Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages

Rac1 is required for FMNL2 localization to cell-cell contacts.

<p><b>(A)</b> Live-cell imaging of FMNL2-/- cells expressing FMNL2-GFP together with mCherry-PA-Rac1 transfected with either control siRNA or siRNA directed against Cdc42. Red arrows highlight FMNL2-GFP localization in filopodia <b>(B)</b> Quantification of cell-cell contact formation as in (A). (<i>n</i> = 53 (si control), <i>n</i> = 43 (si Cdc42), pooled from three independent experiments, ns non-significant, calculated by <i>t</i>-test). <b>(C)</b> Quantification of fluorescence intensity of FMNL2-GFP at cell-cell contacts versus cytoplasm (<i>n</i> = 13 (si control), <i>n</i> = 13 (si Cdc42), pooled from three independent experiments, ns non-significant, determined by <i>t-</i>test.) <b>(D)</b> Time lapse images of FMNL2-/- cells expressing FMNL2-GFP transfected with control, Rac1 or Cdc42 siRNA. <b>(E)</b> Stills of live FMNL2 -/- cells expressing FMNL2-GFP transfected with control, Rac1 or Cdc42 siRNA. Arrows highlight FMNL2-GFP at cell-cell contacts. <b>(F)</b> Quantification of fluorescence intensity at cell-cell contacts in cells treated with siRNA as indicated (<i>n</i> = 8 (si control), <i>n</i> = 12 (si Rac1), <i>n</i> = 6 (si Cdc42), pooled from three independent experiments,*p≤0.01, error bars SEM). siRNA efficiency in MCF10A cells is demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194716#pone.0194716.s001" target="_blank">S1J Fig</a>.</p

A Rac1-FMNL2 signaling module affects cell-cell contact formation independent of Cdc42 and membrane protrusions

<div><p><i>De novo</i> formation of epithelial cell-cell contacts relies on actin-based protrusions as well as tightly controlled turnover of junctional actin once cells encounter each other and adhesion complexes assemble. The specific contributions of individual actin regulators on either protrusion formation or junctional actin turnover remain largely unexplored. Based on our previous findings of Formin-like 2 (FMNL2)-mediated control of junctional actin dynamics, we investigated its potential role in membrane protrusions and impact on newly forming epithelial contacts. CRISPR/Cas9-mediated loss of FMNL2 in human MCF10A cells combined with optogenetic control of Rac1 activity confirmed its critical function in the establishment of intercellular contacts. While lamellipodial protrusion rates remained unaffected, FMNL2 knockout cells were characterized by impaired filopodia formation similar to depletion of the Rho GTPase Cdc42. Silencing of Cdc42, however, failed to affect FMNL2-mediated contact formation. Hence, we propose a cell-cell contact-specific and Rac1-mediated function of FMNL2 entirely independent of Cdc42. Consistent with this, direct visualizations of native epithelial junction formation revealed a striking and specifically Rac1- and not Cdc42-dependent recruitment of FMNL2 to newly forming junctions as well as established cell-cell contacts within epithelial sheets.</p></div

Rac1-induced lamellipodia formation is independent of FMNL2.

<p><b>(A)</b> Single MCF10A cells expressing mCherry-PA-Rac1 were globally photoactivated and monitored over time. <b>(B)</b> Quantification of Rac1-induced cell enlargement in indicated cell lines over time (<i>n</i> = 15 (WT), <i>n</i> = 17 (CRISPR con), <i>n</i> = 15 (FMNL2-/-, <i>n</i> = 11 (FMNL2-/- + FMNL2-GFP), <i>n</i> = 3 (CK-666), data pooled from three independent experiments). Background displays 95% confidence interval of the regression. <b>(C)</b> MCF10A cells expressing mCherry-PA-Rac1 were photoactivated within indicated areas using the 488 nm laser (yellow boxes) and monitored over time. Kymographs were generated as indicated by red lines. <b>(D)</b> Examples of kymographs. <b>(E)</b> Quantification of maximum expansion of lamellipodia (<i>n</i> = 30 (WT), <i>n</i> = 24 (CRIPSR con), <i>n</i> = 18 (FMNL2-/-), <i>n</i> = 18 (FMNL2-/- + FMNL2-GFP), <i>n</i> = 16 (CK-666), <i>n</i> = 13 (not photoactivated), pooled from three independent experiments, *p≤ 0.001). <b>(F)</b> Western blot comparing expression levels of lamellipodial proteins in control and FMNL2-/- cells as indicated.</p

Cdc42 regulates filopodial growth and localization of FMNL2, but does not affect junction formation.

