Cells Respond to ECM Contexts via Integrin-Ligand Lifetimes: Rediscovery of Loading Rate Enhancement through Bayesian Refinement of Force Spectrum Analysis

The phenomenon of loading rate enhancement, where the bond lifetime between protein molecules significantly increases beyond a certain loading rate threshold, has been well-documented across various molecules. However, reports of this phenomenon as a mechanosensing mechanism in cells are scarce, primarily because most reported thresholds exceed physiological norms. In this study, we reevaluate the kinetic properties of integrin-ligand bonds using Single-Molecule Force Spectroscopy and Single-Cell Force Spectroscopy. Our analysis revealed a nonlinear relationship between loading rate and rupture force, with thresholds significantly below the commonly reported 10,000 pN/s. Using a Bayesian-inference-based Micro-interval analysis method, we determined specific loading rate thresholds for integrin α5β1 and α2β1 ligand-binding bonds at 1018.49 pN/s and 2398.23 pN/s, respectively. Given that substrate rigidity correlates with loading rates upon integrins under actomyosin-generated forces, our findings suggest that integrin α5 mediates stronger adhesions on softer substrates compared to integrin α2. These findings demonstrate that integrin-mediated cell adhesion strengthens within physiological loading rate ranges and suggest a mechanism for the subtle regulation of extracellular matrix protein composition in cellular elasticity sensing.