Single Piconewton Forces at Individual Integrins Support Robust Cell Adhesion

Mechanical interactions between cells and the extracellular matrix (ECM) exert a profound influence on cell migration, proliferation, and stem cell differentiation. However, fundamental aspects of how cells detect and generate mechanical forces at the cell-ECM interface remain poorly understood. Here we describe a new technique, termed Molecular Force Microscopy (MFM) that visualizes the forces experienced by single cellular adhesion molecules with nanometer, piconewton, and sub-second resolutions. MFM uses a new class of FRET-based molecular tension sensors that bind to an avidin-coated glass coverslip at one end and present an integrin binding site at the other. Cellular integrins transmit force to the FRET pair, resulting in decreased FRET with increasing load. Unlike previously reported force sensors, MFM sensor molecules allow quantitative FRET imaging at the single molecule level. We found that human foreskin fibroblasts (HFFs) adhered to and spread on surfaces functionalized with the MFM probes, and developed mature focal adhesions as evidenced by paxillin localization and actin stress fiber formation. We observed a bimodal distribution of FRET efficiency values for MFM sensor molecules beneath HFFs, with one peak corresponding to zero load and the other indicating a distribution of forces between 1 and 4 pN. Despite evidence of robust adhesion, the forces we measured were ∼10-fold lower than the force necessary to break individual integrin-ECM bonds. Our data provide the first direct measurement of the tension per integrin molecule necessary to form stable contacts with the ECM. The relatively narrow range of forces that we observed suggests that mechanical tension at individual adhesion molecules is subject to exquisite feedback and control. Ongoing work uses the unique capabilities of MFM to elucidate the mechanical signal transduction mechanisms that underlie cell migration and adhesion.