Advertisement
Short Report
Substrate stiffness-dependent regulation of the SRF−Mkl1 co-activator complex requires the inner nuclear membrane protein Emerin
Margaret K. Willer, Christopher W. Carroll
Journal of Cell Science 2017 130: 2111-2118; doi: 10.1242/jcs.197517

ABSTRACT

The complex comprising serum response factor (SRF) and megakaryoblastic leukemia 1 protein (Mkl1) promotes myofibroblast differentiation during wound healing. SRF−Mkl1 is sensitive to the mechanical properties of the extracellular environment; but how cells sense and transduce mechanical cues to modulate SRF−Mkl1-dependent gene expression is not well understood. Here, we demonstrate that the nuclear lamina-associated inner nuclear membrane protein Emerin stimulates SRF−Mkl1-dependent gene activity in a substrate stiffness-dependent manner. Specifically, Emerin was required for Mkl1 nuclear accumulation and maximal SRF−Mkl1-dependent gene expression in response to serum stimulation of cells grown on stiff substrates but was dispensable on more compliant substrates. Focal adhesion area was also reduced in cells lacking Emerin, consistent with a role for Emerin in sensing substrate stiffness. Expression of a constitutively active form of Mkl1 bypassed the requirement for Emerin in SRF−Mkl1-dependent gene expression and reversed the focal adhesion defects evident in EmdKO fibroblasts. Together, these data indicate that Emerin, a conserved nuclear lamina protein, couples extracellular matrix mechanics and SRF−Mkl1-dependent transcription.

INTRODUCTION

Wound healing requires the differentiation of fibroblasts and other cell types into myofibroblasts, which synthesize and remodel the extracellular matrix (ECM) to promote repair (Desmouliere et al., 1993; Hinz, 2010). Persistent myofibroblast activity, however, leads to tissue stiffening and fibrosis, and has also been linked to the cancer progression (Butcher et al., 2009; Hinz, 2009; Huang et al., 2012). The co-activator complex comprising serum response factor (SRF) and megakaryoblastic leukemia 1 protein (Mkl1; also known as MRTF-A, MAL) (SRF−Mkl1) is critical for myofibroblast differentiation (Crider et al., 2011; Small et al., 2010). Though chemical signals including TGFβ1 induce SRF−Mkl1-dependent gene activation during wound healing, tension in the actin cytoskeleton developed through cell-ECM interactions is required for this response (Gomez et al., 2010; Huang et al., 2012; McGee et al., 2011; O'Connor et al., 2015; Varney et al., 2016). Moreover, a feed-forward loop that couples tissue stiffness and SRF−Mkl1-dependent transcription is thought to underlie persistent myofibroblast activation (Zhou et al., 2013). Thus, determining how mechanical cues modulate SRF−Mkl1-dependent gene expression is critical to understand wound healing and pathologies caused by altered ECM mechanics.

The nuclear lamina is a network of nuclear envelope-associated intermediate filaments and their associated proteins. Recently, the nuclear lamina proteins Lamins A/C and Emerin were shown to promote SRF−Mkl1-dependent transcription (Ho et al., 2014). Actin-generated forces regulate Lamins A/C and Emerin (Buxboim et al., 2014; Guilluy et al., 2014; Swift et al., 2013), but whether the nuclear lamina contributes to the mechanical control of SRF−Mkl1-dependent gene expression has not been tested. Here, we show that Emerin specifically enhances SRF−Mkl1-dependent transcription in cells grown on stiff substrates, thereby linking ECM mechanics to gene expression.

RESULTS AND DISCUSSION

The nuclear lamina promotes Mkl1 nuclear localization in cells grown on stiff substrates

Mkl1 is sequestered in the cytoplasm of resting cells by actin monomers, which both inhibit the nuclear import of Mkl1 and promote its nuclear export (Guettler et al., 2008; Miralles et al., 2003; Mouilleron et al., 2011; Pawłowski et al., 2010; Vartiainen et al., 2007). In response to stimuli that initiate actin polymerization, Mkl1 accumulates in the nucleus and, together with SRF, induces the expression of genes important for myofibroblast differentiation (Cen et al., 2003; Crider et al., 2011; Esnault et al., 2014; Miralles et al., 2003). Mkl1 nuclear localization after stimulation is significantly increased in cells on stiff substrates when compared to cells plated on more compliant substrates (McGee et al., 2011; O'Connor et al., 2015; Varney et al., 2016). We tested whether the nuclear lamina is important for mechanosensing in wild-type (EmdWT) vs Emerin-knockout (EmdKO) mouse embryonic fibroblasts (MEFs) cultured on glass coverslips and collagen-coated polyacrylamide hydrogels with elastic moduli between 0.2−50 kPa, which spans the range observed in vertebrate tissues in vivo (Butcher et al., 2009).

