From Sensor Design to Force Maps: A Systematic Evaluation of FRET-based Vinculin Tension Sensors

Mechanical forces transmitted through focal adhesions regulate cell behavior and disease progression, yet remain difficult to quantify at the molecular level. Genetically encoded FRET-based tension probes enable measurements of piconewton-scale forces across specific proteins in living cells, but their quantitative interpretation is highly sensitive to probe design and measurement modality. Here, we systematically compared vinculin tension sensors under identical experimental conditions, evaluating unloaded reference constructs, fluorophore pairs, mechanical sensor modules, and circularly permuted variants. Unloaded controls established a common no-force baseline and validated force-dependent readout. Among the fluorophore pairs tested, the green-red combination Clover-mScarlet-I yielded a higher unloaded FRET efficiency and hence a broader measurable dynamic range. Comparison of six mechanical sensor modules identified the binary-response sensors FL and CC-S2 as the most responsive, showing the largest force-dependent FRET changes and broadest FRET distributions. At the sub-focal adhesion level, CC-S2 reported the steepest proximal-to-distal tension gradient, indicating that vinculin tension increases sharply along peripheral adhesions and exceeds 10 piconewton. Circular permutation experiments revealed that fluorophore orientation has a strong, module-dependent influence on the measured FRET readout. Together, these results establish a comparative framework for interpreting FLIM-based vinculin tension measurements and provide practical design principles for selecting and engineering molecular tension probes. ### Competing Interest Statement The authors have declared no competing interest. Research Foundation - Flanders, https://ror.org/03qtxy027, G0C2422N, G0A8L24N, G0B9922N, 1S95125N KU Leuven, C14/22/085