DNA-based ForceChrono Probes for Deciphering Single-Molecule Force Dynamics in Living Cells

Accurate measurement of mechanical forces in cells is key to understanding how cells sense and respond to mechanical stimuli, a central aspect of mechanobiology. However, accurately quantifying dynamic forces at the single-molecule level in living cells is a significant challenge. Here, we’ve developed the DNA-based ForceChrono probe to enable in-depth studies of integrin force dynamics at the single-molecule level in living cells. By illuminating two distinct mechanical points and circumventing the inherent fluctuations of single-molecule fluorescence, the ForceChrono probe enables analysis of the complex dynamics of mechanical forces at the single-molecule level, such as loading rates and durations. Our results refine previous broad estimates of cellular loading rates to a more precise range of 0.5 to 2 pN/s, shedding light on the specifics of cellular mechanics. In addition, this study reveals a critical link between the magnitude and duration of integrin forces, consistent with the catch-bond behavior demonstrated in vitro. The ForceChrono probe has distinct advantages, such as precise analysis of single-molecule force dynamics and robust resistance to fluorescence fluctuations, which will significantly advance our understanding of cell adhesion and mechanotransduction at the single-molecule level.