© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 2111-2118 doi:10.1242/jcs.197517

SHORT REPORT Substrate stiffness-dependent regulation of the SRF−Mkl1 co-activator complex requires the inner nuclear membrane Emerin Margaret K. Willer and Christopher W. Carroll*

ABSTRACT transcription is thought to underlie persistent myofibroblast The complex comprising (SRF) and activation (Zhou et al., 2013). Thus, determining how mechanical − megakaryoblastic leukemia 1 protein (Mkl1) promotes myofibroblast cues modulate SRF Mkl1-dependent expression is critical to differentiation during wound healing. SRF−Mkl1 is sensitive to the understand wound healing and pathologies caused by altered ECM mechanical properties of the extracellular environment; but how cells mechanics. sense and transduce mechanical cues to modulate SRF−Mkl1- The nuclear lamina is a network of nuclear envelope-associated dependent gene expression is not well understood. Here, we intermediate filaments and their associated . Recently, the demonstrate that the nuclear lamina-associated inner nuclear nuclear lamina proteins Lamins A/C and Emerin were shown to − membrane protein Emerin stimulates SRF−Mkl1-dependent gene promote SRF Mkl1-dependent transcription (Ho et al., 2014). activity in a substrate stiffness-dependent manner. Specifically, Actin-generated forces regulate Lamins A/C and Emerin (Buxboim Emerin was required for Mkl1 nuclear accumulation and maximal et al., 2014; Guilluy et al., 2014; Swift et al., 2013), but whether the − SRF−Mkl1-dependent gene expression in response to serum nuclear lamina contributes to the mechanical control of SRF Mkl1- stimulation of cells grown on stiff substrates but was dispensable on dependent gene expression has not been tested. Here, we show that − more compliant substrates. Focal adhesion area was also reduced in Emerin specifically enhances SRF Mkl1-dependent transcription cells lacking Emerin, consistent with a role for Emerin in sensing in cells grown on stiff substrates, thereby linking ECM mechanics to substrate stiffness. Expression of a constitutively active form of Mkl1 gene expression. bypassed the requirement for Emerin in SRF−Mkl1-dependent gene expression and reversed the focal adhesion defects evident in EmdKO RESULTS AND DISCUSSION fibroblasts. Together, these data indicate that Emerin, a conserved The nuclear lamina promotes Mkl1 nuclear localization in nuclear lamina protein, couples extracellular matrix mechanics and cells grown on stiff substrates SRF−Mkl1-dependent transcription. Mkl1 is sequestered in the cytoplasm of resting cells by actin monomers, which both inhibit the nuclear import of Mkl1 and KEY WORDS: Nuclear lamina, Lamin A/C, Emerin, promote its nuclear export (Guettler et al., 2008; Miralles et al., Mechanosensitive, SRF, Mkl1 2003; Mouilleron et al., 2011; Pawłowski et al., 2010; Vartiainen et al., 2007). In response to stimuli that initiate actin polymerization, INTRODUCTION Mkl1 accumulates in the nucleus and, together with SRF, induces Wound healing requires the differentiation of fibroblasts and other the expression of important for myofibroblast differentiation cell types into myofibroblasts, which synthesize and remodel the (Cen et al., 2003; Crider et al., 2011; Esnault et al., 2014; Miralles extracellular matrix (ECM) to promote repair (Desmouliere et al., et al., 2003). Mkl1 nuclear localization after stimulation is 1993; Hinz, 2010). Persistent myofibroblast activity, however, leads significantly increased in cells on stiff substrates when compared to tissue stiffening and fibrosis, and has also been linked to the to cells plated on more compliant substrates (McGee et al., 2011; cancer progression (Butcher et al., 2009; Hinz, 2009; Huang et al., O’Connor et al., 2015; Varney et al., 2016). We tested whether the 2012). The co-activator complex comprising serum response factor nuclear lamina is important for mechanosensing in wild-type (SRF) and megakaryoblastic leukemia 1 