Measuring integrin force loading rates using a two-step DNA tension sensor

Abstract

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 (pN) forces exerted by cells 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 integrins 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 a LR probe which is comprised of an engineered DNA structures that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN corresponding to hairpin unfolding and a high force threshold at 56 pN triggered through 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 8 cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a [tau] of 45.6 sec. A small subset of these events (<10%) mature in magnitude to >56pN with a median loading rate of 1.3 pNs-1 with these mechanical ramp events localizing at the periphery of the cell-substrate junction. Importantly, 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 mechanotransduction.