4). Lamin A/C-deficient cells on LP patterns also showed much fewer correlated telomere pairs (mean correlation ∼0.1) compared with control LP cells (Fig. 6K and Fig. S7 E–G). These results suggest Makhija et al.

that active cytoskeletal forces as well as lamin A/C together regulate the correlated dynamics of telomeres. Discussion A critical step in the alteration of genome function is the regulation of nuclear and chromatin dynamics. Chromatin dynamics can be induced by nuclear ATPases such as polymerases, topoisomerases, and chromatin remodeling complexes at millisecond timescales (58–60). However, recent findings suggest that Makhija et al.

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Fig. 6. Actomyosin contractility regulates matrix-associated telomere dynamics. (A and B) Overlap of nucleus periphery and maximum intensity z-projected images of a TRF1-dsRed nucleus at 3-min interval in typical LP and CI cells. Bright spots represent telomeres. (Scale bar, 4 μm.) (C) XYZ trajectories of typical telomeres in LP and CI cells. (D) Normalized histogram of displacement from mean position, combining data for n = 89 telomere trajectories from n = 3 LP cells and n = 68 telomere trajectories from n = 3 CI cells. Black and red curves represent the Gaussian fittings. (E) Full width at half maxima (FWHM) of Gaussian fitting of histograms for displacement from mean position in LP cells, cytochalasin-D–treated LP cells (2 cells, 62 telomeres), CI cells, blebbistatin-treated CI cells (3 cells, 141 telomeres), and laminA/C-deficient CI cells (2 cells, 58 telomeres). (F ) Mean squared displacement vs. time plots for multiple telomeres in control LP and CI cells. (G) Box plot for the exponent α in LP and CI cells obtained by fitting MSD ‹r2(τ)› vs. time lag τ plots to the equation ‹r2(τ)› = Dτα. The bottom and top of the box represent the first and third quartiles, whereas the line and dot inside the box represent the median and mean, respectively. The ends of the whiskers correspond to the lowest/highest data point of the distribution. nLP = 62 telomeres from two cells, and nCI = 141 telomeres from three cells. (H) Mean α values for LP cells, cytochalasin-D–treated LP cells, CI cells, blebbistatin-treated CI cells, and laminA/C-deficient CI cells. Error bars represent SE, and the means are significantly different with **P < 0.01. nLP = 68 telomeres from three cells, nLP+CytoD = 62 telomeres from two cells, nCI = 68 telomeres from three cells, nCI+Blebb. = 141 telomeres from three cells, and nlamin=  MEFs = 58 telomeres from two cells. (I and J) Pearson correlation coefficient calculated between 3D trajectories of 15 telomere pairs (chosen at random) in typical LP and CI cells, respectively. The telomeres are sorted (top to bottom and left to right) in ascending z-position, i.e., basal to apical. (K ) Mean value of the correlation coefficient for LP cells, cytochalasin-D–treated LP cells (3 cells, 89 telomeres), CI cells (3 cells, 68 telomeres), blebbistatin-treated CI cells (3 cells, 141 telomeres), and laminA/C-deficient CI cells (2 cells, 58 telomeres). Error bars represent SE, and the means are significantly different with **P < 0.01. See also Fig. S7.

