Dual regulation of the actin cytoskeleton by CARMIL-GAP

Abstract

CARMIL (Capping protein Arp2/3 Myosin I Linker) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of Capping Protein (CP). Here we characterize CARMIL-GAP, a Dictyostelium CARMIL isoform that contains a ~130 residue insert that, by homology, is a GTPase activating (GAP) domain for Rho-related GTPases. Consistently, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and cells devoid of CARMIL-GAP exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by the re-expression of CARMIL-GAP. Finally, the rescue of CARMIL-GAP null cells with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that while both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of Rho GTPases between their GTP and GDP bound states is often required to drive Rho-dependent biological processes.