Spatio-temporal coordination of Rho GTPase activity patterns in cell migration

Abstract

Cell shape changes are fundamental to processes such as immune cell migration and cancer metastasis and are driven by coordinated cycles of cell protrusion and retraction. These dynamics are regulated by the Rho family GTPases Rac1 and RhoA, which promote protrusion and contraction, respectively. While earlier models proposed that mutual inhibition between Rac1 and RhoA stabilizes cell polarization, recent evidence instead indicates that Rac1 activates RhoA, suggesting a mechanism for dynamic protrusion-retraction cycles rather than stable polarity. However, the molecular basis of this crosstalk and the spatial organization of Rac1 and RhoA signalling remained unclear. This study identifies two complementary mechanisms linking Rac1 to RhoA activation. Arhgef12 is recruited directly to the plasma membrane by active Rac1 through its PH domain, whereas Arhgef11 is recruited near the plasma membrane downstream of Rac1-induced actin polymerization via a unique F-actin-binding motif. These findings establish how Rac1 promotes localized RhoA activation to coordinate protrusion with subsequent retraction. To explain the spatial confinement of Rac1 and RhoA activities, the unconventional Rho GTPase Rnd3 was investigated as a potential regulator. Rapid optogenetic and chemical perturbations confirmed mutual inhibition between RhoA and Rnd3 but unexpectedly revealed that Rac1 is the dominant inhibitor of Rnd3 through p21-activated kinases (PAKs). Functional analyses demonstrated that Rnd3 suppresses ectopic RhoA activity across the cell attachment area while permitting localized contraction at the cell edge. Loss of Rnd3 markedly reduced protrusion-retraction dynamics, whereas its overexpression enhanced these morphodynamic behaviors. Together, these findings establish a revised signalling network in which Rac1 promotes RhoA activation through Arhgef11 and Arhgef12 while simultaneously relieving Rnd3-mediated inhibition at the cell edge. This coordinated Rac1-Rnd3-RhoA circuit explains how localized protrusion-retraction cycles are generated in migrating cells and provides new mechanistic insight into cell morphodynamics relevant to physiological processes and pathological conditions such as cancer metastasis.