Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high-throughput screening. We made this Src-family kinase (SFK) biosensor by derivatizing a monobody specific for activated SFKs with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and changes to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFKs are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 μm from the leading edge.
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We thank C. MacNevin for assistance with dye synthesis, F. Shen for help with imaging studies, D. Renfrew for assistance with computational modeling, A. Nguyen for technical assistance and B. Clarke for administrative assistance. We gratefully acknowledge funding from the American Heart Association (A.G.) and the US National Institutes of Health (GM GM082288 and GM057464 to K.M.H.).
The authors declare no competing financial interests.
Supplementary Results and Supplementary Methods (PDF 1299 kb)
Src activation in fibroblasts with PDGF stimulation (AVI 5283 kb)
Src activation at the cell edge in PDGF stimulated fibroblasts (AVI 482 kb)
Src activation in randomly migrating epithelial cells (AVI 5024 kb)
Src activation at the cell edge and the effect of inhibitor (AVI 865 kb)
Merobody sensor is sensitive to Src activity. (AVI 4690 kb)
Src merobody sensor localization at the cell edge is dependent on Src activity (AVI 649 kb)
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Gulyani, A., Vitriol, E., Allen, R. et al. A biosensor generated via high-throughput screening quantifies cell edge Src dynamics. Nat Chem Biol 7, 437–444 (2011). https://doi.org/10.1038/nchembio.585
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