protein (Mkl1; also known (EmdWT) vs Emerin-knockout (EmdKO) mouse embryonic as MRTF-A, MAL) (SRF−Mkl1) is critical for myofibroblast fibroblasts (MEFs) cultured on glass coverslips and collagen- differentiation (Crider et al., 2011; Small et al., 2010). Though coated polyacrylamide hydrogels with elastic moduli between chemical signals including TGFβ1 induce SRF−Mkl1-dependent 0.2−50 kPa, which spans the range observed in vertebrate tissues gene activation during wound healing, tension in the actin in vivo (Butcher et al., 2009). cytoskeleton developed through cell-ECM interactions is required After serum-starvation, Mkl1 localization − which we quantify for this response (Gomez et al., 2010; Huang et al., 2012; McGee et al., by measuring the nuclear-to-cytoplasmic (N:C) ratio of Mkl1 − was 2011; O’Connor et al., 2015; Varney et al., 2016). Moreover, a feed- similar in EmdWT and EmdKO MEFs, regardless of the underlying forward loop that couples tissue stiffness and SRF−Mkl1-dependent substrate (N:C ratio of ∼1−1.2) (Fig. 1). Mkl1 accumulated in the nucleus within 5 min of serum stimulation in EmdWT MEFs grown Dept. Of Cell Biology, Yale School of Medicine, New Haven, CT 06520, USA. on glass coverslips, reaching a maximum N:C ratio of 3.5±0.2 (Fig. 1A,B). The level of Mkl1 in the nucleus then steadily declined, *Author for correspondence ([email protected]) leading to an intermediate N:C ratio of 1.9±0.1 by 30 min that was C.W.C., 0000-0001-7492-5696 stable for the remainder of the experiment (up to 90 min) (Fig. 1B). The kinetics of Mkl1 nuclear accumulation were not affected by

Received 19 September 2016; Accepted 13 May 2017 substrate stiffness (Fig. S1A), but the maximum level of Mkl1 Journal of Cell Science

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Fig. 1. Emerin promotes Mkl1 nuclear localization in cells grown on stiff substrates. (A,B) Mkl1 localization and nuclear-to-cytoplasmic (N:C) ratio in cells grown on fibronectin-coated glass coverslips at the indicated time after serum stimulation (mean±s.e.m.; n=3). Asterisks indicate a significant difference between control and EmdKO cells at specific time points. (C,D) Mkl1 localization and N:C ratio in cells plated on collagen-coated polyacrylamide hydrogels of the indicated stiffness before or 5 min after serum stimulation (mean±s.e.m., n=3). In D, the black bracket* indicates a significant difference in the N:C ratio between EmdWT and EmdKO MEFs at 50 kPa; the red bracket* indicates a significant difference between EmdWT MEFs grown on 50 kPa and EmdWT MEFs grown on 1 kPa hydrogels. No significant change(purple bracket n.s.) was observed in EmdKO MEFs grown on 50 kPa and EmdKO MEFs grown on 1 kPa hydrogels. (E) Mkl1 N:C ratio in wild- type MEFs treated with control (siControl) or Lamins A/C siRNA (siLA/C) grown on 50 kPa or 1 kPa hydrogels before (Starved) or 5 min after serum stimulation (Stim.) (mean±s.e.m., n=3). (F) Mkl1 N:C ratio in EmdWT and EmdKO cells treated with control (siControl) or Lamins A/C (siLA/C) siRNAs on fibronectin-coated glass before (Starved) or 5 min after serum stimulation (Stim.) (mean±s.e.m, n=3). Scale bars: 20 µm; n.s., not significant; *P<0.05; **P<0.01; ***P<0.001. nuclear accumulation 5 min after serum stimulation was reduced in demonstrating that Mkl1 nuclear accumulation is substrate stiffness- EmdWT MEFs on soft substrates (≤1 kPa) relative to the stiffest dependent (McGee et al., 2011; O’Connor et al., 2015; Varney substrate (50 kPa) (Fig. 1C,D), consistent with previous reports et al., 2016). Emerin was required for maximal Mkl1 nuclear Journal of Cell Science

