Rnd3 regulates cell morphodynamics by spatial restriction of cell contraction signaling

Cell migration is enabled by dynamic changes in cell shape, which are controlled by spatio-temporal activity patterns of the Rho GTPases Rac1 and RhoA. Classical models proposed that these activity patterns are generated by mutual inhibition between the front signal Rac1 and the back signal RhoA, leading to opposing gradients that define the direction of cell migration. However, direct measurements of signal crosstalk showed that Rac1 can activate RhoA, which is incompatible with mutual inhibition. Furthermore, opposing Rac1 and RhoA gradients generated by mutual inhibition would need to overlap at least partially in the cell center. In contrast, both Rac and Rho activities were largely absent in the cell center, and both found to be highly localized near the cell edge in the cell periphery. Here, we hypothesized that these spatio-temporal Rho GTPase activity patterns are generated by the mutual inhibition between RhoA and the unconventional Rho family member Rnd3. Using rapid, optogenetic and chemical perturbations, we confirmed this mutual inhibitory crosstalk. However, we found that this crosstalk does not lead to the expected mutually exclusive spatio-temporal patterns of Rnd3 activity and cell retraction in spontaneously migrating, unperturbed cells. Instead, Rnd3 activity was even slightly elevated during cell retraction and was surprisingly depleted during cell protrusion. We discovered that this depletion is caused by the inhibition of Rnd3 by Rac1 activity, and that this newly identified inhibition is much more pronounced compared to the inhibition of Rnd3 by Rho. By combining rapid optogenetic perturbations with pharmacological manipulations, we found that Rac1 inhibition by Rnd3 is mediated by p21-activated kinases (PAKs). Investigations into the function of Rnd3 showed that it is required for the tight spatio-temporal regulation of the cell contraction/retraction signal Rho, and that it prevents ectopic, highly dynamic, spontaneous Rho activity pulses within the whole cell attachment area. Interestingly, protrusion-retraction dynamics were also severely inhibited in the absence of Rnd3, and its overexpression stimulated this process. Taken together, we show that Rac and not Rho acts as the major Rnd3 inhibitor in cells. Furthermore, our findings support a mechanism, in which Rnd3 acts as a global inhibitor of Rho that spatially restricts Rho activity to regions near the edge of migrating cells.