Measuring Integrin Force Loading Rates Using a Two-Step DNA Tension Sensor

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s^-1 and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.