Recruitment of integrin α ν β 3 to integrin α 5 β 1 -induced clusters enables focal adhesion maturation and cell spreading.

The major fibronectin (FN)-binding alpha(5)beta(1) and alpha(v)beta(3) integrins exhibit cooperativity during cell adhesion, migration and mechanosensing, through mechanisms that are not yet fully resolved. Exploiting mechanically tunable nano-pattemed substrates, and peptidomimetic ligands designed to selectively bind corresponding integrins, we report that focal adhesions (FAs) of endothelial cells assembled on alpha(5)beta(1) integrin-selective substrates rapidly recruit alpha(v)beta(3) integrins, but not vice versa. Blocking of alpha(v)beta(3) integrin hindered FA maturation and cell spreading on alpha(5)beta(1) integrin-selective substrates, indicating a mechanism dependent on extracellular ligand binding and highlighting the requirement of alpha(v)beta(3) integrin engagement for efficient adhesion. Recruitment of alpha(v)beta(3) 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 alpha(v)beta(3) integrin accumulation to adhesion clusters. Our findings highlight a novel mechanism of integrin cooperation and a critical role for alpha(v)beta(3) integrins in promoting cell adhesion on alpha(5)beta(1) integrin-selective substrates.