2112 SHORT REPORT Journal of Cell Science (2017) 130, 2111-2118 doi:10.1242/jcs.197517 accumulation on glass coverslips and on the stiffest hydrogel TAGLN expression in cells exposed to serum on plastic dishes for substrate (maximum N:C ratios in EmdKO MEFs of 2.5±0.1 and 1.8 24 h, which we refer to as steady state (Fig. 2C). Together, these data ±0.1, respectively) (Fig. 1A-D). A similar result was obtained when demonstrate that Emerin promotes Mkl1 localization specifically in EmdKO MEFs were plated on substrates with an elastic modulus of cells grown on stiff substrates and is required to maintain SRF 25 kPa (Fig. 1D), though the data did not rise to the level of −Mkl1-dependent gene activation over a 24 h time period. These statistical significance (P<0.07). On substrates with elastic moduli data agree well with the recent analysis of VCL expression in Lamin of ≤12 kPa, however, the N:C ratio of Mkl1 after serum stimulation A/C null fibroblasts (Ho et al., 2014). In that study, VCL mRNA was comparable between EmdWT and EmdKO cells (Fig. 1C,D). levels were comparable to control cells 1 h after serum stimulation Mkl1 protein levels were identical in EmdWT and EmdKO MEFs but were significantly reduced 5 h later. (Fig. S1B). Further, nuclear accumulation of the Smad 2/3 transcription factors after stimulation with TGFβ1 was similar in Emerin regulates focal adhesion size EmdWT and EmdKO MEFs cultured on glass coverslips or hydrogel A critical mechanism for sensing ECM mechanics occurs through substrates with elastic moduli of 50 or 1 kPa (Fig. S1C-F). Thus, focal adhesions (Schwartz, 2010). We, therefore, investigated Emerin specifically enhances Mkl1 nuclear accumulation in cells whether Emerin is required for focal adhesion assembly. grown on stiff substrates. Complicating these studies was the fact that EmdKO MEFs have a A similar dependence on Lamins A/C for Mkl1 nuclear reduced area and volume compared to control cells (Fig. 3A,B). accumulation has previously been reported in MEFs grown on Hence, we quantified the total focal adhesion area in individual cells glass coverslips (Ho et al., 2014). Consistent with this study, we and the percentage of cell area associated with focal adhesions, found that depletion of Lamins A/C by using specific small which provides an area-normalized measure of focal adhesion size. interfering RNAs (siRNAs) impaired Mkl1 nuclear accumulation EmdKO MEFs on glass coverslips had reduced focal adhesion size 5 min after serum stimulation in MEFs grown on a stiff hydrogel by both metrics at steady state (Fig. 3A,B). Emerin, therefore, plays substrate [50 kPa; N:C ratio: 2.6±0.2 (siControl) vs 1.8±0.2 a critical role in the ability of cells to sense ECM mechanics. (siLamins A/C)] (Fig. 1E, Fig. S2A). On a more compliant Further, the focal adhesion defects persisted in EmdKO cells after substrate, however, the maximum Mkl1 nuclear localization was not 24 h of serum starvation (Fig. 3A,B), which is likely to underlie the significantly different between control and Lamin A/C-depleted deficiency in Mkl1 localization and SRF−Mkl1-dependent gene cells [1 kPa; N:C ratio: 1.7±0.3 (siControl) vs 1.8±0.3 (siLamins activation in response to serum stimulation of cells grown on stiff A/C)] (Fig. 1E, Fig. S2A). The nuclear lamina is, therefore, substrates (Figs 1D, 2A). specifically required to couple ECM mechanics and Mkl1 The reduced focal adhesion size in EmdKO cells correlated with localization following serum stimulation of cells grown on stiff the reduced transcription of SRF−Mkl1 target genes at steady substrates. Interestingly, depletion of Lamins A/C, which anchor state (Figs 2C, 3A,B). In contrast, these parameters were partially Emerin at the inner nuclear membrane (INM), further reduced Mkl1 uncoupled after serum starvation; while differences in focal nuclear accumulation in EmdKO MEFs on glass coverslips from a adhesions remained (Fig. 3A,B), the mRNA levels of VCL, maximum N:C ratio of 2.3±0.1 (siControl) to a N:C ratio of 1.7±0.1 ACTB, and ACTA2 were not significantly different between (siLA/C) (Fig. 1F, see also Fig. S2B). Thus, Lamins A/C are likely control and EmdKO MEFs (Fig. 2B). To explore this paradox to have both Emerin-dependent and Emerin-independent roles in further, we quantified the levels of vinculin, β-actin, SMA and mediating Mkl1 nuclear