Background: Talin mediates RIAM-dependent integrin activation and binds vinculin, which stabilizes adhesions.Results: Structural and biochemical data show that vinculin inhibits RIAM binding to the compact N-terminal region of the talin rod, a region essential for focal adhesion assembly.Conclusion: Talin·RIAM complexes activate integrins at the leading edge, whereas talin·vinculin promotes adhesion maturation.Significance: Talin changes partners in response to force-induced conformational change.

SummaryBackgroundCells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory proteins that, all together, form focal adhesions (FAs). The molecular basis of how these sensing units are regulated, how they are implicated in transducing mechanical stimuli, and how this leads to a spatiotemporal coordination of FAs is unclear.ResultsHere we show that vinculin, through its links to the talin-integrin complex and F-actin, regulates the transmission of mechanical signals from the extracellular matrix to the actomyosin machinery. We demonstrate that the vinculin interaction with the talin-integrin complex drives the recruitment and release of core FA components. The activation state of vinculin is itself regulated by force, as underscored by our observation that vinculin localization to FAs is dependent on actomyosin contraction. Using a variety of vinculin mutants, we establish which components of the cell-matrix adhesion network are coordinated through direct and indirect associations with vinculin. Moreover, using cyclic stretching, we demonstrate that vinculin plays a key role in the transmission of extracellular mechanical stimuli leading to the reorganization of cell polarity. Of particular importance is the actin-binding tail region of vinculin, without which the cell’s ability to repolarize in response to cyclic stretching is perturbed.ConclusionsOverall our data promote a model whereby vinculin controls the transmission of intracellular and extracellular mechanical cues that are important for the spatiotemporal assembly, disassembly, and reorganization of FAs to coordinate polarized cell motility.

The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.