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 CARMIL-GAP null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP null cells. Finally, rescue 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 the Rho GTPase's nucleotide state is often required to drive Rho-dependent biological processes.

Oscillatory dynamics of Rac1 activity inDictyostelium discoideumamoebae

“…Next, we assume that Rac1-specific GAP is recruited to the cell membrane by Rac1T, similar to what has been proposed for Ras1 and Cdc42 [45,46]. Consistent with this, the localization of D. discoideum RhoGAPs Dd5P4 and CARMIL-GAP is largely cytosolic, and they are only transiently recruited to the membrane by interacting with activated Rac1 [47,48], in agreement with data available for mammalian RhoGAPs [49,50].…”

Reconstitution of Arp2/3-Nucleated Actin Assembly with CP, V-1 and CARMIL

“…CPI-motif proteins are diverse; their sequences are completely unrelated to each other except for the presence of the conserved CPI motif 16 . Among CPI-motif proteins, the CARMIL (capping protein, Arp2/3, myosin I linker) was discovered early 19,20 , and the CARMIL protein family has been studied rather extensively [21][22][23][24] .…”