accumulation after serum stimulation. SM22 protein in both control and EmdKO cells grown on plastic. Interestingly, the levels of each protein were significantly reduced Emerin is required for the substrate stiffness-dependent relative to EmdWT MEFs in EmdKO MEFs at both steady state and increase in SRF−Mkl1 activity after serum starvation (Fig. 3C,D). When taken together, these data We next asked how reduced Mkl1 nuclear localization in EmdKO indicate that, after serum deprivation, transcript levels of some cells affected SRF−Mkl1-dependent gene expression. Emerin was genes equalize more rapidly between EmdWT and EmdKO than their required for the full induction of an SRF-specific luciferase reporter protein products do, suggesting that the rate of protein turnover in in cells grown on plastic or 50 kPa hydrogel substrates (Fig. 2A). In the absence of serum influences how cells respond to subsequent contrast, there was no significant difference in reporter activity rounds of serum stimulation. between EmdWT and EmdKO cells grown on substrates with elastic moduli of 1 kPa (Fig. 2A). Furthermore, wild-type MEFs showed Stably expressed constitutively active Mkl1 bypasses the similar levels of Mkl1 accumulation in the nucleus, relative to requirement for Emerin in focal adhesion size EmdKO MEFs, on plastic and stiff hydrogels but showed no If the primary means by which Emerin contributes to sensing ECM significant difference on a more compliant substrate [Fig. 2A, ratio mechanics occur through SRF−Mkl1-dependent gene expression, (EmdWT:EmdKO)], suggesting that Emerin is required to link we would predict that a constitutively active form of Mkl1 (CA- SRF−Mkl1-dependent gene expression to substrate stiffness. Mkl1) can rescue focal adhesion area in EmdKO cells. We, therefore, Surprisingly, the mRNA levels of endogenous SRF−Mkl1 target generated a stable cell line that expressed CA-Mkl1 from a genes, including VCL (which encodes vinculin), ACTB (β-actin), doxycycline-inducible promoter (see Materials and Methods; Cen and ACTA2 (smooth-muscle actin, SMA), were not significantly et al., 2003; Miralles et al., 2003). Although localized throughout different between EmdWT and EmdKO MEFs grown on plastic the cell (Fig. 4A), CA-Mkl1 expression increased the mRNA levels before or 90 min after serum stimulation (Fig. 2B). Moreover, while of all tested SRF−Mkl1 target genes and eliminated the difference TAGLN (smooth muscle protein 22, SM22) mRNA levels were in TAGLN and ACTA2 expression between EmdWT and EmdKO cells lower before and after serum stimulation in EmdKO cells than in in serum-starved cells grown on plastic (Fig. 4B). Similarly, EmdWT cells, the fold-increase in mRNA levels was similar in both CA-Mkl1 induction equalized VCL and TAGLN expression, and cell lines (Fig. 2B). Emerin was, therefore, not required for substantially reduced the difference in ACTA2 expression between endogenous SRF−Mkl1-dependent gene activation after serum control and EmdKO MEFs at steady state (Fig. S3A). The lower KO stimulation. Emerin was, however, required for VCL, ACTA2 and levels of vinculin, actin, SM22 and SMA protein in Emd Journal of Cell Science

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Fig. 2. Emerin promotes SRF−Mkl1 activity in cells grown on stiff substrates. (A) Luciferase reporter activity in EmdWT and EmdKO MEFs grown on plastic or collagen-coated hydrogels of the indicated stiffness. Cells were stimulated with serum for 3 h. The EmdWT:EmdKO ratio (EmdWT/EmdKO) is the relative luciferase activity (RLA) in EmdWT cells (black bars) divided by RLA in EmdKO cells (gray bars). (B) TBP-normalized mRNA levels of SRF−Mkl1 target genes in cells grown on plastic before (Starved) or after stimulation (Stim.) (mean±s.e.m., n=3). (C) TBP-normalized mRNA levels SRF−Mkl1 target genes in MEFs grown with serum on plastic for 24 h (steady state) (mean±s.e.m., n=3). The dotted line shows the level of expression for each gene in serum-starved cells.