chromatin dynamics can also result from matrix remodeling (42, 43). In addition, perturbation of cytoskeletal filaments via RNA interference affects histone diffusion timescales (7). Furthermore, altering cell geometric constraints affects gene expression via redistribution of chromatin remodeling enzymes (61). In this context, to understand how physical forces from the cytoskeleton might regulate genome programs, we performed quantitative analysis of nuclear deformations and chromatin dynamics arising from such active cytoskeletal forces. Centromeres and telomeres are constitutive heterochromatin structures associated with gene regulation and chromosome stability. Hence, we also probed the effect of active cytoskeletal forces on dynamics of these functional chromatin units. We observed an unusual nonmonotonic dependence of actomyosin contractility in the regulation of nuclear dynamics. By altering cell geometric constraints and introducing pharmacological reagents, both actomyosin contractility and microtubule organization could be modulated to investigate this phenomenon. The nonmonotonic dependence on actomyosin contractility suggests that small actin networks cause nuclear fluctuations. Small punctated actin structures were indeed observed in CI and cytochalasin-D–treated LP cells, which show higher nuclear fluctuations (Fig. S8 A and B). These actin structures, which colocalize with myosin (Fig. S8C), have been previously reported as dynamic clusters (62) and could be the key intermediates in the mechanotransduction pathway driving nuclear dynamics. To assess whether forces generated by actomyosin contractility were applied directly on the nucleus, we overexpressed a dominant negative KASH plasmid, which disrupts the link between actin and the nuclear envelope. However, cells with perturbed LINC complex on CI pattern did not show significant change in PNAF compared with control CI cells. Because major changes in microtubule organization were observed when cell geometry was constrained, we induced microtubule depolymerization to assess whether these filaments were involved in the mechanotransduction of force to the nucleus. However, depolymerization of microtubules did not abolish nuclear deformability, suggesting that the small actin networks were exerting forces on the nucleus directly. Because the actin cytoskeleton is physically linked to the lamin meshwork via the LINC complex, we tested whether nuclear stiffness was modulated by altered cell geometry and whether this enhanced the nuclear dynamics. We found that lamin A/C was downregulated as matrix constraints were reduced; an effect similar to the down-regulation of lamin A/C with matrix stiffness (56). This finding prompted us to transiently transfect cells with lamin A/C. Indeed, overexpression of lamin A/C abolished the nuclear fluctuations, and this was in contrast to lamin A/C knockdown, which increased nuclear fluctuations. Taken together, these experiments highlighted an important transcription-dependent mechanoregulatory pathway involving actomyosin contractility that couples matrix properties to nuclear dynamics. A recent study also revealed that Rac-1–mediated nuclear actin also causes nuclear envelope deformations (63). Next, we wanted to test whether the dynamics of chromatin remodeling were affected by the enhanced nuclear deformability. Heterochromatin structures have been shown to be stabilized in cells that are strongly adhered to the extracellular matrix (64). We hypothesized that a reduction in the number of physical links to the extracellular matrix may disrupt heterochromatin integrity, thus making chromatin more permissive. Consistent with this, heterochromatin dynamics increased and correlation between heterochromatin foci trajectories decreased in cells on constrained isotropic geometry. The dynamic correlations of heterochromatin in cells of elongated polarized geometry were position dependent. Here the apical and basal foci were highly correlated with other apical and basal foci, respectively. Also, the motion of telomeres nearer to the nuclear membrane was more correlated to envelope fluctuations than that of a more interiorly positioned telomere (Fig. S9), suggesting that an elaborate structural network inside

the nucleus may be modulated by changes in actomyosin contractility. The pharmacological inhibitor washout experiments revealed that dynamic correlation in heterochromatin organization and its apical–basal grouping, which was reduced upon actin depolymerization, was restored after the actin depolymerizing agent was washed out. This suggests that a structural memory in the spatial organization of heterochromatin exists and highlights the importance of mechanical homeostatic balance for higher-order chromatin organization in living cells. Our studies also revealed an important role for actomyosin contractility in maintaining telomere positioning and dynamics. The enhanced dynamics of telomeres with actin depolymerization suggests that the telomere ends have to be protected during various cellular processes including its migration. Because these ends are susceptible to DNA damage, the cytoskeletal control of telomere stability is important to genomic integrity. In conclusion, our data systematically reveal an important link between cytoskeletal components and nuclear and chromatin dynamics (Fig. 7). We suggest that the mechanical changes within the extracellular matrix tune the epigenetic states of chromatin dynamics via actomyosin contractility to regulate cellular functions including transcription, genome integrity, migration, and cellular homeostasis. Materials and Methods Cell Culture, Pharmacological Perturbations, and Plasmid Transfections. Wildtype NIH 3T3 fibroblasts and NIH 3T3 fibroblasts stably expressing H2B-EGFP were cultured in low-glucose Dulbecco’s Modified Eagle Medium (Gibco; Life Technologies) supplemented with 10% (vol/vol) FBS (Gibco; Life Technologies) and 1% penicillin-streptomycin (Gibco; Life Technologies) at 37 °C and 5% CO2 in humid conditions. Cells were trypsinized (Gibco; Life Technologies) and seeded on fibronectin (Sigma) micropatterned dishes for 3 h before imaging. Cytochalasin-D (Sigma) was used at 500 nM working concentration, and cells were imaged either immediately or 30 min after treatment. Jasplakinolide (Gene Ethics) was used at concentration of 200 nM, and cells were imaged