After serum-starvation, Mkl1 localization − which we quantify by measuring the nuclear-to-cytoplasmic (N:C) ratio of Mkl1 − was similar in EmdWT and EmdKO MEFs, regardless of the underlying 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 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, leading to an intermediate N:C ratio of 1.9±0.1 by 30 min that was stable for the remainder of the experiment (up to 90 min) (Fig. 1B). The kinetics of Mkl1 nuclear accumulation were not affected by substrate stiffness (Fig. S1A), but the maximum level of Mkl1 nuclear accumulation 5 min after serum stimulation was reduced in EmdWT MEFs on soft substrates (≤1 kPa) relative to the stiffest substrate (50 kPa) (Fig. 1C,D), consistent with previous reports demonstrating that Mkl1 nuclear accumulation is substrate stiffness-dependent (McGee et al., 2011; O'Connor et al., 2015; Varney et al., 2016). Emerin was required for maximal Mkl1 nuclear accumulation on glass coverslips and on the stiffest hydrogel substrate (maximum N:C ratios in EmdKO MEFs of 2.5±0.1 and 1.8±0.1, respectively) (Fig. 1A-D). A similar result was obtained when EmdKO MEFs were plated on substrates with an elastic modulus of 25 kPa (Fig. 1D), though the data did not rise to the level of statistical significance (P<0.07). On substrates with elastic moduli of ≤12 kPa, however, the N:C ratio of Mkl1 after serum stimulation was comparable between EmdWT and EmdKO cells (Fig. 1C,D). Mkl1 protein levels were identical in EmdWT and EmdKO MEFs (Fig. S1B). Further, nuclear accumulation of the Smad 2/3 transcription factors after stimulation with TGFβ1 was similar in EmdWT and EmdKO MEFs cultured on glass coverslips or hydrogel substrates with elastic moduli of 50 or 1 kPa (Fig. S1C-F). Thus, Emerin specifically enhances Mkl1 nuclear accumulation in cells grown on stiff substrates.

Fig. 1.
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.

A similar dependence on Lamins A/C for Mkl1 nuclear accumulation has previously been reported in MEFs grown on glass coverslips (Ho et al., 2014). Consistent with this study, we found that depletion of Lamins A/C by using specific small interfering RNAs (siRNAs) impaired Mkl1 nuclear accumulation 5 min after serum stimulation in MEFs grown on a stiff hydrogel substrate [50 kPa; N:C ratio: 2.6±0.2 (siControl) vs 1.8±0.2 (siLamins A/C)] (Fig. 1E, Fig. S2A). On a more compliant substrate, however, the maximum Mkl1 nuclear localization was not significantly different between control and Lamin A/C-depleted cells [1 kPa; N:C ratio: 1.7±0.3 (siControl) vs 1.8±0.3 (siLamins A/C)] (Fig. 1E, Fig. S2A). The nuclear lamina is, therefore, specifically required to couple ECM mechanics and Mkl1 localization following serum stimulation of cells grown on stiff substrates. Interestingly, depletion of Lamins A/C, which anchor Emerin at the inner nuclear membrane (INM), further reduced Mkl1 nuclear accumulation in EmdKO MEFs on glass coverslips from a maximum N:C ratio of 2.3±0.1 (siControl) to a N:C ratio of 1.7±0.1 (siLA/C) (Fig. 1F, see also Fig. S2B). Thus, Lamins A/C are likely to have both Emerin-dependent and Emerin-independent roles in mediating Mkl1 nuclear accumulation after serum stimulation.