MEFs grown on plastic was also reversed by CA-Mkl1 somewhat muted, effect was observed in cells at steady state (Fig. expression (Fig. 4C,D, Fig. S3B,C). CA-Mkl1, therefore, S3D,E). Thus, CA-Mkl1 expression largely suppressed the focal effectively bypassed the requirement for Emerin in SRF−Mkl1- adhesion defect in EmdKO MEFs, suggesting that Emerin contributes dependent gene activation. to the assembly of an actin-based mechanosensory apparatus by CA-Mkl1 did not rescue the spread area or volume deficiencies promoting SRF−Mkl1-dependent transcription. evident in EmdKO MEFs (Fig. 4E,F, Fig. S3D,E). The total focal A crucial question is how Emerin specifically promotes SRF– adhesion area after CA-Mkl1 expression was similar in serum-starved Mkl1-dependent gene activation in cells grown on stiff substrates. EmdWT and EmdKO cells grown on glass, which resulted in a greater Emerin binds to actin and promotes actin polymerization in vitro percentage of the cell area in EmdKO MEFs being associated with (Holaska and Wilson, 2007; Holaska et al., 2004), and focal adhesions than in control cells (Fig. 4E,F). A similar, although that disrupt actin binding do not support SRF−Mkl1-dependent Journal of Cell Science

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Fig. 3. Emerin is required for focal adhesion size. (A) Vinculin and F-actin in EmdWT and EmdKO MEFs on fibronectin-coated glass coverslips at steady state or after 24 h of serum starvation (Starved). (B) Quantification of cell area, volume, total FA area per cell and percentage of cell area associated with focal adhesions (FA), in cells grown on fibronectin-coated glass coverslips. Steady state (SS); serum starved (Star.); n=30 cells from three independent experiments were quantified. Box and whiskers span the first to third quartiles and 5−95% of all data points, respectively. (C,D) Western blot and protein levels from EmdWT and EmdKO MEFs cultured to steady state (SS) or under starvation (Star.) conditions (mean±s.e.m.; n=4). Scale bar: 20 µm. *P<0.05, **P<0.01, ****P<0.0001. transcription (Ho et al., 2014). The ability of Emerin to promote actin Lamins A/C promote Mkl1 nuclear localization in cells grown on stiff polymerization can, therefore, be linked to ECM mechanics. Indeed, substrates. We have demonstrated recently that nuclear envelope- Emerin is tyrosine phosphorylated in cells grown on stiff substrates spanning linker of nucleoskeleton and cytoskeleton (LINC) and was recently shown to move from the INM to the outer nuclear complexes, which couple F-actin, microtubules and intermediate membrane (ONM) after exposure to biaxial strain (Guilluy et al., filaments to the nuclear lamina, promote focal adhesion assembly 2014; Le et al., 2016); although we did not observe a change in through a transcription-independent mechanism (Thakar et al., 2017). Emerin localization at the INM or ONM on hydrogel substrates of Thus, Lamins A/C could enhance Mkl1 nuclear accumulation differing stiffness in this study (Fig. S4A,B). Further studies through LINC complexes in EmdKO cells. exploring the relationship between Emerin, substrate stiffness and These studies have important implications for our understanding actin dynamics will be crucial to address these issues. Equally of fibrosis and cancer, both of which are linked to persistent important is to discover how the Emerin-independent functions of myofibroblast activity. Specifically, our results suggest that Emerin Journal of Cell Science

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Fig. 4. CA-Mkl1 activity rescues cytoskeletal defects in EmdKO MEFs. (A) Localization of constitutively active Mkl1 tagged to GFP (CA-Mkl1−GFP) in EmdWT and EmnKO cells. (B) mRNA levels in serum-starved cells on plastic with or without (−) CA-Mkl1. Dashed line indicates expression levels in steady-state cells. (C, D) Representative western blot and protein levels in serum-starved EmdWT and EmdKO MEFs grown on plastic with or without (−) CA-Mkl1. (E) Representative images of vinculin and F-actin in starved cells grown on fibronectin-coated glass with or without (−) CA-Mkl1. (F) Quantification of cell area, volume and total focal adhesion (FA) area per cell, and the percentage of cell area associated with the focal adhesions in starved EmdWT and EmnKO MEFs grown on fibronectin-coated glass coverslips. n=30 cells from three independent experiments were quantified. Box and whiskers span the first to third quartiles and 5−95% of all data points, respectively. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Scale bars: 20 µm. Journal of Cell Science

2116 SHORT REPORT Journal of Cell Science (2017) 130, 2111-2118 doi:10.1242/jcs.197517 is a crucial component of the mechanical feed-forward loop that Acknowledgements sustains SRF−Mkl1-dependent gene expression in response to The authors thank Christopher May (Yale University, New Haven, CT) for comments on the manuscript. We also thank the King, Lusk, Yao, and Bahnmanyar labs for tissue stiffening (Duffield et al., 2013; Small et al., 2010; Zhou helpful comments on the project, Jan Lammerding (Cornell University, Ithaca, NY) et al., 2013). The nuclear lamina may, therefore, drive pathologies for the EmdWT and EmdKO MEFs, Diane Krause (Yale University, New Haven, CT) that involve altered ECM mechanics. for Mkl1 and pRenilla plasmids, and Carson Thoreen (Yale University, New Haven, CT) for lentiviral vectors.

