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Focal adhesions (FAs) are large, integrin-based protein complexes that link cells to the extracellular matrix (ECM). 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 adaptor 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, purified ABS3 binds F-actin only weakly in solution. We used a single molecule optical trap assay to examine how and whether ABS3 binds F-actin under physiologically relevant mechanical loads. We find that ABS3 forms a catch bond with F-actin when force is applied toward the pointed end of the actin filament, with binding lifetimes >100-fold longer than when force is applied toward the barbed end. Long-lived bonds to F-actin under load require the ABS3 C-terminal dimerization domain, whose cleavage has been reported to regulate FA turnover. Our results support a mechanism in which talin ABS3 preferentially binds to 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.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair. How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Here authors show that force- FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair.

Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell–extracellular matrix adhesions at the plasma membrane. Its C-terminal rod domain, which contains 13 helical bundles, plays important roles in mechanosensing during cell adhesion and spreading. However, how the structural stability and transition kinetics of the 13 helical bundles of talin are utilized in the diverse talin-dependent mechanosensing processes remains poorly understood. Here we report the force-dependent unfolding and refolding kinetics of all talin rod domains. Using experimentally determined kinetics parameters, we determined the dynamics of force fluctuation during stretching of talin under physiologically relevant pulling speeds and experimentally measured extension fluctuation trajectories. Our results reveal that force-dependent stochastic unfolding and refolding of talin rod domains make talin a very effective force buffer that sets a physiological force range of only a few pNs in the talin-mediated force transmission pathway. Talin is a mechanosensing cytoplasmic adaptor that links integrin cell adhesion receptors to the actin cytoskeleton. Here the authors measure the force-dependent folding and refolding kinetics of all talin rod domains to propose that talin can function as a force buffer under physiologically relevant conditions.

Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin’s interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function. Waterman and colleagues use super-resolution microscopy and biosensor technology to characterize the spatiotemporal regulation of the protein interactions within focal adhesions that control vinculin activation and function during focal adhesion maturation.

Multicellular organisms have well-defined, tightly regulated mechanisms for cell adhesion. Heterodimeric αβ integrin receptors play central roles in this function and regulate processes for normal cell functions, including signaling, cell migration, and development, binding to the extracellular matrix, and senescence. They are involved in hemostasis and the immune response, participate in leukocyte function, and have biological implications in angiogenesis and cancer. Proper control of integrin activation for cellular communication with the external environment requires several physiological processes. Perturbation of these equilibria may lead to constitutive integrin activation that results in bleeding disorders. Furthermore, integrins play key roles in cancer progression and metastasis in which certain tumor types exhibit higher levels of various integrins. Thus, the integrin-associated signaling complex is important for cancer therapy development. During inside-out signaling, the cytoskeletal protein talin plays a key role in regulating integrin affinity whereby the talin head domain activates integrin by binding to the cytoplasmic tail of β-integrin and acidic membrane phospholipids. To understand the mechanism of integrin activation by talin, we determined the crystal structure of the talin head domain bound to the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), allowing us to design a lipid-binding–deficient talin mutant. Our confocal microscopy with talin knockout cells suggests that the talin–cell membrane interaction seems essential for focal adhesion formation and stabilization. Basal integrin activation in Chinese hamster ovary cells suggests that the lipidbinding–deficient talin mutant inhibits integrin activation. Thus, membrane attachment of talin seems necessary for integrin activation and focal adhesion formation.

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. The mechanosensitive proteins talin and vinculin mediate the linkage between integrin-bound extracellular matrix and the actin cytoskeleton. Here the authors dissect distinct roles for two actin-binding sites within talin on adhesion complex assembly and maturation, which are regulated by vinculin binding to talin.

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals. Yamaguchi et al. report an approach to measure stresses during cell migration in the zebrafish embryo in vivo, and suggest that collectively migrating cells may be pushed forward by their rear cells.

The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule-calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages following cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton forces and bear, on average, 7–10 pN during cell adhesion depending on their association with F-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing. Austen et al.  generated talin biosensors to study integrin-based force transduction. They report that extracellular rigidity sensing requires talin’s mechanical engagement and find talin isoform-dependent effects in integrin-mediated mechanosensing.

Continuous contraction–relaxation cycles of the heart require strong and stable connections of cardiac myocytes (CMs) with the extracellular matrix (ECM) to preserve sarcolemmal integrity. CM attachment to the ECM is mediated by integrin complexes localized at the muscle adhesion sites termed costameres. The ubiquitously expressed cytoskeletal protein talin (Tln) is a component of muscle costameres that links integrins ultimately to the sarcomere. There are two talin genes, Tln1 and Tln2. Here, we tested the function of these two Tln forms in myocardium where Tln2 is the dominant isoform in postnatal CMs. Surprisingly, global deletion of Tln2 in mice caused no structural or functional changes in heart, presumably because CM Tln1 became up-regulated. Tln2 loss increased integrin activation, although levels of the musclespecific β1D-integrin isoform were reduced by 50%. With this result, we produced mice that had simultaneous loss of both CM Tln1 and Tln2 and found that cardiac dysfunction occurred by 4 wk with 100% mortality by 6 mo. β1D integrin and other costameric proteins were lost from the CMs, and membrane integrity was compromised. Given that integrin protein reduction occurred with Tln loss, rescue of the phenotype was attempted through transgenic integrin overexpression, but this could not restore WT CM integrin levels nor improve heart function. Our results show that CM Tln2 is essential for proper β1D-integrin expression and that Tln1 can substitute for Tln2 in preserving heart function, but that loss of all Tln forms from the heart-muscle cell leads to myocyte instability and a dilated cardiomyopathy.