Molecular Tension Sensors for Probing the Mechanical and Chemical Roles of Distinct Integrin Classes

Abstract

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to the extracellular matrix (ECM). Mammalian cells express numerous α/β-integrin heterodimers, each with a unique combination of affinities for individual ECM components and distinct roles in intracellular signal transduction. Notably, αvβ3, αvβ5, and α5β1 integrins recognize structurally similar RGD-based binding motifs, but play distinct, and central, roles in cancer metastasis (αvβ3 integrin), septic shock (αvβ5 integrin), and angiogenic sprouting (α5β1 integrin). In this study, we used Forster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) that report on the mechanical forces exerted by individual integrin heterodimers to probe the roles of αvβ3, αvβ5, and α5β1 integrins in cell adhesion and traction force generation. We developed MTS variants that incorporated a minimal RGD sequence derived from fibronectin (MTSRGD), an RGD sequence derived from vitronectin (MTSVN), or the complete ninth and tenth type-III domains from fibronectin (MTSFN). Using primary dermal fibroblasts, we found that both MTSRGD and MTSVN recruited αvβ3 and αvβ5 integrins to large, force-generating adhesions at the cell periphery. In contrast, MTSFN supported the recruitment of α5β1 integrin to force-producing adhesions. However, measurements at the single-molecule level revealed that the forces experienced by individual MTSRGD molecules were on average larger than those experienced by MTSFN. These and other data suggest that force production at the level of individual integrins is regulated by the interaction of the integrin with its extracellular ligand. More broadly, our results imply that αv-containing and α5β1 integrins play distinct roles within adhesions, and that ligand affinity and traction force generation are not necessarily coupled at the level of individual integrins.