Imaging Mechanical Force Transmission at Single Integrins in Living Cells

Abstract

Mechanical interactions between cells and the extracellular matrix (ECM) exert a profound influence on cell migration, proliferation, and stem-cell differentiation. However, how cells generate and detect mechanical force remains poorly understood, in part due to a lack of methods that visualize molecular-scale forces in living cells. Here we describe a Forster resonance energy transfer (FRET)-based molecular tension sensor (MTS) that allows us to directly visualize cellular forces at the single molecule level. We designed a MTS that binds to a glass coverslip via avidin or covalent bond at one end and presents an integrin binding site at the other. Cellular integrins bind surface immobilized MTSs and transmit force to the FRET module, resulting in decreased FRET with increasing load. In agreement with previous work, we find that force generation is largely confined to dense integrin-containing assemblies termed focal adhesions (FAs). The enhanced spatial resolution of our measurement in comparison to previous techniques allows us to directly visualize the distribution of forces within FAs. We observe localized force generation that is only weakly correlated with paxillin recruitment, a standard marker for FA maturity, consistent with the proposal that FAs are structurally heterogeneous on the submicron length scale. FRET values measured for single MTS molecules are consistent with tensions ranging from 1 to 5 pN, substantially less than those required to rupture integrin-ligand bonds. These relatively modest tensions suggest that the collective contribution of numerous weak integrin-binding interactions can be sufficient to drive robust cell adhesion, and by extension mechanotransduction.