Pagano, S.; Sanchez, C.; Cox-Cammer, N.; Bhalla, R. M.; Ravishankar, R.; Segura-Villalobos, D.; Muesch, A.; Aguirre-Ghiso, J.; Danuser, G.; Hodgson, L.

Cell migration requires the precise coordination of signaling pathways that regulate cytoskeletal dynamics and adhesion turnover. Rho GTPase-activating proteins (RhoGAPs) play critical roles in shaping these processes by controlling the spatial and temporal activity of small GTPases. ARHGAP20 is a RhoA-specific GAP, a downstream target of the Ras-related GTPase Rap1, and has been implicated in cancer cell motility, yet its functional role in coordinating migration-associated signaling remains poorly understood. Here, we investigated the role of ARHGAP20 in cell migration and its impact on the coordination between adhesion- and contractility-associated signaling pathways regulated by Rap1A and RhoA respectively. Using loss-of-function approaches in MTLn3 cells, we show that depletion of ARHGAP20 impairs both directed and random migration, leading to reduced cell velocity and displacement, and increased cell adhesion. To explore the underlying signaling mechanisms, we developed a genetically encoded FRET biosensor to monitor Rap1A activity and combined it with a near-infrared RhoA biosensor to simultaneously analyze their spatiotemporal dynamics in living cells. We found that Rap1A and RhoA activities are negatively coordinated during leading-edge dynamics and that ARHGAP20 depletion enhances this local anticorrelation. To further define where ARHGAP20 regulates Rap1A-RhoA signaling, we applied a microdomain-based analytical framework to quantify local signaling clusters. This analysis revealed that ARHGAP20 selectively modulates Rap1A-RhoA coordination outside focal adhesion regions, while leaving signaling correlations and overlap within focal adhesions unchanged. Subcellular localization analyses further reveal that ARHGAP20 is largely excluded from focal adhesions but associates with microtubules, the endoplasmic reticulum, the Golgi apparatus, and multiple Rab-positive vesicular compartments, supporting a trafficking-dependent mechanism for its spatial targeting. Together, our results identify ARHGAP20 as a regulator of cell migration that modulates the coordination between Rap1A and RhoA signaling through intracellular vesicular trafficking, highlighting the GAP's role in organizing the spatial coupling between signaling pathways in adhesion-associated regions required for efficient cell migration.