We are interested in the molecular processes underlying mechanical force transduction in cells. To investigate these processes, we developed a microscopy technique that allows the quantification of mechanical forces with piconewton-sensitivity in living cells. The method is based upon Fӧrster resonance energy transfer (FRET)–based biosensors, in which two fluorophores are connected by a mechanosensitive linker peptide that elongates in response to mechanical tension. Peptide extension leads to a decrease in FRET efficiency, which can be quantified with advanced microscopy methods such as fluorescence lifetime microscopy (FLIM). To allow quantitative measurements, we calibrated our probes using single-molecule force spectroscopy to determine the sensors’ force thresholds, their folding characteristics, and loading-rate sensitivities. In total, we generated four different tension sensor modules with force sensitivities at 1-6 piconewton (pN), 3-5 pN, 6-8 pN and 9-11 pN. By genetically inserting these individual biosensors into proteins of interest, mechanical tension across the respective target protein can be quantified. We applied our methods to study molecular forces across cell adhesion proteins like vinculin, talin and desmoplakin. Independent research labs adapted our method to investigate processes of force transduction in other subcellular structures like the cortical actin network, cell-cell junctions, and the glycocalxy. In summary, our technique can be utilized to investigate subcellular mechanical processes in living cells with piconewton sensitivity and in a spatio-temporally resolved manner.
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