Spatiotemporal coordination of Rac1 and Cdc42 at the whole cell level during cell ruffling

Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.