MATERIALS AND METHODS Competing interests Cell culture The authors declare no competing or financial interests. EmdWT and EmdKO (Emd+/Y and Emd−/Y, respectively) MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% Author contributions fetal bovine serum (FBS; Sigma, St Louis, MO) and starved in serum-free Conceptualization: M.K.W., C.W.C.; Methodology: M.K.W.; Software: C.W.C.; medium containing 0.2% FBS or in 0.5% fatty acid-free BSA (Sigma) for Investigation: M.K.W.; Data curation: M.K.W.; Writing - original draft: M.K.W., C.W.C.; 16-24 h. Following starvation, MEFs were stimulated with 10% FBS. Cells Writing - review & editing: C.W.C.; Funding acquisition: C.W.C. at steady state were kept in a constant serum concentration for 24 h. Cell lines expressing doxycycline-inducible constitutively active Mkl1 tagged to Funding GFP (CA-Mkl1−GFP) (residues 81−929 of mouse Mkl1) that lacks the This work was supported in part by an Innovator Award from the Progeria Research Foundation Award PRF2013-49 (C.W.C.). N-terminal actin-binding domain, were generated as described (Thoreen et al., 2012). CA-Mkl1 expression was induced with 1 μg/ml doxycycline Supplementary information for 24 h before cells were used. Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.197517.supplemental Indirect immunofluorescence and confocal microscopy Coverslips were either coated with 50 μg/ml fibronectin or with References Butcher, D. T., Alliston, T. and Weaver, V. M. (2009). A tense situation: forcing collagen pre-coated polyacrylamide hydrogels (Matrigen, Brea, CA). tumour progression. Nat. Rev. Cancer 9, 108-122. Immunofluorescence was performed as described (Carroll et al., 2009). To Buxboim, A., Swift, J., Irianto, J., Spinler, K. R., Dingal, P. C. D. P., Athirasala, assess Emerin localization, fixed cells were permeabilized with 0.25% A., Kao, Y.-R. C., Cho, S., Harada, T., Shin, J.-W. et al. (2014). Matrix elasticity Triton-X 100 or 0.003% digitonin. Images were captured on a Nikon regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin. TE2000-S by using a 40× oil immersion objective or a Leica SP5 laser Curr. Biol. 24, 1909-1917. scanning confocal microscope by using a 100× oil immersion objective. Carroll, C. W., Silva, M. C. C., Godek, K. M., Jansen, L. E. T. and Straight, A. F. Image analysis and quantification was performed in ImageJ. N:C ratios (2009). Centromere assembly requires the direct recognition of CENP-A were calculated by dividing the total fluorescence signal in the nucleus by nucleosomes by CENP-N. Nat. Cell Biol. 11, 896-902. Cen, B., Selvaraj, A., Burgess, R. C., Hitzler, J. K., Ma, Z., Morris, S. W. and the total fluorescence signal in a ring of cytoplasm surrounding the nucleus Prywes, R. (2003). Megakaryoblastic leukemia 1, a potent transcriptional after accounting for differences in the measurement areas. 100 cells were coactivator for serum response factor (SRF), is required for serum induction of quantified for each condition in each experiment. Emerin localization to SRF target genes. Mol. Cell. Biol. 23, 6597-6608. the INM or ONM was quantified by measuring the intensity levels of Crider, B. J., Risinger, G. M., Haaksma, C. J., Howard, E. W. and Tomasek, J. J. Emerin fluorescence at the nuclear envelope in Triton X-100 or digitonin- (2011). -related transcription factors A and B are key regulators of TGF- permeabilized cells, respectively (Le et al., 2016). Focal adhesions were β1-induced fibroblast to myofibroblast differentiation. J. Invest. Dermatol. 