β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments.

How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of β1- and αv-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with β1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of αv-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with αv-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked αv-class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and α5β1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5β1integrins accomplish force generation, whereas αv-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.