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Eukaryotic Cell, June 2003, p. 474-485, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.474-485.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

RacB Regulates Cytoskeletal Function in Dictyostelium spp.

Eunkyung Lee,1,{dagger} David J. Seastone,2 Ed Harris,2 James A. Cardelli,2 and David A. Knecht1*

Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269,1 Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, Louisiana 711302

Received 4 November 2002/ Accepted 1 March 2003

Thus far, 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. To investigate the specific function of RacB, we have conditionally expressed wild-type (WT-RacB), dominant negative (N17-RacB), and constitutively activated (V12-RacB) versions of the protein. On induction, cells expressing V12-RacB stopped growing, detached from the surface, and formed numerous spherical surface protrusions while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin-driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin II activity, which distinguishes them from blebs formed by wild-type cells under stress. Finally, we examined the functional consequences of expression of wild-type and mutant RacB. Phagocytosis, endocytosis, and fluid phase efflux rates were reduced in all cell lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin but the consequences of activation appear to be different from other Dictyostelium Rac proteins so far investigated, resulting in different morphological and functional changes in cells.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269. Phone: (860) 486-2200. Fax: (860) 486-4331. E-mail: knecht{at}uconn.edu.

{dagger} Present address: Department of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06510.

Eukaryotic Cell, June 2003, p. 474-485, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.474-485.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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