Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin/F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin via its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed end of the actin filament resulted in a bond that was maximally stable at 8 pN, with a mean lifetime (12 s) 10-fold longer than the mean lifetime when force was applied toward the barbed end. A computational model of lamellipodial actin dynamics suggested that the directionality of the vinculin/F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may provide a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.

Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin/F-actin bond is unclear. Using a singlemolecule optical trap assay, we found that vinculin forms a force-dependent catch bond with Factin via its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed end of the actin filament resulted in a bond that was maximally stable at 8 pN, with a mean lifetime (12 s) 10-fold longer than the mean lifetime when force was applied toward the barbed end. A computational model of lamellipodial actin dynamics suggested that the directionality of the vinculin/F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may provide a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.Cadherin-and integrin-based protein assemblies link cells to each other and to the extracellular matrix (ECM), respectively, and together provide the physical basis for the organization of multicellular tissues (1). Both classes of adhesion complexes are exquisitely sensitive to mechanical load, and change rapidly in size and composition in order to maintain the physical integrity of living tissues (2). These adhesions are also essential in defining the physical asymmetries that underlie both individual and collective cell migration in the context of embryonic development (3), wound healing (4), and cancer metastasis (5). However, the molecular basis of how cadherin-and integrin-based adhesions respond to The protein vinculin is a component of both cadherin-and integrin-based adhesion complexes, and is rapidly recruited to both types of adhesions in response to mechanical load through its interactions with α-catenin and talin, respectively (6-8). Vinculin plays a key role in maintaining tissue integrity (9, 10); for example, loss of vinculin in mice results in the death of the developing embryo owing to defects in neural tube closure and heart development (11). Importantly, vinculin is required for persistent directional cell migration, suggestive of a role in generating a polarized connection between adhesions and the actin cytoskeleton (12, 13). Although vinculin is also recruited to cadherin-based adhesions in a force-dependent manner (6, 14), comparatively little is known about how it might regulate actin organization and dynamics at those sites.Vinculin binds directly to filamentous (F-) actin through its actin-binding tail domain (Vt) (15, 16), but how and whether this bond may be regulated by mechanical load is not known. Defining this mechanism is critical to understanding the role of vinculin as a reinforcing link between adhesion complexes and the actin cytoskeleton. We modified a previously developed optical trap (OT)-based assay (17) to define the load dependence of the binding interaction between vinculin and F-actin (Fig. 1A). Actin binding to full-length, wild-t...

Significance Talin is a mechanosensitive adaptor protein that links integrins to the actin cytoskeleton at cell–extracellular matrix adhesions. Although the C-terminal actin-binding domain ABS3 of talin is required for function, it binds weakly to actin in solution. We show that ABS3 binds actin strongly only when subjected to mechanical forces comparable to those generated by the cytoskeleton. Moreover, the interaction between ABS3 and actin depends strongly on the direction of force in a manner predicted to organize actin to facilitate adhesion growth and efficient cytoskeletal force generation. These characteristics can explain how force sensing by talin helps to nucleate adhesions precisely when and where they are required to transmit force between the cytoskeleton and the extracellular matrix.

Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via β-catenin, α- catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, where it contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. Further, load is distributed asymmetrically among complexes, such that only one bears the majority of applied load. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization.

Focal adhesions (FAs) are large, integrin-based adhesion complexes that link cells to the extracellular matrix (ECM). Previous work demonstrates that FAs form only when and where they are necessary to transmit force between the cellular cytoskeleton and the ECM, but how this occurs remains poorly understood. Talin is a 270 kDa adapter protein that links integrins to filamentous (F)-actin and recruits additional components during FA assembly in a force-dependent manner. Cell biological and developmental data demonstrate that the third, and C-terminal, F-actin binding site (ABS3) of talin is required for normal FA formation. However, ABS3 binds F-actin only weakly in in vitro, biochemical assays. We used a single-molecule optical trap assay to examine how and whether ABS3 binds F-actin under physiologically relevant, pN mechanical loads. We find that ABS3 forms a directional catch bond with F-actin when force is applied towards the pointed end of the actin filament, with binding lifetimes more than 100-fold longer than when force is applied towards the barbed end. Long-lived bonds to F-actin under load require the ABS3 C-terminal dimerization domain, whose cleavage is known to regulate focal adhesion turnover. Our results support a mechanism in which talin ABS3 preferentially binds and orients actin filaments with barbed ends facing the cell periphery, thus nucleating long-range order in the actin cytoskeleton. We suggest that talin ABS3 may function as a molecular AND gate that allows FA growth only when sufficient integrin density, F-actin polarization, and mechanical tension are simultaneously present.