Emerin is required for the substrate stiffness-dependent increase in SRF−Mkl1 activity

We next asked how reduced Mkl1 nuclear localization in EmdKO cells affected SRF−Mkl1-dependent gene expression. Emerin was required for the full induction of an SRF-specific luciferase reporter in cells grown on plastic or 50 kPa hydrogel substrates (Fig. 2A). In contrast, there was no significant difference in reporter activity between EmdWT and EmdKO cells grown on substrates with elastic moduli of 1 kPa (Fig. 2A). Furthermore, wild-type MEFs showed similar levels of Mkl1 accumulation in the nucleus, relative to EmdKO MEFs, on plastic and stiff hydrogels but showed no significant difference on a more compliant substrate [Fig. 2A, ratio (EmdWT:EmdKO)], suggesting that Emerin is required to link SRF−Mkl1-dependent gene expression to substrate stiffness.

Fig. 2.
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.

Surprisingly, the mRNA levels of endogenous SRF−Mkl1 target genes, including VCL (which encodes vinculin), ACTB (β-actin), and ACTA2 (smooth-muscle actin, SMA), were not significantly different between EmdWT and EmdKO MEFs grown on plastic before or 90 min after serum stimulation (Fig. 2B). Moreover, while TAGLN (smooth muscle protein 22, SM22) mRNA levels were lower before and after serum stimulation in EmdKO cells than in EmdWT cells, the fold-increase in mRNA levels was similar in both cell lines (Fig. 2B). Emerin was, therefore, not required for endogenous SRF−Mkl1-dependent gene activation after serum stimulation. Emerin was, however, required for VCL, ACTA2 and TAGLN expression in cells exposed to serum on plastic dishes for 24 h, which we refer to as steady state (Fig. 2C). Together, these data demonstrate that Emerin promotes Mkl1 localization specifically in cells grown on stiff substrates and is required to maintain SRF−Mkl1-dependent gene activation over a 24 h time period. These data agree well with the recent analysis of VCL expression in Lamin A/C null fibroblasts (Ho et al., 2014). In that study, VCL mRNA levels were comparable to control cells 1 h after serum stimulation but were significantly reduced 5 h later.

Emerin regulates focal adhesion size

A critical mechanism for sensing ECM mechanics occurs through focal adhesions (Schwartz, 2010). We, therefore, investigated whether Emerin is required for focal adhesion assembly. Complicating these studies was the fact that EmdKO MEFs have a reduced area and volume compared to control cells (Fig. 3A,B). Hence, we quantified the total focal adhesion area in individual cells and the percentage of cell area associated with focal adhesions, which provides an area-normalized measure of focal adhesion size. EmdKO MEFs on glass coverslips had reduced focal adhesion size by both metrics at steady state (Fig. 3A,B). Emerin, therefore, plays a critical role in the ability of cells to sense ECM mechanics. Further, the focal adhesion defects persisted in EmdKO cells after 24 h of serum starvation (Fig. 3A,B), which is likely to underlie the deficiency in Mkl1 localization and SRF−Mkl1-dependent gene activation in response to serum stimulation of cells grown on stiff substrates (Figs 1D, 2A).

Fig. 3.
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.

The reduced focal adhesion size in EmdKO cells correlated with the reduced transcription of SRF−Mkl1 target genes at steady state (Figs 2C, 3A,B). In contrast, these parameters were partially uncoupled after serum starvation; while differences in focal adhesions remained (Fig. 3A,B), the mRNA levels of VCL, ACTB, and ACTA2 were not significantly different between control and EmdKO MEFs (Fig. 2B). To explore this paradox further, we quantified the levels of vinculin, β-actin, SMA and SM22 protein in both control and EmdKO cells grown on plastic. Interestingly, the levels of each protein were significantly reduced relative to EmdWT MEFs in EmdKO MEFs at both steady state and after serum starvation (Fig. 3C,D). When taken together, these data indicate that, after serum deprivation, transcript levels of some genes equalize more rapidly between EmdWT and EmdKO than their protein products do, suggesting that the rate of protein turnover in the absence of serum influences how cells respond to subsequent rounds of serum stimulation.

