Ligand binding initiates single-molecule integrin conformational activation

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single -molecule fluorescence dynamics show that ligand binding to the bent -closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high -affinity integrin conformation. The extended -closed integrin conformation is not an intermediate but can be directly accessed from the extended -open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin b -subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside -in signaling and followed by inside -out signaling. Our results further imply that talin binding is insufficient for inside -out integrin activation and that tensile force transmission through the ligand-integrintalin-actin cytoskeleton complex is required.