20 min after treatment. Blebbistatin (Merck) was used at concentration of 25 μM, and cells were imaged 30 min after treatment. SMIFH2 (ChemBridge Corporation) was used at a concentration of 20 μM, and cells were imaged an hour after treatment. Nocodazole (Sigma) was used at a concentration of 10 μg/mL, and cells were imaged an hour after treatment. All transfections were carried out using jetPRIME (Polyplus transfection). Real-Time PCR. Real-time PCR was used to quantify the fold change in lamin A/C expression in CI and LP cells. mRNA was extracted using RNeasy Mini kit (Qiagen). Five hundred nanograms of total mRNA extracted was subjected to cDNA synthesis using iScript cDNA Synthesis kit (Bio-Rad). qPCR was performed using SsoFast qPCR (Bio-Rad) for 40 cycles in a Bio-Rad CFX96. The relative fold change in levels of lamin A/C was then measured with respect to GAPDH levels. Primer sequences used were LaminA (fwd-GTACAACCTGCGCTCACGCACCGT; rev-CACTGCGGAAGCTTCGAGTGACT); and GAPDH (fwd-GACCAGGTTGTCTCCTGCGACTT; rev-CCATGAGGTCCACCACCCTGTT). Preparation of PDMS Stamps, Microcontact Printing, and Cell Seeding on Patterns. To make stamps, PDMS (Sylgard 184; Dow Corning) precursor and curing agent were mixed homogeneously in 10:1 ratio and poured over the silicon wafer which had large polarized (LP) or constrained isotropic (CI) micropatterned wells. After degassing in the desiccator for 30 min to remove air bubbles from the PDMS mixture, the silicon wafer with the PDMS mixture was cured in the oven at 80 °C for 2 h. Solidified PDMS was then peeled from the wafer and cut into ∼1 cm × 1 cm stamps. These stamps were oxidized using plasma for 4 min, and then 15 μL of 100 μg/mL fibronectin solution (mixed with Alexa Fluor 647 dye; Sigma) was poured over each stamp. Extra solution was wiped with a tissue, and the stamp was allowed to dry for 10 min. The stamp was then checked under the microscope for complete drying between the micropatterned structures, after which it was inverted carefully onto the surface of an uncoated hydrophobic 35-mm dish (Ibidi). The stamp was gently removed after 2 min, and the stamping on the dish was checked by visualizing Alexa 647 fluorescence in the farred channel in the epifluorescence microscope. To passivate the nonpatterned surface of the dish, it was then treated with 2 mg/mL pluronic F-127 for 5 min and washed twice with PBS and cell culture medium before seeding single cells. Imaging, Image Processing, and 3D Rendering. All imaging was carried out using a 100× objective on a NikonA1R Confocal microscope. Time lapse imaging was done in either widefield or confocal mode with 30-s, 60-s, or 90-s time intervals for up to 60 min in each condition. The z-depth for confocal imaging was 0.5 μm. H2B-EGFP images were thresholded, projected nuclear area was calculated, and time lapse images of the nuclear periphery were generated using custom written code in MATLAB. Merged images of the nuclear periphery at different time points were generated in ImageJ. To generate edge kymographs, time series of nuclear periphery images were analyzed in IMARIS as a z-stack (Fig. S1D) and then fitted with a surface. Three-dimensional rendering of actin, microtubules, and the nucleus was also done using IMARIS. Line kymographs across the nucleus for visualizing heterochromatin foci dynamics were generated in ImageJ.

Fig. 7. A model summarizing cytoskeletal and nucleoskeletal regulation of nuclear and chromatin dynamics. The actin is shown in purple, the microtubules are shown in green, the lamin A/C is shown as blue (time t) and red (time t + Δt) outline of the nucleus, and heterochromatin foci are shown as blue (time t) and red (time t + Δt) structures of DNA. Briefly, in LP cells the long apical actin stress fibers press on the nucleus, making it flat and elongated. The lamin A/C expression levels are higher. The nuclear periphery, as well as heterochromatin foci, is less dynamic. In contrast, in CI cells, long actin stress fibers are absent, and actin exists as meshwork of short filaments and punctae. The zoom-in of actin structures shows actin, myosin, and formin asters as reported earlier (62). Lamin A/C expression levels are lower. We speculate that active forces from the dynamic actin–myosin–formin asters, as well as decreased rigidity because of lower lamin A/C expression levels, make the nuclear periphery and heterochromatin foci more dynamic in CI cells. See also Fig. S8.

