GTPase activating protein DLC1 spatio-temporally regulates Rho signaling

Abstract

Tightly regulated spatio-temporal Rho GTPase activity patterns regulate morphogenetic processes such as cell migration. Emerging evidence suggests that binding of Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) to the cytoskeleton or adhesions mediate feedback regulation to spatio-temporal Rho GTPase activation. To explore such feedback regulation, we study the Rho specific GAP Deleted in Liver Cancer 1 (DLC1) which binds to focal adhesions (FAs) through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to global increase in Rho activity and contractility throughout the cell without affecting a striking lamellar RhoA activity band in fibroblasts. Although RhoA activity may appear steady at the population level, individual molecules are continually activated by GEFs, inactivated by GAPs, and diffusing between subcellular regions, a dynamic turnover referred to as "signaling flux". To directly measure this flux, we built a genetic circuit combining an optogenetic actuator to control Rho activity with a Rho activity biosensor. In spreading cells at steady state, optogenetic manipulation of Rho activity reveals that DLC1 controls the rate of Rho activation rather than duration, both at FAs and at the plasma membrane (PM). Local and reversible optogenetic control of contractility shows that DLC1 associates/dissociates with FAs during their reinforcement/relaxation. This might provide positive feedback that locally increases the rate of Rho activation at FAs that experience local tension to facilitate FA disassembly. Our results indicate that DLC1 operates both at the PM and at FAs to regulate global Rho activity levels at steady state, or to amplify local Rho activity at FAs experiencing a strong mechanical input, presumably to induce robust FA disassembly. This provides new insights in the complexity of spatio-temporal Rho GTPase signaling.