The study investigates the spatiotemporal coordination of Rho GTPase activities during cell protrusion. Using biosensors and a computational multiplexing approach, the researchers found that RhoA is activated synchronously with the advancement of the cell edge, while Cdc42 and Rac1 are activated 2 μm behind the edge with a 40-second delay. This indicates that RhoA operates in the initial stages of protrusion, while Rac1 and Cdc42 are involved in the reinforcement and stabilization of the expanded protrusions. The findings suggest that Rho GTPases are not only temporally but also spatially coordinated, with RhoA activation being closely linked to the leading edge dynamics, and Rac1 and Cdc42 activation propagating in both anterior and posterior directions. The study also highlights the role of RhoA in actin polymerization and microtubule stabilization, and the antagonistic relationship between RhoA and Rac1.The study investigates the spatiotemporal coordination of Rho GTPase activities during cell protrusion. Using biosensors and a computational multiplexing approach, the researchers found that RhoA is activated synchronously with the advancement of the cell edge, while Cdc42 and Rac1 are activated 2 μm behind the edge with a 40-second delay. This indicates that RhoA operates in the initial stages of protrusion, while Rac1 and Cdc42 are involved in the reinforcement and stabilization of the expanded protrusions. The findings suggest that Rho GTPases are not only temporally but also spatially coordinated, with RhoA activation being closely linked to the leading edge dynamics, and Rac1 and Cdc42 activation propagating in both anterior and posterior directions. The study also highlights the role of RhoA in actin polymerization and microtubule stabilization, and the antagonistic relationship between RhoA and Rac1.