RacG regulates morphology, phagocytosis, and chemotaxis.
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 nucleotide-dependent 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 microM 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 GTPgammaS 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.
Somesh
Baggavalli P
BP
Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany.
Vlahou
Georgia
G
Iijima
Miho
M
Insall
Robert H
RH
Devreotes
Peter
P
Rivero
Francisco
F
D000199:Actins / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002449:Cell Aggregation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology* / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects; D015870:Gene Expression; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015003:Yeasts / Q000166:cytology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D000596:Amino Acids; D002867:Chromones; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000242:Cyclic AMP; D020741:rho GTP-Binding Proteins