Molecular Tension Sensors Measure Forces Generated By Single Integrin Molecules in Living Cells

Disruption in the ability of cells to sense the mechanical properties of their surroundings represents a hallmark of many diseases, including muscular dystrophy, arteriosclerosis, cardiomyopathies, and cancer. Although cells have numerous mechanisms for detecting mechanical inputs, one of the most prominent is through integrins, a class of heterodimeric transmembrane proteins that cluster into micron-sized assemblies termed focal adhesions (FAs).  FAs link the cell cytoskeleton to the surrounding extracellular matrix (ECM), transmitting and responding to mechanical force. Force transmission through integrins is essential for cell migration and adhesion, while force sensing at FAs regulates numerous cellular processes including cancer cell proliferation and stem cell differentiation. Despite intense interest, how force is generated within individual FAs remains poorly understood due to a lack of methods that directly visualize molecular-scale forces in living cells.

We engineered Förster resonance energy transfer (FRET)-based molecular tension sensors to measure cell-generated forces at single integrins. This approach allows us to directly visualize the distribution of mechanical forces within FAs, a measurement that is not feasible using conventional approaches such as traction force microscopy. We find that regions of low FRET localize primarily to the cell periphery and coincide with paxillin recruitment, a canonical FA marker, showing that force generation is largely localized to FAs. However, we observe strikingly complex distributions of tensions within individual FAs. In addition, FRET values at single probe molecules indicate that the majority of integrins generate tensions in the range of 1-4 pN, considerably less than the forces required to break integrin-ECM bonds. These observations are consistent with a model in which the number of engaged integrins within a FA controls the total force exerted while the force per integrin remains bracketed in a relatively narrow range. In ongoing work, we have generalized the molecular tension sensor design for use in wide variety of cell adhesion and cell-ligand interactions, providing a potentially powerful tool for elucidating the molecular mechanisms underlying cellular mechanotransduction.

Figure 1. FRET-based molecular force sensors measure forces at single integrin adhesions. (a) Sensors are site-specifically labeled with biotin (Avitag), a FRET donor (KC tag), and FRET acceptor (ACP tag), and present the RGD sequence from fibronectin to promote adhesion. The (GPGGA)₈ sequence acts as an entropic spring that is stretched upon the application of force. (b) Sensors are attached to a coverslip via biotin and Neutravidin; the biotinylated PEG brush prevents nonspecific cell and sensor attachment. Integrin heterodimers attach to the RGD domain and apply load generated by the cell cytoskeleton. (c) Brightfield image of a spreading fibroblast on a sensor-functionalized surface. (d) Corresponding FRET map showing areas of high force localized at the cell periphery. Color bar represents FRET index (blue: low FRET/high force; red: high FRET/low force). Scale bar: 10 μm.