Stably expressed constitutively active Mkl1 bypasses the requirement for Emerin in focal adhesion size

If the primary means by which Emerin contributes to sensing ECM mechanics occur through SRF−Mkl1-dependent gene expression, we would predict that a constitutively active form of Mkl1 (CA-Mkl1) can rescue focal adhesion area in EmdKO cells. We, therefore, generated a stable cell line that expressed CA-Mkl1 from a doxycycline-inducible promoter (see Materials and Methods; Cen et al., 2003; Miralles et al., 2003). Although localized throughout the cell (Fig. 4A), CA-Mkl1 expression increased the mRNA levels of all tested SRF−Mkl1 target genes and eliminated the difference in TAGLN and ACTA2 expression between EmdWT and EmdKO cells in serum-starved cells grown on plastic (Fig. 4B). Similarly, CA-Mkl1 induction equalized VCL and TAGLN expression, and substantially reduced the difference in ACTA2 expression between control and EmdKO MEFs at steady state (Fig. S3A). The lower levels of vinculin, actin, SM22 and SMA protein in EmdKO MEFs grown on plastic was also reversed by CA-Mkl1 expression (Fig. 4C,D, Fig. S3B,C). CA-Mkl1, therefore, effectively bypassed the requirement for Emerin in SRF−Mkl1-dependent gene activation.

Fig. 4.
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.

CA-Mkl1 did not rescue the spread area or volume deficiencies evident in EmdKO MEFs (Fig. 4E,F, Fig. S3D,E). The total focal adhesion area after CA-Mkl1 expression was similar in serum-starved EmdWT and EmdKO cells grown on glass, which resulted in a greater percentage of the cell area in EmdKO MEFs being associated with focal adhesions than in control cells (Fig. 4E,F). A similar, although somewhat muted, effect was observed in cells at steady state (Fig. S3D,E). Thus, CA-Mkl1 expression largely suppressed the focal adhesion defect in EmdKO MEFs, suggesting that Emerin contributes to the assembly of an actin-based mechanosensory apparatus by promoting SRF−Mkl1-dependent transcription.

A crucial question is how Emerin specifically promotes SRF−Mkl1-dependent gene activation in cells grown on stiff substrates. Emerin binds to actin and promotes actin polymerization in vitro (Holaska and Wilson, 2007; Holaska et al., 2004), and mutations that disrupt actin binding do not support SRF−Mkl1-dependent transcription (Ho et al., 2014). The ability of Emerin to promote actin polymerization can, therefore, be linked to ECM mechanics. Indeed, Emerin is tyrosine phosphorylated in cells grown on stiff substrates and was recently shown to move from the INM to the outer nuclear membrane (ONM) after exposure to biaxial strain (Guilluy et al., 2014; Le et al., 2016); although we did not observe a change in Emerin localization at the INM or ONM on hydrogel substrates of differing stiffness in this study (Fig. S4A,B). Further studies exploring the relationship between Emerin, substrate stiffness and actin dynamics will be crucial to address these issues. Equally important is to discover how the Emerin-independent functions of Lamins A/C promote Mkl1 nuclear localization in cells grown on stiff substrates. We have demonstrated recently that nuclear envelope-spanning linker of nucleoskeleton and cytoskeleton (LINC) complexes, which couple F-actin, microtubules and intermediate filaments to the nuclear lamina, promote focal adhesion assembly through a transcription-independent mechanism (Thakar et al., 2017). Thus, Lamins A/C could enhance Mkl1 nuclear accumulation through LINC complexes in EmdKO cells.

These studies have important implications for our understanding of fibrosis and cancer, both of which are linked to persistent myofibroblast activity. Specifically, our results suggest that Emerin is a crucial component of the mechanical feed-forward loop that sustains SRF−Mkl1-dependent gene expression in response to tissue stiffening (Duffield et al., 2013; Small et al., 2010; Zhou et al., 2013). The nuclear lamina may, therefore, drive pathologies that involve altered ECM mechanics.

MATERIALS AND METHODS

Cell culture

EmdWT and EmdKO (Emd+/Y and Emd−/Y, respectively) MEFs were maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS; Sigma, St Louis, MO) and starved in serum-free medium containing 0.2% FBS or in 0.5% fatty acid-free BSA (Sigma) for 16-24 h. Following starvation, MEFs were stimulated with 10% FBS. Cells at steady state were kept in a constant serum concentration for 24 h. Cell lines expressing doxycycline-inducible constitutively active Mkl1 tagged to GFP (CA-Mkl1−GFP) (residues 81−929 of mouse Mkl1) that lacks the N-terminal actin-binding domain, were generated as described (Thoreen et al., 2012). CA-Mkl1 expression was induced with 1 μg/ml doxycycline for 24 h before cells were used.