131, quantified as described (Thakar et al., 2017). The area of all FAs from each 2378-2385. Desmouliere, A., Geinoz, A., Gabbiani, F. and Gabbiani, G. (1993). Transforming cell was summed and then divided by the cell area to determine total FA growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation area per cell and the percent of vinculin-positive area per cell, respectively. tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell. The radius of spherical cells after trypsinization, measured with a Biol. 122, 103-111. Cellometer Auto T4 cell counter (Nexcelom Bioscience, Lawrence, Duffield, J. S., Lupher, M., Thannickal, V. J. and Wynn, T. A. (2013). Host MA), was used to calculate cell volume. Actin filaments were visualized responses in tissue repair and fibrosis. Annu. Rev. Pathol. Mech. Dis. 8, 241-276. with phalloidin (Life Technologies, Eugene, OR). Antibodies are listed in Esnault, C., Stewart, A., Gualdrini, F., East, P., Horswell, S., Matthews, N. and Table S1. Treisman, R. (2014). Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts. Genes Dev. 28, 943-958. Protein and RNA methods Gomez, E. W., Chen, Q. K., Gjorevski, N. and Nelson, C. M. (2010). Tissue MEFs were lysed directly in 1× Laemmli buffer for western blots. mRNA geometry patterns epithelial-mesenchymal transition via intercellular for quantitative PCR (qPCR) was isolated by using the RNeasy Mini Kit mechanotransduction. J. Cell. Biochem. 110, 44-51. (Qiagen, Germany). cDNA was synthesized from 100 ng mRNA with Guettler, S., Vartiainen, M. K., Miralles, F., Larijani, B. and Treisman, R. (2008). RPEL motifs link the serum response factor cofactor MAL but not myocardin to SuperScript III First-Strand synthesis system (Invitrogen, Carlsbad, CA) Rho signaling via actin binding. Mol. Cell. Biol. 28, 732-742. using an oligo dT primer. qPCR was performed using SYBR green Guilluy, C., Osborne, L. D., Van Landeghem, L., Sharek, L., Superfine, R., detection in a CFX96 Real-Time System (Bio-Rad, Hercules, CA). mRNA Garcia-Mata, R. and Burridge, K. (2014). Isolated nuclei adapt to force and levels were calculated from qPCR data by using the dCT method after reveal a mechanotransduction pathway in the nucleus. Nat. Cell Biol. 16, 376-381. normalization to TATA-box binding protein (TBP) mRNA. Oligonucleotids Hinz, B. (2009). Tissue stiffness, latent TGF-beta1 activation, and mechanical for qPCR are listed in Table S2. signal transduction: implications for the pathogenesis and treatment of fibrosis. Curr. Rheumatol. Rep. 11, 120-126. Hinz, B. (2010). The myofibroblast: paradigm for a mechanically active cell. Luciferase assays J. Biomech. 43, 146-155. Cells were transfected with a 50:1 ratio of 5xSRF-RE (Promega, Madison, Ho, C. Y., Jaalouk, D. E., Vartiainen, M. K. and Lammerding, J. (2014). Lamin A/C WI) and pRenilla plasmids (gift from D. Krause). Four hours later, the and emerin regulate MKL1–SRF activity by modulating actin dynamics. Nature medium was replaced with serum-free medium for 24 h. Cells were then 497, 507-511. harvested or stimulated for 3 h with FBS. Luciferase activity was measured Holaska, J. M. and Wilson, K. L. (2007). An emerin “Proteome”: purification of using the Dual Luciferase Reporter Kit (Promega, Madison, WI). Values for distinct emerin-containing complexes from HeLa cells suggests molecular basis for diverse roles including gene regulation, mRNA splicing, signaling, each experimental condition are the average ratio of firefly-to-Renilla mechanosensing, and nuclear architecture †. Biochemistry 46, 8897-8908. luciferase activity in three technical replicates. The specificity of the Holaska, J. M., Kowalski, A. K. and Wilson, K. L. (2004). Emerin caps the pointed luciferase reporter was verified using cytochalasin D and latrunculin B as end of actin filaments: evidence for an actin cortical network at the nuclear inner described (Vartiainen et al., 2007). membrane. PLoS Biol. 2, e231. Journal of Cell Science

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