Molecular Tension Sensors Reveal a Minimally Tensioned Integrin State in Living Cells

Abstract

Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms. Despite this biological prominence, fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. We used Forster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) to measure the distribution of forces exerted by individual integrin heterodimers in living cells. Taking advantage of the sensors’ modular nature, we engineered MTS variants that display a minimal RGD sequence derived from fibronectin (MTSRGD) or the 9th and 10th type III domains of fibronectin (MTSFN), and are sensitive to low (0-7 pN) or intermediate (8-11 pN) loads. Using these sensors, we found that a large fraction of integrins in living cells exert forces <3 pN, while a minority subpopulation experiencing substantially higher loads was enriched in large adhesion complexes. Treatment with the filamentous (F)-actin disruptor cytochalasin D revealed that only the high-load integrin population required an intact actin cytoskeleton, and that the low-load (<3 pN) population was sufficient to mediate cell adhesion on short, ∼15 minute timescales. Integrin engagement with the fibronectin synergy site, a secondary binding site specifically for α5β1 integrin, led to increased recruitment of α5β1 integrin to adhesions, but not to an increase in overall cellular traction generation. Consistent with previous reports, the presence of the synergy site did, however, increase the resistance of cells to detachment via externally applied load. Based on these data, we suggest that a large pool of engaged, but minimally tensioned integrins may provide a synergy site-dependent adhesive reserve that imparts cells and tissues with mechanical integrity in the presence of widely varying mechanical loads. In ongoing work, we take advantage of the unique capabilities of MTSs to determine how subpopulations of load-bearing integrins are altered in response to external mechanical perturbations, and how (and whether) distinct integrin subtypes bear differing levels of mechanical load.