Indirect immunofluorescence and confocal microscopy

Coverslips were either coated with 50 μg/ml fibronectin or with collagen pre-coated polyacrylamide hydrogels (Matrigen, Brea, CA). Immunofluorescence was performed as described (Carroll et al., 2009). To assess Emerin localization, fixed cells were permeabilized with 0.25% Triton-X 100 or 0.003% digitonin. Images were captured on a Nikon TE2000-S by using a 40× oil immersion objective or a Leica SP5 laser scanning confocal microscope by using a 100× oil immersion objective. Image analysis and quantification was performed in ImageJ. N:C ratios were calculated by dividing the total fluorescence signal in the nucleus by the total fluorescence signal in a ring of cytoplasm surrounding the nucleus after accounting for differences in the measurement areas. 100 cells were quantified for each condition in each experiment. Emerin localization to the INM or ONM was quantified by measuring the intensity levels of Emerin fluorescence at the nuclear envelope in Triton X-100 or digitonin-permeabilized cells, respectively (Le et al., 2016). Focal adhesions were quantified as described (Thakar et al., 2017). The area of all FAs from each cell was summed and then divided by the cell area to determine total FA area per cell and the percent of vinculin-positive area per cell, respectively. The radius of spherical cells after trypsinization, measured with a Cellometer Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA), was used to calculate cell volume. Actin filaments were visualized with phalloidin (Life Technologies, Eugene, OR). Antibodies are listed in Table S1.

Protein and RNA methods

MEFs were lysed directly in 1× Laemmli buffer for western blots. mRNA for quantitative PCR (qPCR) was isolated by using the RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 100 ng mRNA with SuperScript III First-Strand synthesis system (Invitrogen, Carlsbad, CA) using an oligo dT primer. qPCR was performed using SYBR green detection in a CFX96 Real-Time System (Bio-Rad, Hercules, CA). mRNA levels were calculated from qPCR data by using the dCT method after normalization to TATA-box binding protein (TBP) mRNA. Oligonucleotids for qPCR are listed in Table S2.

Luciferase assays

Cells were transfected with a 50:1 ratio of 5xSRF-RE (Promega, Madison, WI) and pRenilla plasmids (gift from D. Krause). Four hours later, the medium was replaced with serum-free medium for 24 h. Cells were then harvested or stimulated for 3 h with FBS. Luciferase activity was measured using the Dual Luciferase Reporter Kit (Promega, Madison, WI). Values for each experimental condition are the average ratio of firefly-to-Renilla luciferase activity in three technical replicates. The specificity of the luciferase reporter was verified using cytochalasin D and latrunculin B as described (Vartiainen et al., 2007).

Acknowledgements

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 helpful comments on the project, Jan Lammerding (Cornell University, Ithaca, NY) for the EmdWT and EmdKO MEFs, Diane Krause (Yale University, New Haven, CT) for Mkl1 and pRenilla plasmids, and Carson Thoreen (Yale University, New Haven, CT) for lentiviral vectors.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Author contributions

    Conceptualization: M.K.W., C.W.C.; Methodology: M.K.W.; Software: C.W.C.; Investigation: M.K.W.; Data curation: M.K.W.; Writing - original draft: M.K.W., C.W.C.; Writing - review & editing: C.W.C.; Funding acquisition: C.W.C.

  • Funding

    This work was supported in part by an Innovator Award from the Progeria Research Foundation Award PRF2013-49 (C.W.C.).

  • Supplementary information

    Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.197517.supplemental

  • Received September 19, 2016.
  • Accepted May 13, 2017.