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Data Analysis and Statistical Tests. Absolute projected nuclear area (in μm2) was first measured by thresholding either the widefield nucleus images or maximum intensity projection of confocal z-slices of the nucleus. This projected area was then plotted as a function of time and fitted with thirdorder polynomial (or exponential for LP+CytoD condition) curves in ORIGIN. The residual values were divided by the value of the polynomial (or exponential) at each time point and multiplied by 100 to obtain the percentage normalized residual area fluctuations or the PNAF. Such PNAFs were combined from multiple cells and time points for each condition to obtain a normal distribution. SD (σ) of such distribution indicates the amplitude (in percentage) of area fluctuations. To determine whether the difference in σ of PNAF distribution for various conditions is statistically significant, two-sample F-test for variance was performed in ORIGIN. P values were less than 0.001 in all cases. Heterochromatin Foci and Telomere Trajectory Correlation Analysis. Time lapse confocal stacks of H2B-EGFP nuclei were opened in IMARIS. Nucleus

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  !  P ! ~ AðtÞ − Amean   ·   BðtÞ − ~ Bmean vector  correlation  coefficient = rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffirffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi. 2  2  ~ ~ −~ BðtÞ − ~ Bmean AðtÞ Amean Various trajectories were simulated to verify that the vector correlation coefficient yields high correlation values between trajectories of particles moving in same direction and low correlation values between those moving

ACKNOWLEDGMENTS. We thank Marco Foiani, Jacques Prost, and Madan Rao for useful discussions. We thank Steven Wolf for critical reading of the manuscript, Zhang Bo for preparing the summary cartoon, and Mallika Nagrajan for helping with TRF transfections. Lamin A/C knockdown MEFs and Nesprin DN-KASH plasmid were kind gifts from the Colin Stewart and Brian Burke laboratories. We thank the Mechanobiology Institute, National University of Singapore, Singapore, and Ministry of Education Tier-3 Grant Program for funding.

1. Isermann P, Lammerding J (2013) Nuclear mechanics and mechanotransduction in health and disease. Curr Biol 23(24):R1113–R1121. 2. Woringer M, Darzacq X, Izeddin I (2014) Geometry of the nucleus: A perspective on gene expression regulation. Curr Opin Chem Biol 20:112–119. 3. Burke B, Stewart CL (2014) Functional architecture of the cell’s nucleus in development, aging, and disease. Curr Top Dev Biol 109:1–52. 4. Mazumder A, Shivashankar GV (2010) Emergence of a prestressed eukaryotic nucleus during cellular differentiation and development. J R Soc Interface 7(Suppl 3): S321–S330. 5. Broers JL, et al. (2004) Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: Implications for the development of laminopathies. Hum Mol Genet 13(21):2567–2580. 6. Shumaker DK, Kuczmarski ER, Goldman RD (2003) The nucleoskeleton: Lamins and actin are major players in essential nuclear functions. Curr Opin Cell Biol 15(3): 358–366. 7. Ramdas NM, Shivashankar GV (2015) Cytoskeletal control of nuclear morphology and chromatin organization. J Mol Biol 427(3):695–706. 8. Guilluy C, et al. (2014) Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus. Nat Cell Biol 16(4):376–381. 9. Pajerowski JD, Dahl KN, Zhong FL, Sammak PJ, Discher DE (2007) Physical plasticity of the nucleus in stem cell differentiation. Proc Natl Acad Sci USA 104(40):15619–15624. 10. Talwar S, Kumar A, Rao M, Menon GI, Shivashankar GV (2013) Correlated spatiotemporal fluctuations in chromatin compaction states characterize stem cells. Biophys J 104(3):553–564. 11. Meshorer E, et al. (2006) Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev Cell 10(1):105–116. 12. Bhattacharya D, Talwar S, Mazumder A, Shivashankar GV (2009) Spatio-temporal plasticity in chromatin organization in mouse cell differentiation and during Drosophila embryogenesis. Biophys J 96(9):3832–3839. 13. Wang N, Tytell JD, Ingber DE (2009) Mechanotransduction at a distance: Mechanically coupling the extracellular matrix with the nucleus. Nat Rev Mol Cell Biol 10(1):75–82. 14. Versaevel M, et al. (2014) Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites. Sci Rep 4:7362. 15. Maniotis AJ, Chen CS, Ingber DE (1997) Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci USA 94(3):849–854. 16. Osmanagic-Myers S, Dechat T, Foisner R (2015) Lamins at the crossroads of mechanosignaling. Genes Dev 29(3):225–237. 17. Swift J, Discher DE (2014) The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue. J Cell Sci 127(Pt 14):3005–3015. 18. Chambliss AB, et al. (2013) The LINC-anchored actin cap connects the extracellular milieu to the nucleus for ultrafast mechanotransduction. Sci Rep 3:1087. 19. Sims JR, Karp S, Ingber DE (1992) Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape. J Cell Sci 103(Pt 4): 1215–1222. 20. Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T (2007) Dynamic genome architecture in the nuclear space: Regulation of gene expression in three dimensions. Nat Rev Genet 8(2):104–115. 21. Talwar S, Jain N, Shivashankar GV (2014) The regulation of gene expression during onset of differentiation by nuclear mechanical heterogeneity. Biomaterials 35(8): 2411–2419. 22. Shivashankar GV (2011) Mechanosignaling to the cell nucleus and gene regulation. Annu Rev Biophys 40:361–378. 23. Wang L, et al. (2013) Actin polymerization negatively regulates p53 function by impairing its nuclear import in response to DNA damage. PLoS One 8(4):e60179. 24. Zuchero JB, Belin B, Mullins RD (2012) Actin binding to WH2 domains regulates nuclear import of the multifunctional actin regulator JMY. Mol Biol Cell 23(5): 853–863. 25. Kumar A, Shivashankar GV (2012) Mechanical force alters morphogenetic movements and segmental gene expression patterns during Drosophila embryogenesis. PLoS One 7(3):e33089. 26. Gundersen GG, Worman HJ (2013) Nuclear positioning. Cell 152(6):1376–1389. 27. Harada T, et al. (2014) Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival. J Cell Biol 204(5):669–682.

