A Rho GTPase-effector ensemble governs cell migration behavior.

How can a cell navigate its environment without any external cues? Since such cues are not always present in the environment, cells rely on internal machinery to explore their surroundings. Although Rho GTPases are known for orchestrating cell motility, the intrinsic Rho GTPase-effector mechanisms governing spontaneous migration remain incompletely understood. Here we show an imaging-based method that profiles protein-protein interactions (PPIs) through phase-separated condensates. By applying this method to hundreds of interaction profiles between Rho small GTPases and their effector proteins, we uncovered two intrinsic mechanisms governing cell migration. Formin-like protein (FMNL) determines the front of the cell by restricting Cdc42 activity, establishing front-rear polarity. In contrast, Rac1-ROCK-interaction-mediated arc stress fiber formation at the front inherently enables spontaneous directional changes and enhances cellular responses to external cues. Our findings elucidate the intricate roles of the Rho GTPase-effector ensemble that governs cell migration behavior, revealing an intrinsic program for efficient motility strategies.