References

  1. ↵
    1. Butcher, D. T.,
    2. Alliston, T. and
    3. Weaver, V. M.
    (2009). A tense situation: forcing tumour progression. Nat. Rev. Cancer 9, 108-122. doi:10.1038/nrc2544
    OpenUrlCrossRefPubMedWeb of Science
  2. ↵
    1. Buxboim, A.,
    2. Swift, J.,
    3. Irianto, J.,
    4. Spinler, K. R.,
    5. Dingal, P. C. D. P.,
    6. Athirasala, A.,
    7. Kao, Y.-R. C.,
    8. Cho, S.,
    9. Harada, T.,
    10. Shin, J.-W. et al.
    (2014). Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin. Curr. Biol. 24, 1909-1917. doi:10.1016/j.cub.2014.07.001
    OpenUrlCrossRefPubMed
  3. ↵
    1. Carroll, C. W.,
    2. Silva, M. C. C.,
    3. Godek, K. M.,
    4. Jansen, L. E. T. and
    5. Straight, A. F.
    (2009). Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N. Nat. Cell Biol. 11, 896-902. doi:10.1038/ncb1899
    OpenUrlCrossRefPubMedWeb of Science
  4. ↵
    1. Cen, B.,
    2. Selvaraj, A.,
    3. Burgess, R. C.,
    4. Hitzler, J. K.,
    5. Ma, Z.,
    6. Morris, S. W. and
    7. Prywes, R.
    (2003). Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes. Mol. Cell. Biol. 23, 6597-6608. doi:10.1128/MCB.23.18.6597-6608.2003
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Crider, B. J.,
    2. Risinger, G. M.,
    3. Haaksma, C. J.,
    4. Howard, E. W. and
    5. Tomasek, J. J.
    (2011). Myocardin-related transcription factors A and B are key regulators of TGF-β1-induced fibroblast to myofibroblast differentiation. J. Invest. Dermatol. 131, 2378-2385. doi:10.1038/jid.2011.219
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    1. Desmouliere, A.,
    2. Geinoz, A.,
    3. Gabbiani, F. and
    4. Gabbiani, G.
    (1993). Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell. Biol. 122, 103-111. doi:10.1083/jcb.122.1.103
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Duffield, J. S.,
    2. Lupher, M.,
    3. Thannickal, V. J. and
    4. Wynn, T. A.
    (2013). Host responses in tissue repair and fibrosis. Annu. Rev. Pathol. Mech. Dis. 8, 241-276. doi:10.1146/annurev-pathol-020712-163930
    OpenUrlCrossRef
  8. ↵
    1. Esnault, C.,
    2. Stewart, A.,
    3. Gualdrini, F.,
    4. East, P.,
    5. Horswell, S.,
    6. Matthews, N. and
    7. Treisman, R.
    (2014). Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts. Genes Dev. 28, 943-958. doi:10.1101/gad.239327.114
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Gomez, E. W.,
    2. Chen, Q. K.,
    3. Gjorevski, N. and
    4. Nelson, C. M.
    (2010). Tissue geometry patterns epithelial-mesenchymal transition via intercellular mechanotransduction. J. Cell. Biochem. 110, 44-51. doi:10.1002/jcb.22545
    OpenUrlCrossRefPubMed
  10. ↵
    1. Guettler, S.,
    2. Vartiainen, M. K.,
    3. Miralles, F.,
    4. Larijani, B. and
    5. Treisman, R.
    (2008). RPEL motifs link the serum response factor cofactor MAL but not myocardin to Rho signaling via actin binding. Mol. Cell. Biol. 28, 732-742. doi:10.1128/MCB.01623-07
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Guilluy, C.,
    2. Osborne, L. D.,
    3. Van Landeghem, L.,
    4. Sharek, L.,
    5. Superfine, R.,
    6. Garcia-Mata, R. and
    7. Burridge, K.
    (2014). Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus. Nat. Cell Biol. 16, 376-381. doi:10.1038/ncb2927
    OpenUrlCrossRefPubMedWeb of Science
  12. ↵
    1. Hinz, B.
    (2009). Tissue stiffness, latent TGF-beta1 activation, and mechanical signal transduction: implications for the pathogenesis and treatment of fibrosis. Curr. Rheumatol. Rep. 11, 120-126. doi:10.1007/s11926-009-0017-1
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hinz, B.
    (2010). The myofibroblast: paradigm for a mechanically active cell. J. Biomech. 43, 146-155. doi:10.1016/j.jbiomech.2009.09.020
    OpenUrlCrossRefPubMedWeb of Science
  14. ↵
    1. Ho, C. Y.,
    2. Jaalouk, D. E.,
    3. Vartiainen, M. K. and
    4. Lammerding, J.
    (2014). Lamin A/C and emerin regulate MKL1–SRF activity by modulating actin dynamics. Nature 497, 507-511. doi:10.1038/nature12105
    OpenUrlCrossRef
  15. ↵
    1. Holaska, J. M. and
    2. Wilson, K. L.
    (2007). An emerin “Proteome”: purification of distinct emerin-containing complexes from HeLa cells suggests molecular basis for diverse roles including gene regulation, mRNA splicing, signaling, mechanosensing, and nuclear architecture †. Biochemistry 46, 8897-8908. doi:10.1021/bi602636m
    OpenUrlCrossRefPubMed
  16. ↵
    1. Holaska, J. M.,
    2. Kowalski, A. K. and
    3. Wilson, K. L.
    (2004). Emerin caps the pointed end of actin filaments: evidence for an actin cortical network at the nuclear inner membrane. PLoS Biol. 2, e231. doi:10.1371/journal.pbio.0020231
    OpenUrlCrossRefPubMed
  17. ↵
    1. Huang, X.,
    2. Yang, N.,
    3. Fiore, V. F.,
    4. Barker, T. H.,
    5. Sun, Y.,
    6. Morris, S. W.,
    7. Ding, Q.,
    8. Thannickal, V. J. and