28. Ribeiro AJ, Khanna P, Sukumar A, Dong C, Dahl KN (2014) Nuclear stiffening inhibits migration of invasive melanoma cells. Cell Mol Bioeng 7(4):544–551. 29. Worman HJ, Ostlund C, Wang Y (2010) Diseases of the nuclear envelope. Cold Spring Harb Perspect Biol 2(2):a000760. 30. Folker ES, Ostlund C, Luxton GW, Worman HJ, Gundersen GG (2011) Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement. Proc Natl Acad Sci USA 108(1):131–136. 31. Lee JS, et al. (2007) Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration. Biophys J 93(7):2542–2552. 32. Li Q, Kumar A, Makhija E, Shivashankar GV (2014) The regulation of dynamic mechanical coupling between actin cytoskeleton and nucleus by matrix geometry. Biomaterials 35(3):961–969. 33. Solon J, Levental I, Sengupta K, Georges PC, Janmey PA (2007) Fibroblast adaptation and stiffness matching to soft elastic substrates. Biophys J 93(12):4453–4461. 34. Zemel A, Rehfeldt F, Brown AE, Discher DE, Safran SA (2010) Optimal matrix rigidity for stress fiber polarization in stem cells. Nat Phys 6(6):468–473. 35. Chen CS (2008) Mechanotransduction - A field pulling together? J Cell Sci 121(Pt 20): 3285–3292. 36. Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689. 37. Kilian KA, Bugarija B, Lahn BT, Mrksich M (2010) Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci USA 107(11): 4872–4877. 38. Roca-Cusachs P, et al. (2008) Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation. Biophys J 94(12): 4984–4995. 39. Wang D, et al. (2014) Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment. Sci Rep 4:6160. 40. Khatau SB, et al. (2009) A perinuclear actin cap regulates nuclear shape. Proc Natl Acad Sci USA 106(45):19017–19022. 41. Kim DH, Wirtz D (2015) Cytoskeletal tension induces the polarized architecture of the nucleus. Biomaterials 48:161–172. 42. Iyer KV, Pulford S, Mogilner A, Shivashankar GV (2012) Mechanical activation of cells induces chromatin remodeling preceding MKL nuclear transport. Biophys J 103(7):1416–1428. 43. Poh YC, et al. (2012) Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells. Nat Commun 3:866. 44. Mazumder A, Shivashankar GV (2007) Gold-nanoparticle-assisted laser perturbation of chromatin assembly reveals unusual aspects of nuclear architecture within living cells. Biophys J 93(6):2209–2216. 45. Dahl KN, Engler AJ, Pajerowski JD, Discher DE (2005) Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures. Biophys J 89(4): 2855–2864. 46. Makatsori D, et al. (2004) The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope. J Biol Chem 279(24):25567–25573. 47. Raz V, et al. (2006) Changes in lamina structure are followed by spatial reorganization of heterochromatic regions in caspase-8-activated human mesenchymal stem cells. J Cell Sci 119(Pt 20):4247–4256. 48. Blackburn EH (2001) Switching and signaling at the telomere. Cell 106(6):661–673. 49. Zhang Q, Ragnauth C, Greener MJ, Shanahan CM, Roberts RG (2002) The nesprins are giant actin-binding proteins, orthologous to Drosophila melanogaster muscle protein MSP-300. Genomics 80(5):473–481. 50. Lombardi ML, et al. (2011) The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J Biol Chem 286(30):26743–26753. 51. Roux KJ, et al. (2009) Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization. Proc Natl Acad Sci USA 106(7):2194–2199. 52. Sun D, Leung CL, Liem RK (2001) Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): Identification of a novel group of microtubule associated proteins. J Cell Sci 114(Pt 1):161–172. 53. Waterman-Storer C, et al. (2000) Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport. J Cell Biol 150(2):361–376.

