Focal Adhesion Architecture and Composition Regulate Cell Adhesion and Traction Generation Independent of Integrin Class

Integrins are a class of heterodimeric transmembrane proteins that mediate attachment to the extracellular matrix (ECM) and serve as templates for focal adhesions (FAs), sophisticated protein complexes that regulate mechanical and chemical signals. At present, the mechanisms by which specific focal adhesion architectures affect traction generation and cellular adhesion remain poorly understood. Here, we use Förster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) to measure the distribution of mechanical forces exerted by individual integrin heterodimers in living cells at the single-molecule level. We engineered two MTS variants: MTS_low, to probe a low force range (0-8 pN), and MTS_int, for examining intermediate forces (8-12 pN). We find the majority of bound integrins bear forces <12 pN and that integrins within defined adhesion complexes exert tractions that fall into distinct subpopulations. Using integrin knock-out lines rescued with specific integrin classes, we find that the single-molecule force distributions remain remarkably similar, indicating that these subpopulations are not primarily determined by the integrin class. Furthermore, we find that although integrin linkages bearing more than 6 pN are actin mediated, they are not required to support cell adhesion. In ongoing work, we investigate how perturbations to FA architecture and composition alter traction on the molecular and cellular levels.