Single-molecule force spectroscopy reveals a high-force requirement for dissociation of integrin transmembrane domains.

Integrins are bidirectional mechanochemical receptors that transmit signals upon ligand binding to the cytoskeleton (outside-in) and cytoskeletal forces back across the membrane (inside-out) to the integrin-ligand bond. Integrins are activated prior to ligand binding, which involves large conformational rearrangements across the extracellular, transmembrane (TM), and cytoplasmic regions. While the conformational and energetic basis of outside-in activation is increasingly well defined, the mechanical forces required for separating the tightly packed αβ TM helices during inside-out signaling remain largely unknown. Here, we directly quantify the forces required to dissociate integrin TM domains (TMDs) in a lipid environment. Engineered α5β1 polypeptides consisting of TMDs and cytoplasmic tails were reconstituted into lipid nanodiscs and probed using single-molecule optical tweezers. Mechanical marker domains on each cytoplasmic tail verified correct vectorial force application, and fluorescent lipids confirmed nanodisc integrity. We find that the heterodimeric TM complex is a mechanically robust unit. In wild-type constructs, no TMD separation was observed in repeated pulls up to ∼35 pN. Point mutations in the β1-TMD (G744L, L748R) that weaken TMD interactions revealed discrete splitting events. The high mechanical forces necessary for TMD separation support a "ratchet-like" role for mechanical forces in inside-out signaling: rather than actively opening closed TMDs, they prevent re-closing of spontaneously split TMDs, thus keeping them open and activated.