    9. Zhou, Y.
    (2012). Matrix stiffness–induced myofibroblast differentiation is mediated by intrinsic mechanotransduction. Am. J. Respir. Cell Mol. Biol. 47, 340-348. doi:10.1165/rcmb.2012-0050OC
    OpenUrlCrossRefPubMedWeb of Science
  18. ↵
    1. Le, H. Q.,
    2. Ghatak, S.,
    3. Yeung, C.-Y. C.,
    4. Tellkamp, F.,
    5. Günschmann, C.,
    6. Dieterich, C.,
    7. Yeroslaviz, A.,
    8. Habermann, B.,
    9. Pombo, A.,
    10. Niessen, C. M. et al.
    (2016). Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment. Nat. Cell Biol. 18, 864-875. doi:10.1038/ncb3387
    OpenUrlCrossRef
  19. ↵
    1. McGee, K. M.,
    2. Vartiainen, M. K.,
    3. Khaw, P. T.,
    4. Treisman, R. and
    5. Bailly, M.
    (2011). Nuclear transport of the serum response factor coactivator MRTF-A is downregulated at tensional homeostasis. scientific report. EMBO Rep. 12, 963-970. doi:10.1038/embor.2011.141
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Miralles, F.,
    2. Posern, G.,
    3. Zaromytidou, A.-I. and
    4. Treisman, R.
    (2003). Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113, 329-342. doi:10.1016/S0092-8674(03)00278-2
    OpenUrlCrossRefPubMedWeb of Science
  21. ↵
    1. Mouilleron, S.,
    2. Langer, C. A.,
    3. Guettler, S.,
    4. McDonald, N. Q. and
    5. Treisman, R.
    (2011). Structure of a pentavalent G-actin*MRTF-A complex reveals how G-Actin controls nucleocytoplasmic shuttling of a transcriptional coactivator. Sci. Signal. 4, ra40. doi:10.1126/scisignal.2001750
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. O'Connor, J. W.,
    2. Riley, P. N.,
    3. Nalluri, S. M.,
    4. Ashar, P. K. and
    5. Gomez, E. W.
    (2015). Matrix rigidity mediates TGFβ1-induced epithelial-myofibroblast transition by controlling cytoskeletal organization and MRTF-a localization. J. Cell. Physiol. 230, 1829-1839. doi:10.1002/jcp.24895
    OpenUrlCrossRefPubMed
  23. ↵
    1. Pawłowski, R.,
    2. Rajakylä, E. K.,
    3. Vartiainen, M. K. and
    4. Treisman, R.
    (2010). An actin-regulated importin α/β-dependent extended bipartite NLS directs nuclear import of MRTF-A. EMBO J. 29, 3448-3458. doi:10.1038/emboj.2010.216
    OpenUrlCrossRefPubMedWeb of Science
  24. ↵
    1. Schwartz, M. A.
    (2010). Integrins and extracellular matrix in mechanotransduction. Cold Spring Harbor Perspect. Biol. 2, a005066. doi:10.1101/cshperspect.a005066
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Small, E. M.,
    2. Thatcher, J. E.,
    3. Sutherland, L. B.,
    4. Kinoshita, H.,
    5. Gerard, R. D.,
    6. Richardson, J. A.,
    7. DiMaio, J. M.,
    8. Sadek, H.,
    9. Kuwahara, K. and
    10. Olson, E. N.
    (2010). Myocardin-related transcription factor-A controls myofibroblast activation and fibrosis in response to myocardial infarction. Circ. Res. 107, 294-304. doi:10.1161/CIRCRESAHA.110.223172
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Swift, J.,
    2. Ivanovska, I. L.,
    3. Buxboim, A.,
    4. Harada, T.,
    5. Dingal, P. C. D. P.,
    6. Pinter, J.,
    7. Pajerowski, J. D.,
    8. Spinler, K. R.,
    9. Shin, J. W.,
    10. Tewari, M. et al.
    (2013). Nuclear lamin-a scales with tissue stiffness and enhances matrix-directed differentiation. Science 341, 1240104. doi:10.1126/science.1240104
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Thakar, K.,
    2. May, C. K.,
    3. Rogers, A. and
    4. Carroll, C. W.
    (2017). Opposing roles for distinct LINC complexes in regulation of the small GTPase RhoA. Mol. Biol. Cell 28, 182-191. doi:10.1091/mbc.E16-06-0467
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Thoreen, C. C.,
    2. Chantranupong, L.,
    3. Keys, H. R.,
    4. Wang, T.,
    5. Gray, N. S. and
    6. Sabatini, D. M.
    (2012). A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109-113. doi:10.1038/nature11083
    OpenUrlCrossRefPubMedWeb of Science
  29. ↵
    1. Varney, S. D.,
    2. Betts, C. B.,
    3. Zheng, R.,
    4. Wu, L.,
    5. Hinz, B.,
    6. Zhou, J. and
    7. Van De Water, L.
    (2016). Hic-5 is required for myofibroblast differentiation by regulating mechanically dependent MRTF-A nuclear accumulation. J. Cell. Sci. 129, 774-787. doi:10.1242/jcs.170589
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Vartiainen, M. K.,
    2. Guettler, S.,
    3. Larijani, B. and
    4. Treisman, R.
    (2007). Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science 316, 1749-1752. doi:10.1126/science.1141084
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Zhou, Y.,
    2. Huang, X.,
    3. Hecker, L.,
    4. Kurundkar, D.,
    5. Kurundkar, A.,
    6. Liu, H.,
    7. Jin, T.-H.,
    8. Desai, L.,
    9. Bernard, K. and
    10. Thannickal, V. J.
    (2013). Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis. J. Clin. Investig. 123, 1096-1108. doi:10.1172/JCI66700
    OpenUrlCrossRefPubMedWeb of Science
View Abstract
Back to top

