Recruitment of α_νβ₃ integrin to α₅β₁ integrin-induced clusters enables focal adhesion maturation and cell spreading

The major fibronectin (FN)-binding α₅β₁ and α_vβ₃ integrins exhibit cooperativity during cell adhesion, migration and mechanosensing, through mechanisms that are not yet fully resolved. Exploiting mechanically tunable nano-patterned substrates, and peptidomimetic ligands designed to selectively bind corresponding integrins, we report that focal adhesions (FAs) of endothelial cells assembled on α₅β₁ integrin-selective substrates rapidly recruit α_vβ₃ integrins, but not vice versa. Blocking of α_vβ₃ integrin hindered FA maturation and cell spreading on α₅β₁ integrin-selective substrates, indicating a mechanism dependent on extracellular ligand binding and highlighting the requirement of α_vβ₃ integrin engagement for efficient adhesion. Recruitment of α_vβ₃ integrins additionally occurred on hydrogel substrates of varying mechanical properties, above a threshold stiffness that supports FA formation. Mechanistic studies revealed the need for soluble factors present in serum to allow recruitment, and excluded exogenous, or endogenous, FN as the ligand responsible for α_vβ₃ integrin accumulation to adhesion clusters. Our findings highlight a novel mechanism of integrin cooperation and a critical role for α_vβ₃ integrins in promoting cell adhesion on α₅β₁ integrin-selective substrates.