Clustering of α₅β₁ integrins determines adhesion strength whereas α_vβ₃ and talin enable mechanotransduction

A key molecular link between cells and the extracellular matrix is the binding between fibronectin and integrins α₅β₁ and α_vβ₃. However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of fibronectin-integrin-cytoskeleton linkages by applying physiological nanonewton forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin domains within 40 nm led to integrin α₅β₁ recruitment, and increased the ability to sustain force by over six-fold. This force was supported by α ₅β₁ integrin clusters. Importantly, we did not detect a role of either integrin α_vβ₃ or talin 1 or 2 in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered α₅β₁ integrins, while less stable links to α_vβ₃ integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.