Abstract Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter‐ and intra‐molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin‐membrane distance of 2.5 nm. We demonstrate that QD‐FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub‐nanometer accuracy.
