Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

Abstract

Integrins are transmembrane receptors that, upon activation, bind extracellular matrix (ECM) or cell surface ligands and link them to the actin cytoskeleton to mediate cell adhesion and migration1,2. One model for the structural transitions mediating integrin activation termed “the cytoskeletal force hypothesis” posits that force transmitted from the cytoskeleton to ligand-bound integrins acts as an allosteric stabilizer of the extended-open, high-affinity state3. Since cytoskeletal forces in migrating cells are generated by centripetal “retrograde flow” of F-actin from the cell leading edge, where integrin-based adhesions are initiated4,5, this model predicts that F-actin flow should align and orient activated, ligand-bound integrins in integrin-based adhesions. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrin chimeras in migrating fibroblasts shows that integrins are aligned with respect to the axis of FAs and the direction of F-actin flow, and this alignment requires binding immobilized ligand and talin-mediated linkage to a flowing cytoskeleton. Polarization imaging and Rosetta modelling of chimeras engineered to orient GFP differentially with respect to the integrin headpiece suggest that ligand-bound αVβ3 integrin may be markedly tilted by the force of F-actin flow. These results show that actin cytoskeletal forces actively sculpt an anisotropic molecular scaffold in FAs that may underlie the ability of cells to sense directional ECM and physical cues.