<p><b>(A)</b> MCF10A cells co-expressing GFP-Fascin and mCherry-PA-Rac1. <b>(B)</b> Examples of filopodia being formed in WT or FMNL2-/- cells facing each other. <b>(C)</b> Quantification of filopodia length in indicated cell lines (<i>n</i> = 345 (WT), <i>n</i> = 448 (CRIPSR con), <i>n</i> = 364 (FMNL2-/-), <i>n</i> = 501 (FMNL2-/- + FMNL2-GFP), <i>n</i> = 408 (FMNL2-/- + FMNL2-NT-GFP), <i>n</i> = 185 (BFP-CDC42 N17), pooled from three independent experiments, *p≤0.001). <b>(D and E)</b> Quantification of filopodial length comparing FMNL2 or Cdc42 depleted cells to cells treated with control siRNA (<i>n</i> = 182 (si control), <i>n</i> = 61 (si FMNL2#1), <i>n</i> = 112 (si FMNL2#2), <i>n</i> = 274 (si control), <i>n</i> = 234 (si Cdc42), pooled from three independent experiments, *p≤0.01). Western blot demonstrating FMNL2 siRNA and Cdc42 siRNA efficiency is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194716#pone.0194716.s001" target="_blank">S1G and S1J Fig</a>.</p

CRISPR/Cas9-mediated knockout of FMNL2 affects lumen formation in MCF10A cells.

<p><b>(A)</b> Simplified structure of the human FMNL2 locus. <b>(B)</b> Cartoon illustrating the FMNL2 protein. Red asterisk (*) indicates the region targeted by Cas9. bp, base pairs; aa, amino acid; GBD, GTPase binding domain; FH, formin homology domain; DAD, diaphanous autoinhibitory domain. <b>(C)</b> Western Blot comparing FMNL1, 2, 3 expression in WT and FMNL2-/- cells. Antibodies were applied as indicated. <b>(D)</b> Colorimetric cell viability assay shows no differences in proliferation between generated cell lines (N = 3, triplicates for each time point, error bars SD, ns non-significant). <b>(E)</b> WT and FMNL2-/- cells were grown in hydrogel for 14 d before staining for F-actin and DAPI. Graphs depict quantifications of successful lumen formation and the overall area covered by spheroids (N = 3, *p ≤0.0001, <i>n</i> = 8, error bars SD, ns indicates no significance).</p

Impairment in cell-cell contact formation in FMNL2-/- cells can be rescued by reintroduction of FMNL2-GFP.

<p><b>(A)</b> Live wildtype MCF10A cells expressing mCherry-PA-Rac1 and GFP-α-catenin were imaged over time to visualize adhesion junction formation upon Rac1 activation. Small inset shows mCherry-PA-Rac1 expression. <b>(B)</b> Comparison of cell-cell contact formation dynamics upon PA-Rac1 activation in indicated cell lines. Dashed circles highlight regions of interest. <b>(C)</b> Western blot comparing endogenous FMNL2 and exogenous FMNL2-GFP expression. Antibodies were applied as indicated. <b>(D)</b> Quantification of cell-cell contact establishment after Rac1 activation as in (B) (<i>n</i> = 60 (WT), <i>n</i> = 65 (FMNL2-/-), <i>n</i> = 64 (FMNL2-/- + FMNL2-GFP), pooled from three independent experiments, error bars SD, *p ≤0.05,). <b>(E)</b> Quantification of cell-cell contact formation in cells treated either with control or Cdh1 siRNA (<i>n</i> = 43 (si control), <i>n</i> = 44 (si Cdh1) pooled from three independent experiments, error bars SD, *p≤0.5 calculated by <i>t</i>-test). Western blot demonstrating siRNA efficiency in MCF10A cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194716#pone.0194716.s001" target="_blank">S1E Fig</a>. <b>(F)</b> Live FMNL2-/- cells co-expressing FMNL2-GFP or FMNL2-NT-GFP with PA-Rac1 (upper small image) were imaged. Stills show protrusion dynamics and contact formation over time. Zoom image depict magnification of filopodia. Asterisks highlight areas of contact sites. <b>(G)</b> Co-immunoprecipitation of HEK cells expressing GFP or E-Cadherin CT-GFP with flag-tagged FMNL2 derivatives. Flag-tagged proteins were precipitated and bound proteins were detected using GFP-HRP antibody. Please note, E-Cadherin CT-GFP is pulled down only with flag-FMNL2 CT. In immunoprecipitates of cells expressing high levels of flag-FMNL2 CT, a band at 60 kD is observed likely deriving from a truncation product.</p