Cell motility requires the orchestration of many dynamic cellular systems, which can only be fully understood through quantitation of protein activity in living cells and animals. This talk will describe new tools to visualize and manipulate signaling in vivo, using Rho family GTPase networks and cell motility as test beds. Multiplexed imaging of biosensors and computational image analysis will be used to examine the coordination of  GTPases. The role of specific protein activation events regulating coordination will be probed using engineered allosteric switches and different methods to control the dynamics of protein activity with light. New technologies will include methods to direct activated kinases to specific targets, broadly applicable methods to control sequestration of proteins at  intracellular membranes with light, and improvements in biosensor design for applications in animals.