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...

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.

Summary Cells regulate adhesion in response to internally-generated and externally-applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin–integrin α5β1 bond switch the bond from a short-lived state with 1-s lifetime to a long-lived state with 100-s lifetime. We term this phenomenon “cyclic mechanical reinforcement” as the bond strength remembers the history of force application, accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin–integrin α5β1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor–ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.

Engineered biointerfaces covered with biomimetic motifs, including short bioadhesive ligands, are a promising material-based strategy for tissue repair in regenerative medicine. Potentially useful coating molecules are ligands for the integrins, major extracellular matrix receptors that require both ligand binding and nanoscale clustering for maximal signaling efficiency. We prepared coatings consisting of well-defined multimer constructs with a precise number of recombinant fragments of fibronectin (monomer, dimer, tetramer, and pentamer) to assess how nanoscale ligand clustering affects integrin binding, stem cell responses, tissue healing, and biomaterial integration. Clinical-grade titanium was grafted with polymer brushes that presented monomers, dimers, trimers, or pentamers of the α5β1 integrin–specific fibronectin III (7 to 10) domain (FNIII7–10). Coatings consisting of trimers and pentamers enhanced integrin-mediated adhesion in vitro, osteogenic signaling, and differentiation in human mesenchymal stem cells more than did surfaces presenting monomers and dimers. Furthermore, ligand clustering promoted bone formation and functional integration of the implant into bone in rat tibiae. This study establishes that a material-based strategy in which implants are coated with clustered bioadhesive ligands can promote robust implant-tissue integration.