A toolkit of customised protein sensors for interrogating mechanical forces in the cell

Mechanical forces play a role in a wide variety of cellular processes, such as activation of ion channels upon mechanical stimulation, force-bearing proteins in the muscle and extracellular matrix, and spindle tension in chromosome segregation. Specific proteins must, therefore, be capable of sensing mechanical signals and giving biological outputs. The understanding of mechanical stimuli at the molecular level has been limited to date, though, due to the lack of tools that can accurately measure low forces in vivo. Moreover, these force sensors must be customised for each specific use, to cover the appropriate force regime. Here we report a toolkit for measuring different ranges of forces in the cell, made possible by the striking spring-like properties of the tandem-repeat protein class and their exceptional amenability to rational design. We design a panel of Fluorescence Resonance Energy Transfer (FRET)-based tension sensors using consensus tetratricopeptide (CTPR) proteins as the mechanosensitive linkers. These are flanked by the fluorescent protein mCherry and contain an engineered a tetracysteine motif in the protein sequence, which then binds tightly to the fluorescein arsenical hairpin binder dye (FlAsH), resulting in a FRET pair. Optical tweezers experiments on the CTPR linkers show that the proteins respond to physiological forces in a highly distinctive manner. We dissect the mechanics of the proteins by mutationally creating and then characterising a series of variants with systematically modified properties, to build create a toolbox of customisable, calibrated force sensors. Our toolkit is capable of probing the varied mechanotransduction processes within the cell beyond what is currently possible.