Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis inDictyostelium discoideum

Abstract

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis inDictyostelium.

Dynacortin Is a Novel Actin Bundling Protein That Localizes to Dynamic Actin Structures

“…These same IQGAP proteins or other related proteins could modulate dynacortin's localization and activity. Other Rac family proteins may also regulate dynacortin recruitment and/or actin cross-linking in other cortical structures such as actin crowns and phagocytic cups (27,28). Finally, since dynacortin is a novel protein with such an unusual amino acid composition, discerning its actin bundling mechanism will be of interest.…”

Nadrin, a Novel Neuron-specific GTPase-activating Protein Involved in Regulated Exocytosis

“…5B). To test biochemically whether the endogenous Nadrin was associated with the cytoskeletal components, cell lysates were incubated with Triton X-100 to solubilize membranes, and the Triton-insoluble cytoskeleton was pelleted by centrifugation (57,58). Nadrin was predominantly present in the 100,000 ϫ g-precipitated fraction, and the Triton-treatment did not release endogenous Nadrin into the supernatant, whereas 0.5 M KCl treatment readily released Nadrin (Fig.…”

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…Actin cross-linking proteins and small guanosine triphosphatases (GTPases) of the Rac and Rho family that regulate and organise the actin cytoskeleton are important in the process of ruffle extension in D. discoideum (Seastone et al 1998;Seastone et al 1999). A subset of actin-binding proteins associate transiently with the phagocytic cup of amoebae such as talin (Niewohner et al 1997) and coronin, which is thought to regulate the G-to F-actin equilibrium (Maniak et al 1995).…”

“…Cells expressing a Rab11 dominant negative mutant show a swollen contractile vacuole reticular network and are impaired in contractile vacuole function (Bush et al 1998;Mol Bioi Cell 9: 464a). Together these observations indicate that RabD may regulate transport of vesicles rich in proton pumps between endosomes and the contractile vacuole complex.…”