Cells experience force and, therefore, need to mechanically respond to stabilize cell junctions with both neighboring cells and the underlying extracellular matrix. Cadherins are the adhesion proteins composing the adherens junctions at cell-cell contacts while cadherin-associated proteins and the actin cytoskeleton provide stability and structural support between neighboring cells. The E-cadherin complex was identified as a mechanosensor at cell-cell contacts where applying force on the extracellular domain of E-cadherin resulted in vinculin-dependent cell stiffening . Vinculin-dependent cell stiffening was also observed at integrin junctions [2, 3], suggesting a similar role for vinculin at both force-bearing cell-cell and cell-matrix junctions. Thus, vinculin may be a key protein in generating tension at cell-cell contacts in response to external forces from neighboring cells.
Vinculin was originally identified as a protein associated at the ends of actin fibers terminating at the plasma membrane . Along with F-actin [5, 6], additional binding partners to vinculin at focal adhesions include talin [7, 8], paxillin , α-actinin , and phospholipids [11, 12]. Vinculin is composed of a head and tail domain that is linked together by a proline-rich linker region and exists in either an open, activated state or a closed, auto-inhibited state where the head and tail domains interact [13–15]. In the open state, previously hidden sites for vinculin binding partners are exposed. Vinculin activation is achieved through interacting with one of several vinculin binding partners [14, 16–18].
The association of vinculin with integrins at focal adhesions has been well examined, where vinculin binds to talin and paxillin, two integrin-binding proteins [7–9]. At focal adhesions, vinculin is involved in mechano-coupling between the integrins bound to the underlying extracellular matrix, and the actin cytoskeleton [3, 19, 20]. In this position, vinculin plays a major role in force-generating processes such as cell migration on a two-dimensional surface  and cell invasion in a three-dimensional matrix . Vinculin regulates actomyosin force generation in response to external cues through the vinculin tail domain . Although vinculin at focal adhesions has been well studied, the role of vinculin at cell-cell contacts has not.
In biochemical assays with purified proteins, a direct interaction occurs between the vinculin head domain and the cadherin-associated protein α-catenin [23–25], with the vinculin binding site on α-catenin located between aa 326-509 [14, 24]. The additional vinculin interactions with β-catenin [26, 27] or myosin VI  have also been reported. Interestingly, vinculin recruitment to cell-cell contacts is decreased by the myosin II inhibitor blebbistatin in some epithelial cell lines [1, 25, 29], thus supporting the role of vinculin in responding to increased tension at cell-cell contacts. An inhibitory region for vinculin binding was identified on α-catenin (aa 510-697) and suggested α-catenin existing in either a closed conformation with the inhibitory domain occluding the vinculin binding site, or in an open conformation under increased tension with the vinculin binding site exposed .
MDCK cells are the prototypical polarized epithelial cell model, yet the interaction of vinculin with the E-cadherin complex in MDCK cells is different from other cell lines. This has been attributed to low tension in MDCK cell monolayers under normal conditions possibly due to the unique cadherin distribution of MDCK cell-cell contacts , thus resulting in the lack of zonula adherens with vinculin accumulation that is observed in other epithelial cell lines . However, vinculin accumulation increases at adherens junctions lining the wound edge in the MDCK wound-healing model, a process inhibited by blebbistatin [25, 29]. Additionally, an α-catenin antibody recognizing a sequence near the vinculin binding site was localized at the junctions along the wound edge, therefore, indicating the availability of vinculin binding sites on α-catenin at cell-cell contacts under increased tension . Also, in HGF-treated highly migratory MDCK cells, increased vinculin recruitment to cell-cell contacts is reversed with the inclusion of blebbistatin, suggesting myosin II-dependence . This indicates that vinculin is recruited under conditions of increased tension to cell-cell contacts in a myosin II activity-dependent manner. Interestingly, in the MDCK cell monolayer with low tension cell-cell contacts, vinculin still localizes to cell-cell contacts. Therefore, additional factors (myosin II-independent) may be recruiting vinculin to sites of cell-cell contacts, but the mechanisms by which vinculin accumulates under these conditions remain unclear.
The aim of this study was to investigate the role of vinculin at force-bearing adherens junctions and its recruitment to sites of cell-cell contact. In this report, using vinculin knockdown and GFP-vinculin expressing cells in wound-healing assays, we demonstrate that vinculin is required for proper wound closure and vinculin-deficiency results in a reduced rate of wound closure. Additionally, we show that recruitment of vinculin to cell-cell contacts is increased with F-actin stabilization.