Nanoscale Architecture of Tension Generation within Focal Adhesions

Understanding the molecular mechanisms by which cells respond to physical cues such as extracellular matrix (ECM) stiffness and ligand density constitutes a critical bottleneck in the field of tissue engineering. Cells adhere to the ECM via integrins, a class of heterodimeric, transmembrane proteins that physically link the cell cytoskeleton to ECM proteins. The cytoplasmic domains of integrins in turn recruit hundreds of proteins that collectively comprise focal adhesions (FAs), highly complex, dynamic assemblies that mediate “outside-in” mechanosensing and “inside-out” traction force generation. How these assemblies mediate force sensing and force transmission is thus the subject of great interest. Current techniques lack the spatial resolution to directly visualize mechanical forces within individual FAs, posing a critical technical limitation in addressing this general question. We engineered Förster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) to directly visualize the mechanical force exerted by individual integrins in living cells. Simultaneous super-resolution light imaging of MTSs and GFP-tagged cellular proteins results in maps of the force-producing structures within FAs with <100 nm spatial resolution, allowing us to, for the first time, directly correlate local force generation with the presence of specific integrins and cytoskeletal proteins. We find that α_vβ₃ integrin localizes to high force regions, whereas α₅β₁ integrin localization is more diffuse. The canonical FA proteins paxillin, vinculin, talin, and α-actinin colocalize with force production to varying degrees. Surprisingly, paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles in mechanotransduction. In addition, simultaneous time-lapse imaging of either GFP-paxillin or GFP­-α-actinin with MTS-measured local traction forces reveals that paxillin and tension are closely related in both space and time in assembling and disassembling adhesions, while α-actinin exhibits a more complex relationship with tension. The high degree of spatial correlation of both paxillin and α_vβ₃ integrin with mechanical tension suggests that these proteins may play direct roles in cellular mechanotransduction.