Recruitment of integrin ανβ3 to integrin α5β1-induced clusters enables focal adhesion maturation and cell spreading

The major fibronectin (FN) binding integrins α5β1 and αvβ3 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 integrin α5β1-selective substrates, rapidly recruit αvβ3 integrins, but not vice versa. Blocking of integrin αvβ3 hindered FA maturation and cell spreading on α5β1-selective substrates, indicating a mechanism dependent on extracellular ligand binding and highlighting the requirement of αvβ3 engagement for efficient adhesion. Recruitment of αvβ3 integrins additionally occurred on hydrogel substrates of varying mechanical properties, above a threshold stiffness supporting FA formation. Mechanistic studies revealed the need for soluble factors present in serum to allow recruitment, and excluded exogenous, or endogenous, FN as the responsible ligand for integrin αvβ3 accumulation to adhesion clusters. Our findings highlight a novel mechanism of integrin co-operation and the critical role for αvβ3 integrins in promoting cell adhesion on α5β1-selective substrates.