The Vinculin C-terminal Hairpin Mediates F-actin Bundle Formation, Focal Adhesion, and Cell Mechanical Properties

Abstract

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...

Binding of the WASP/N-WASP-Interacting Protein WIP to Actin Regulates Focal Adhesion Assembly and Adhesion

“…This was associated with a concomitant decrease in the mRNA expression of the genes that encode these FA proteins. Both vinculin and VASP have been demonstrated to be important for FA assembly and cell adhesion (25,50), suggesting that deficiencies in these FA proteins contribute to defective FA assembly in WIP Ϫ/Ϫ fibroblasts. In contrast, zyxindeficient fibroblasts show enhanced adhesion (19), suggesting that zyxin has important effects on cell adhesion other than those that contribute to FA assembly.…”

Analysis of key genes and pathways involved in acute lung injury in a mouse model

“…The protein product of Vcam1 mediates leukocyte-endothelial cell adhesion and signal transduction (Lobb et al, 1995). Ezrin and Vinculin are also known to play critical roles in cell adhesion, migration, and organization (Hiscox and Jiang, 1999;Poullet et al, 2001;Carisey and Ballestrem, 2011;Shen et al, 2011).…”

Differential lipid binding of vinculin isoforms promotes quasi-equivalent dimerization

“…Vinculin oligomerization amplifies its interactions with other adhesion proteins, yet the observed PIP 2 -induced oligomerization (26,(39)(40)(41) might have been the result of an artifact of crosslinking and the physiological lipid-induced vinculin oligomer seems to be the dimer (42). In the Vt/PIP 2 crystal structure, one PIP 2 molecule is sandwiched between three Vt molecules, whereby two Vt subunits engage in a domain swap-like arrangement of their C-terminal coiled-coil regions (38).…”