Measuring Integrin\nForce Loading Rates Using a Two-Step\nDNA Tension Sensor

Cells apply forces to extracellular matrix (ECM) ligands\nthrough\ntransmembrane integrin receptors: an interaction which is intimately\ninvolved in cell motility, wound healing, cancer invasion and metastasis.\nThese small (piconewton) integrin-ECM forces have been studied by\nmolecular tension fluorescence microscopy (MTFM), which utilizes a\nforce-induced conformational change of a probe to detect mechanical\nevents. MTFM has revealed the force magnitude for integrin receptors\nin a variety of cell models including primary cells. However, force\ndynamics and specifically the force loading rate (LR) have important\nimplications in receptor signaling and adhesion formation and remain\npoorly characterized. Here, we develop an LR probe composed of an\nengineered DNA structure that undergoes two mechanical transitions\nat distinct force thresholds: a low force threshold at 4.7 pN (hairpin\nunfolding) and a high force threshold at 47 pN (duplex shearing).\nThese transitions yield distinct fluorescence signatures observed\nthrough single-molecule fluorescence microscopy in live cells. Automated\nanalysis of tens of thousands of events from eight cells showed that\nthe bond lifetime of integrins that engage their ligands and transmit\na force >4.7 pN decays exponentially with a τ of 45.6 s.\nA subset\nof these events mature in magnitude to >47 pN with a median loading\nrate of 1.1 pN s<sup>–1</sup> and primarily localize at the\nperiphery of the cell–substrate junction. The LR probe design\nis modular and can be adapted to measure force ramp rates for a broad\nrange of mechanoreceptors and cell models, thus aiding in the study\nof molecular mechanotransduction in living systems.