Myosin II activity dependent and independent vinculin recruitment to the sites of E-cadherin-mediated cell-cell adhesion

Abstract

BackgroundMaintaining proper adhesion between neighboring cells depends on the ability of cells to mechanically respond to tension at cell-cell junctions through the actin cytoskeleton. Thus, identifying the molecules involved in responding to cell tension would provide insight into the maintenance, regulation, and breakdown of cell-cell junctions during various biological processes. Vinculin, an actin-binding protein that associates with the cadherin complex, is recruited to cell-cell contacts under increased tension in a myosin II-dependent manner. However, the precise role of vinculin at force-bearing cell-cell junctions and how myosin II activity alters the recruitment of vinculin at quiescent cell-cell contacts have not been demonstrated.ResultsWe generated vinculin knockdown cells using shRNA specific to vinculin and MDCK epithelial cells. These vinculin-deficient MDCK cells form smaller cell clusters in a suspension than wild-type cells. In wound healing assays, GFP-vinculin accumulated at cell-cell junctions along the wound edge while vinculin-deficient cells displayed a slower wound closure rate compared to vinculin-expressing cells. In the presence of blebbistatin (myosin II inhibitor), vinculin localization at quiescent cell-cell contacts was unaffected while in the presence of jasplakinolide (F-actin stabilizer), vinculin recruitment increased in mature MDCK cell monolayers.ConclusionThese results demonstrate that vinculin plays an active role at adherens junctions under increased tension at cell-cell contacts where vinculin recruitment occurs in a myosin II activity-dependent manner, whereas vinculin recruitment to the quiescent cell-cell junctions depends on F-actin stabilization.

E-cadherin is under constitutive actomyosin-generated tension that is increased at cell–cell contacts upon externally applied stretch

“…Force-induced conformation changes within the cadherin/catenin complex could elicit downstream signaling, for example, by modulating binding affinity to direct or indirect binding partners such as EPLIN or vinculin (17-21, 25, 27-29). Protein accumulation at adhesion sites could thus promote changes in cell and tissue mechanical properties and cell adhesive and migratory behavior (18,25,26,28,29). Over longer timescales, this may also regulate gene expression and likewise have important roles during development and disease.…”

“…External forces applied to cells through cadherins reveal a mechanical coupling between the membrane and the cytoskeleton that requires the cadherin cytoplasmic domain (24) and may cause changes in cellular mechanical properties or protrusive activity (25,26). Conformational changes in α-catenin and/or the recruitment of cytoskeleton-binding proteins to sites of cadherin-mediated adhesion may be involved (17,18,(27)(28)(29). However, there is no direct evidence at the molecular level that cadherins are under actomyosin-dependent tension or that cadherins transmit extracellular forces to the cytoskeleton through the catenin-binding cytoplasmic domain.…”

“…In addition, mechanical stimulation of cells through the extracellular domain of cadherins results in vinculin-dependent cell stiffening (25), and membrane protrusion reorientation via a mechanism involving γ-catenin and intermediate filaments (26). Conversely, actomyosin activity is proposed to induce conformational changes in α-catenin and the recruitment of vinculin to cellcell contacts (27), which provides resistance to cell-cell contact disruption (28,29). Moreover, cadherins can transmit cell-generated forces to the extracellular environment in a stiffness-dependent manner (30)(31)(32)(33).…”

Mechanosensitive systems at the cadherin–F-actin interface

“…Interestingly, it has been shown that vinculin can localize to apical cell-cell junctions in a force-dependent and force-independent manner. The latter is mediated by an increased stabilization of F-actin, and could thus rely on the actin-binding activity of vinculin, rather than on its interaction with a-catenin (Sumida et al, 2011). EPLIN also localizes to the actin cytoskeleton outside of cell-cell junctions [ (Abe and Takeichi, 2008;Song et al, 2002) and our unpublished observations].…”

Self-contact elimination by membrane fusion

“…Interestingly, however, vinculin localization was minimal at self-contacts ( Fig. S9 and Movie S7), suggesting that self-contacts are relatively force-free, although vinculin accumulation does not necessarily correlate with myosin activity in MDCK cells (29).…”