Abstract
Integrins are mechanosensitive adhesion receptors that transduce extracellular forces into intracellular signals through conformational changes. However, the dynamic nature of these force-induced transitions remains poorly understood. Here, we employed single-molecule Förster resonance energy transfer (smFRET) combined with magnetic tweezers to directly visualize and quantify conformational changes in purified integrin αVβ3 under applied tensile forces. By site-specifically labeling integrin with donor and acceptor fluorophores at positions that report on the hybrid domain swing-out and headpiece opening, we monitored real-time FRET efficiency changes in response to controlled forces ranging from 0 to 40 pN. Our results reveal that integrins undergo a stepwise activation pathway: an initial low-force (∼5 pN) transition to an extended conformation, followed by a high-force (∼20 pN) transition to a fully open headpiece, with intermediate states lasting tens of milliseconds. Force-dependent rate constants derived from dwell-time analysis show that force exponentially accelerates the opening rates while decelerating closure, consistent with a catch-bond mechanism. Additionally, we observed that ligand binding and Mn2+ ions shift the equilibrium toward the open state, lowering the force threshold. These findings provide a quantitative framework for understanding integrin mechanotransduction at the single-molecule level.