Digital and Tunable Genetically Encoded Tension Sensors Based on Engineered Coiled-Coils

Abstract

Genetically encoded tension sensors (GETSs) allow for quantifying forces experienced by intracellular proteins involved in mechanotransduction. The vast majority of GETSs are comprised of a FRET pair flanking an elastic “spring-like” domain that gradually extends in response to force. Because of ensemble averaging, the FRET signal generated by such analog sensors conceals forces that deviate from the average, and hence it is unknown if a subset of proteins experience greater magnitudes of force. We address this problem by developing digital GETSs comprised of coiled-coils (CCs) with tunable mechanical thresholds. We validate the mechanical response of CC digital probes using thermodynamic stability prediction, AlphaFold2 modeling, steered molecular dynamics simulations, and single molecule force microscopy. Live cell measurements using optimized CC tension sensors that are inserted into vinculin demonstrate that 13% of this mechanosensor experiences forces > 9.9 pN within focal adhesions. This reveals greater magnitudes of vinculin force than had previously been reported and demonstrates that coiled-coil tension sensors enable more facile and precise tension measurements in living systems.