A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions

Abstract

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrincytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or ␣-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to K d ϳ 10 ؊7 is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.Integrins mediate transmembrane connections between the actin cytoskeleton and extracellular matrix (1, 2). These connections are organized into discrete clusters such as focal complexes (3) and focal adhesions (3, 4) in adherent cells. Focal adhesions serve dual, opposing functions in cell motility, acting both in transmission of traction forces to generate displacement of the cell body and as anchors that resist detachment from the substratum (5). Thus, dynamic regulation of focal adhesion structure, and the integrin-cytoskeleton associations contained therein, plays a central role in balancing adhesive and migratory stimuli in the cell.Two key proteins implicated in the physical connections between integrins and F-actin are the actin-binding proteins talin and vinculin. In vitro, talin binds to ␤ integrin tails through its FERM domain (6) and induces conformational changes in integrin associated with increased binding to the extracellular matrix (7). In cells, exposure of activated epitopes on ␤ 1 and ␤ 3 integrins and fibronectin binding are strongly inhibited by knockdown of talin expression (8). Moreover, talin-1 null cell lines are deficient in the formation of mechanical linkages between fibronectin, ␤ 3 integrin, and the cytoskeleton as shown by loss of a transient molecular slip bond that can sustain up to 2 piconewtons of force (9).Interestingly, the seminal report that talin binds directly to ␣ 5 ␤ 1 integrin also demonstrated that integrin, talin, and vincu...

S100A4 downregulates filopodia formation through increased dynamic instability

“…Importantly, kinetic analyses have recently demonstrated the involvement of myosin II in the timely maturation of FX and FA. 7,10,11 The biological mechanisms deciphering how myosin II functions in FA maturation remain to be fully unraveled, but could be through either the generation of tension, which directly affects the conformation of proteins in the adhesion complex 12 or its cross-linking activity. 11,13 Consequently, factors that can regulate both myosin II's cross-linking activities and contraction are good candidates to govern FA formation.…”

“…17 Inhibiting the molecular motor properties of myosin IIA would led to a loss of tension on actin stress fibers, resulting in significant changes in the conformation of proteins in the adhesion complex. 12 Alternatively S100A4 has been shown to impair myosin polymerization and its organization into highly defined filaments. 17 The aggregation of myosin IIA into fibers is thought to induce cross-linking activities on adjacent actin filaments, a process that is also required for FX maturation.…”

Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

“…Vinculin binds to both filamentous actin and talin, which is another integrin binding protein that also cross-links actin. The conformation of vinculin can be reversibly regulated between an open state in which it can bind to actin filaments as well as to talin and ␣-actinin, and a closed conformation in which it does not bind to either of these proteins, thus enabling it to reversibly form connections between actin filaments and membrane adhesion sites (24,29). Vinculin binds to paxillin and is recruited with paxillin to the adhesion complexes of tracheal smooth muscle cells during contractile stimulation (95).…”

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

“…The conformational state of the vinculin molecule can be reversibly regulated between an activated "open" state and an inactive "autoinhibited" state (14,15,18,19,25,27). In the open state, binding sites for talin and ␣-actinin on the head domain of vinculin and binding sites for F-actin on the tail domain are exposed, enabling vinculin to form connections between the actin cytoskeleton and integrin adhesion complexes (14,19,26,28).…”