A novel high content analysis tool reveals Rab8-driven actin and FA reorganization through Rho GTPases and calpain/MT1

Abstract

Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. A high content analysis (HCA) tool developed to dissect Rab8-mediated actin and focal adhesion (FA) reorganization revealed that Rab8 activation significantly induced Rac1/Tiam1 to mediate cortical actin (CA) formation and RhoA-dependent stress fibre (SF) disassembly. Rab8 activation increased Rac1 activity, while its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with FA, promoting their disassembly in a microtubule dependent manner. This Rab8 effect involved Calpain, MT1-MMP and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of FA, and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.