This study investigates the coordination of Rho GTPase activities during cell protrusion. Using biosensors and computational multiplexing, the researchers observed that RhoA is activated at the cell edge synchronous with edge advancement, while Cdc42 and Rac1 are activated 2 micrometers behind the edge with a 40-second delay. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, with RhoA playing a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways involved in reinforcement and stabilization of newly expanded protrusions. The computational multiplexing approach allows for the alignment of GTPase activation timings using protrusion/retraction events as a reference. The study found that GTPase activities are regulated most prominently within a few micrometers from the leading edge, supporting the hypothesis that these fluctuations are linked to cell edge movements. The results show that GTPases are activated over a fixed time interval relative to the dynamics of the leading edge. The study also found that RhoA activation is synchronous with forward movements of the cell edge, while Rac1 and Cdc42 activation lags behind. These findings suggest that RhoA may play a role in initiating protrusion, while Rac1 and Cdc42 are involved in reinforcing and stabilizing protrusions. The study further shows that the activation of Rac1 and Cdc42 propagates in anterior and posterior directions, losing close coordination with edge movements. The study also validates the time shifts between GTPase activations predicted by computational multiplexing through direct observation of two signaling activities in the same cell. The results confirm that direct visualization and indirect inference of signaling relationships yield the same result. The study presents two complementary approaches to in situ analysis of cellular pathways: simultaneous imaging and computational multiplexing. These methods enable the exploration of the relationship between two pathways and the correlation of many signaling activities. The computational multiplexing approach is potentially less perturbing than methods that rely on acute cell stimulation to initiate a signaling cascade. The study also supports the notion that Rac1 operates as an antagonist to RhoA, with RhoA activity being rapidly suppressed as Rac1 reaches its maximum activation. The findings suggest that RhoA may play a role in initiating protrusion, while Rac1 and Cdc42 are involved in reinforcing and stabilizing protrusions. The study also highlights the importance of spatial and temporal coordination in cell protrusion and retraction. The results provide insights into the complex interplay between GTPase activities and cell morphology during cell movement.This study investigates the coordination of Rho GTPase activities during cell protrusion. Using biosensors and computational multiplexing, the researchers observed that RhoA is activated at the cell edge synchronous with edge advancement, while Cdc42 and Rac1 are activated 2 micrometers behind the edge with a 40-second delay. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, with RhoA playing a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways involved in reinforcement and stabilization of newly expanded protrusions. The computational multiplexing approach allows for the alignment of GTPase activation timings using protrusion/retraction events as a reference. The study found that GTPase activities are regulated most prominently within a few micrometers from the leading edge, supporting the hypothesis that these fluctuations are linked to cell edge movements. The results show that GTPases are activated over a fixed time interval relative to the dynamics of the leading edge. The study also found that RhoA activation is synchronous with forward movements of the cell edge, while Rac1 and Cdc42 activation lags behind. These findings suggest that RhoA may play a role in initiating protrusion, while Rac1 and Cdc42 are involved in reinforcing and stabilizing protrusions. The study further shows that the activation of Rac1 and Cdc42 propagates in anterior and posterior directions, losing close coordination with edge movements. The study also validates the time shifts between GTPase activations predicted by computational multiplexing through direct observation of two signaling activities in the same cell. The results confirm that direct visualization and indirect inference of signaling relationships yield the same result. The study presents two complementary approaches to in situ analysis of cellular pathways: simultaneous imaging and computational multiplexing. These methods enable the exploration of the relationship between two pathways and the correlation of many signaling activities. The computational multiplexing approach is potentially less perturbing than methods that rely on acute cell stimulation to initiate a signaling cascade. The study also supports the notion that Rac1 operates as an antagonist to RhoA, with RhoA activity being rapidly suppressed as Rac1 reaches its maximum activation. The findings suggest that RhoA may play a role in initiating protrusion, while Rac1 and Cdc42 are involved in reinforcing and stabilizing protrusions. The study also highlights the importance of spatial and temporal coordination in cell protrusion and retraction. The results provide insights into the complex interplay between GTPase activities and cell morphology during cell movement.