To better understand signals underlying rapid and dynamic cellular events, i.e. cell motility and cytoskeletal dynamics, we have been designing novel tools to visualize and manipulate protein behavior in living cells. For protein manipulation with subsecond and microns resolution we have developed genetically encoded caged proteins, undergoing reversible activation upon irradiation with light of 450-500 nm. Caged Rac has revealed divergent roles for Pak, ROCK and MLCK signaling during Rac-induced motility. A general strategy for drug-induced kinase activation will be described, applied to decipher the role of FAK in cell protrusion and adhesion dynamics. New biosensors based upon FRET ‘antenna systems' can be used to visualize endogenous Pak, Src and Cdc42 activation with genetically encoded sensors. Dye-based and FRET-based biosensors from the above studies are used in the same cell for high resolution studies of Rho GTPase coordination during cell motility.

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