Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actininduced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.The ability of cells to sense and respond to environmental cues such as mechanical forces is critical for multiple cellular processes including embryogenesis and wound healing (1-3). To transmit forces across the cell membrane in both directions, the actin cytoskeleton couples transmembrane receptors (integrin or cadherin), through points of cell adhesion consisting of multiple protein complexes that form cell-extracellular matrix (focal adhesions) and cell-cell (adherens junctions) contacts (3, 4). Vinculin is an abundant protein found in both focal adhesions and adherens junctions, and plays a key role in regulating cell morphology, cell motility, and force transduction (2, 5). Vinculin is essential during development as vinculin knockout (KO) mouse embryos fail to survive beyond day E10 with extensive defects in myocardial and endocardial structures (6). Consistent with a role in muscle structure, vinculin KO mice are predisposed to stress-induced cardiomyopathy (7). Moreover, mutations and deletions in the tail domain of metavinculin, a splice isoform of vinculin, are associated with dilated cardiomyopathy (8 -10). Vinculin also possesses tumor suppressor properties as vinculin KO cells are less adherent, have a rounded morphology, reduced lamellipodial stability, increased motility (6, 11), and are resistant to apoptosis and anoikis (12).Vinculin is a highly conserved cytoskeletal protein that has 1066 residues and contains a globular head, a flexible prolinerich linker, and a tail domain (13). Each discrete vinculin domain recognizes multiple binding partners: the vinculin head (Vh) 4 binds to talin, ␣-actinin, and IpaA (14 -16); the prolinerich linker inte...

SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling.

Forces on JAM-A activate RhoA to increase cell stiffness. Activation of RhoA requires GEF-H1 and p115 RhoGEF activation downstream of FAK/ERK and Src family kinases, respectively.

N-glycosylation is identified as a novel regulator of JAM-A function. Human JAM-A carries a single N-glycan at N185, which regulates the protein’s role in barrier function, migration, and leukocyte binding.

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