RacG Regulates Morphology, Phagocytosis, and Chemotaxis

Abstract

RacG is an unusual member of the complex family of Rho GTPases in Dictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotidedependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 M LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTP␥S stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or more Dictyostelium Rho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

A dual role for Rac1 GTPases in the regulation of cell motility

“…Biological roles of some Dictyostelium Rho GTPases have been assessed by genetic knockout and overexpression experiments, as well as localization studies, and a number of their effectors have been identified . RacE, for instance, plays a role in the regulation of cytokinesis (Larochelle et al, 1997), RacB is involved in the control of directional sensing (Park et al, 2004), whereas RacG is important for cell shape, motility and phagocytosis (Somesh et al, 2006). However, direct evidence for the sites of activity and dynamics of RhoGTPases in Dictyostelium has been lacking so far.…”

Costars, aDictyosteliumprotein similar to the C-terminal domain of STARS, regulates the actin cytoskeleton and motility

“…Of the more characterized Rac-related GTPases, several are linked to chemotaxis-related functions. Rac1A controls filopodia formation and cell motility (Dumontier et al, 2000); RacB regulates actin polymerization and chemotaxis, and is controlled by the PI3K pathway (Park et al, 2004); RacC stimulates WASPdependent F-actin assembly, and is required for PI3K activation and proper chemotaxis (Han et al, 2006); and RacG induces filopodia formation and actin polymerization, and regulates chemotaxis (Somesh et al, 2006). It is of interest to check whether Costars function depends on any of these D. discoideum Rac-related GTPases.…”

Filopodia: Complex models for simple rods

“…Cdc42-subfamily members such as Wrch1 (Aspenstrom et al, 2004, Ruusala andAspenstrom, 2008), or RhoD and Rif (Ellis andMellor, 2000, Pellegrin andMellor, 2005). In Dictyostelium, filopodia can be induced by at least four GTPases, Rac1A, B, C, and G (Dumontier et al, 2000, Somesh et al, 2006, but not all Rac GTPases expressed in this organism have been thoroughly examined. Clearly, future efforts to improve our knowledge on the commonalities and specificities of signalling pathways driving filopodia formation will have to include attempts to abolish these structures by interference with multiple GTPases simultaneously.…”