stFRET, a novel tool to study molecular force in living cells and animals
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Mechanical stress is intimately involved in biology from organ function such as heart, lungs, muscle, and the cochlea and vestibular system. However, mechanical stress is involved subtly in cell biology, in familiar processes as cell migration less familiar ones such as stem cell development. Little is known about how mechanics alters signal transduction in vivo . To begin dissecting the distribution of stress in time and space we developed stFRET, a molecular force sensor based on fluorescence energy transfer. stFRET consists of green fluorescence protein (GFP) mutants Cerulean and Venus with a stable a-helix peptide as linker. The FRET efficiency of the cassette protein was a function of the length of the linker, the angles of the fluorophores, temperature and urea denaturation and protease treatment. The linking helix was stable to 80C°, unfolded in 8M urea and was rapidly digested by proteases. In all cases, the fluorophores were unaffected. To vary the angles and distance between the donor and acceptor we modified the a-helix linker by adding and subtracting residues. Assuming the linker and fluorophores were rigid, we calculated its geometry. We tested the strain sensitivity by linking both ends to a rubber sheet subjected to equibiaxial stretch and FRET decreased proportional to strain. stFRET could be expressed in cultured cells. For unknown reasons the free protein localized to the nucleus and nucleoli. However when stFRET was incorporated into host proteins, it was localized similarly to unlabelled hosts. We incorporated stFRET into various locations within the cytoskeleton proteins actinin, filamin, spectrin and collagen-19 in C. elegans . The host proteins were generally under resting tension. In migrating cells, the hosts were stressed at the leading edge and relaxed at the trailing edge. Disassembling actin filaments or disrupting focal attachments eliminated the strain in actinin, filamin and spectrin, suggesting the dominant stress is pulling on adhesion plaques through actin. stFRET also sensed strain from the external forces exerted on C. elegans . We stretched transgenic worms and showed reversible changes in FRET as we stretched and bent the worm. This study opens the door to examining how mechanical forces affect the function of specific proteins.