Keywords

 Download PDF

Email
Short Report
Substrate stiffness-dependent regulation of the SRF−Mkl1 co-activator complex requires the inner nuclear membrane protein Emerin
Margaret K. Willer, Christopher W. Carroll
Journal of Cell Science 2017 130: 2111-2118; doi: 10.1242/jcs.197517
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Alerts

Article navigation

Other journals from The Company of Biologists

Advertisement

2020 at The Company of Biologists

Despite the challenges of 2020, we were able to bring a number of long-term projects and new ventures to fruition. While we look forward to a new year, join us as we reflect on the triumphs of the last 12 months.

Mole – The Corona Files

"This is not going to go away, 'like a miracle.' We have to do magic. And I know we can."

Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.

Cell scientist to watch – Christine Faulkner

In an interview, Christine Faulkner talks about where her interest in plant science began, how she found the transition between Australia and the UK, and shares her thoughts on virtual conferences.

Read & Publish participation extends worldwide

“The clear advantages are rapid and efficient exposure and easy access to my article around the world. I believe it is great to have this publishing option in fast-growing fields in biomedical research.”

Dr Jaceques Behmoaras (Imperial College London) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 60 institutions in 12 countries taking part – find out more and view our full list of participating institutions.

JCS and COVID-19

For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.

If you have any questions or concerns, please do not hestiate to contact the Editorial Office.