How vinculin regulates force transmission

Abstract

Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculinnull cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V H ) and tail domains. V H enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with V H , implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V H has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V H , correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.cell adhesion | fibronectin I ntegrin-mediated adhesion to ECM provides mechanical anchorage and signals that direct cell migration, proliferation, and differentiation (1, 2), processes central to tissue organization, maintenance, and repair. After ligand binding, integrins cluster into focal adhesion (FA) complexes that transmit adhesive and traction forces (3-6). FAs consist of integrins and actins separated by a ∼40 nm core that includes cytoskeleton (CSK) elements, such as vinculin and talin, and signaling molecules, including focal adhesion kinase and paxillin (7). FAs mediate responses to internal and external stresses by modulating force transfer between integrins and the CSK (8-10). This function has been likened to a "mechanical clutch" between an engine and transmission (11).On the basis of its structure and binding partners, vinculin represents an attractive candidate for orchestrator of clutch function. Vinculin consists of a globular head (V H ) linked to a tail domain (V T ) by a proline-rich strap (12). V H contains talin, α-actinin, and α-and β-catenin binding sites; actin, paxillin, and phosphatidylinositol 4,5-bisphosphate (PIP2) binding sites are in V T ; and vasodilator-stimulated phosphoprotein (VASP), actinrelated protein 2/3 (Arp2/3), and vinexin binding sites reside in the proline-rich region. Interactions with these partners are regulated by an autoinhibited conformation arising from high-affinity intramolecular head-tail binding (13,14). Activation of vinculin can occur by simultaneous binding to talin and actin or α-catenin and actin (15,16). Vinculin is activated when localized t...

Talin determines the nanoscale architecture of focal adhesions

“…2, we observed a similar hierarchical organization of FA proteins along the z-dimension, with the N-C polarized orientation of talin that seems to span the FA core, linking with actin stress fibers. These results suggest that vinculin is dispensable for the stratified nanoscale architecture of FAs, consistent with previous studies documenting FA assembly and maturation in the absence of vinculin (33,34).…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…The interaction of vinculin with acidic phospholipids and ␣-actinin may also contribute to the conversion of vinculin to an open conformation (33)(34)(35). Mechanical forces exerted on cells may also promote stabilization of the activated open conformation of vinculin (36). Changes in the conformation of vinculin enable it to act as a molecular switch for connections between cytoskeletal actin filaments and membrane adhesion protein complexes during stimulation of the cell and thus regulate the transmission of mechanical forces between the cytoskeleton and the extracellular matrix during contraction, adhesion, and migration.…”

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

“…To better understand the relative contribution of these two interfaces to FA force transfer as well as the force transfer pathway within FAs, we examined the traction force and adhesions strength of two vinculin mutants: (1) a molecule comprising only V H (1-851) and lacking most of the proline-rich strap and actinbinding tail, and (2) a full-length vinculin (T12) with mutations in several key residues responsible for the strong head-tail interaction, promoting an active confirmation of the protein capable of readily binding talin and actin. 3 We found that V H does not increase traction force whereas T12 increased traction force by 2-fold over vinculin-null cells and 40% over wild-type vinculin, suggesting that connectivity between extracellular matrixintegrin clusters and the actin CSK is required for the generation of strong traction forces. These vinculin-mediated increases in traction force could be mediated by direct and indirect interactions with the actin CSK.…”

“…Indeed, the study of force-FA relationships is the central focus of our lab. [2][3][4][5][6] FAs regulate their size, shape, and molecular composition in response to a variety of mechanical stimuli, including substrate stiffness, externally applied forces, and internally generated contractile forces. 7,8 The molecular mechanisms that govern FA mechanoresponses remain, however, largely unknown.…”

“…4,5 Having established the contribution of endogenous vinculin to adhesion strength, our most recent experiments focused on elucidating the relative contributions of the structural domains of vinculin to force transfer at FAs -a key output of FA mechanotransduction. 3 Using vinculin-null cells expressing vinculin mutants, we first analyzed the contributions of vinculin to two quantitative measures of cellular force: (1) traction force and (2) adhesion strength. Traction force measures the magnitude and direction of internally generated forces as they are applied through FAs to the extracellular matrix.…”