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in opposite directions (Fig. S5B). Vector correlation coefficients of the simulated trajectories (Fig. S5C) were calculated using custom written codes in MATLAB. The vector correlation is highest (+1) when the angle between trajectories is 0°, is lowest (−1) when the angle between trajectories is 180°, and has values between −1 and +1 for angles between 0° and 180°. Using this approach, vector correlation coefficients were then calculated between pairs of heterochromatin foci (and telomeres), and their correlation matrices were generated using custom written codes in MATLAB.

BIOPHYSICS AND COMPUTATIONAL BIOLOGY

trajectory was corrected for translation and rotation shift. Bright spots corresponding to heterochromatin foci/telomere were picked using surface thresholding, and their centroid xyz trajectories were obtained. To calculate correlation between the xyz trajectories of heterochromatin foci (or telomeres), Pearson correlation coefficient was used, and the ! ! scalar variables A(t) and B(t) were replaced by vectors AðtÞ and BðtÞ as shown below:

54. De Vos WH, et al. (2010) Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins. Biochim Biophys Acta 1800(4):448–458. 55. Lammerding J, et al. (2006) Lamins A and C but not lamin B1 regulate nuclear mechanics. J Biol Chem 281(35):25768–25780. 56. Swift J, et al. (2013) Nuclear lamin-A scales with tissue stiffness and enhances matrixdirected differentiation. Science 341(6149):1240104. 57. Sullivan T, et al. (1999) Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J Cell Biol 147(5):913–920. 58. Zidovska A, Weitz DA, Mitchison TJ (2013) Micron-scale coherence in interphase chromatin dynamics. Proc Natl Acad Sci USA 110(39):15555–15560. 59. Hameed FM, Rao M, Shivashankar GV (2012) Dynamics of passive and active particles in the cell nucleus. PLoS One 7(10):e45843.

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60. Sinha DK, Banerjee B, Maharana S, Shivashankar GV (2008) Probing the dynamic organization of transcription compartments and gene loci within the nucleus of living cells. Biophys J 95(11):5432–5438. 61. Jain N, Iyer KV, Kumar A, Shivashankar GV (2013) Cell geometric constraints induce modular gene-expression patterns via redistribution of HDAC3 regulated by actomyosin contractility. Proc Natl Acad Sci USA 110(28): 11349– 11354. 62. Luo W, et al. (2013) Analysis of the local organization and dynamics of cellular actin networks. J Cell Biol 202(7):1057–1073. 63. Navarro-Lérida I, et al. (2015) Rac1 nucleocytoplasmic shuttling drives nuclear shape changes and tumor invasion. Dev Cell 32(3):318–334. 64. Brown SW (1966) Heterochromatin. Science 151(3709):417–425.

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