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. 2017 Jul 1;130(13):2111-2118.
doi: 10.1242/jcs.197517. Epub 2017 Jun 2.

Substrate stiffness-dependent regulation of the SRF-Mkl1 co-activator complex requires the inner nuclear membrane protein Emerin

Margaret K Willer  1 , Christopher W Carroll  2
Affiliations

Substrate stiffness-dependent regulation of the SRF-Mkl1 co-activator complex requires the inner nuclear membrane protein Emerin

Margaret K Willer et al. J Cell Sci. .

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.

Keywords: Emerin; Lamin A/C; Mechanosensitive; Mkl1; Nuclear lamina; SRF.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

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.
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.
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.
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.
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