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Eukaryotic Cell, October 2006, p. 1648-1663, Vol. 5, No. 10
1535-9778/06/$08.00+0     doi:10.1128/EC.00221-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

RacG Regulates Morphology, Phagocytosis, and Chemotaxis{triangledown} ,{dagger}

Baggavalli P. Somesh ,1,{ddagger},§ Georgia Vlahou,1,{ddagger} Miho Iijima,3 Robert H. Insall,2 Peter Devreotes,3 and Francisco Rivero1*

Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany,1 School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,2 Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, Maryland 212053

Received 12 July 2006/ Accepted 17 August 2006

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 µM 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{gamma}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.

* Corresponding author. Mailing address: Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany. Phone: 49 221 478 6987. Fax: 49 221 478 6979. E-mail: francisco.rivero{at}uni-koeln.de.

{triangledown} Published ahead of print on 1 September 2006.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} B.P.S. and G.V. contributed equally to this study.

§ Present address: Cancer Research UK, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, London, United Kingdom.

Eukaryotic Cell, October 2006, p. 1648-1663, Vol. 5, No. 10
1535-9778/06/$08.00+0     doi:10.1128/EC.00221-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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