Dictyostelium RacH Regulates Endocytic Vesicular Trafficking and is Required for Localization of Vacuolin

Abstract

Dictyostelium RacH localizes predominantly to membranes of the nuclear envelope, endoplasmic recticulum and Golgi apparatus. To investigate the role of this protein, we generated knockout and overexpressor strains. RacH-deficient cells displayed 50% reduced fluid-phase uptake and a moderate exocytosis defect, but phagocytosis was unaffected. Detailed examination of the endocytic pathway revealed defective acidification of early endosomes and reduced secretion of acid phosphatase in the presence of sucrose. The distribution of the postlysosomal marker vacuolin was altered, with a high proportion of cells showing a diffuse vesicular pattern in contrast to the wild-type strain, where few intensely stained vacuoles predominate. Cytokinesis, cell motility, chemotaxis and development appeared largely unaffected. In a cell-free system, RacH stimulates actin polymerization, suggesting that this protein is involved in actin-based trafficking of vesicular compartments. We also investigated the determinants of subcellular localization of RacH by expression of green-fluorescent-proteintagged chimeras in which the C-terminus of RacH and the plasma-membrane-targeted RacG were exchanged, the insert region was deleted or the net positive charge of the hypervariable region was increased. We show that several regions of the molecule, not only the hypervariable region, determine targeting of RacH. Overexpression of mistargeted RacH mutants did not recapitulate the phenotypes of a strain overexpressing nonmutated RacH, indicating that the function of this protein is in great part related to its subcellular localization.

Phosphoinositides differentially regulate bacterial uptake and Nramp1-induced resistance toLegionellainfection inDictyostelium

“…In this paper, we have provided several lines of evidence in favor of this hypothesis: (1) engulfment by wild-type isolates, which are unable to macropinocytose, is ten-times lower than with axenic AX2 cells; (2) uptake by axenic strains under shaking is negligible; (3) the partial PI3K dependence of uptake; and (4) the defective uptake in racH-null cells. This also evidence that ingestion occurs by macropinocytosis, because bacterial phagocytosis is independent of PI3K (Peracino et al, 1998;Rupper et al, 2001b) and macropinocytosis, but not phagocytosis, is defective in the racH-null mutant (Somesh et al, 2006); (5) Legionella binding to Dictyostelium cells and ingestion under shaking are dramatically improved by treating the bacteria with sodium periodate. Periodate treatment leads to oxidative removal of distal carbohydrate residues from the lipopolysaccharide (LPS) coat, exposing internal core sugar residues linked to the lipid A backbone.…”

“…2C). In the racH-null mutant, which was previously shown to be normal for E. coli phagocytosis (Somesh et al, 2006), Legionella ingestion dropped to about 30% of that in the control. Inhibition in the ptennull mutant was not statistically significant.…”

“…To test this hypothesis, we took advantage of previous studies showing that macropinocytosis, in contrast to phagocytosis, is partially sensitive to PI3K inactivation, is strongly defective in racH-null cells (Somesh et al, 2006) and is quite negligible in wild-type strains (Kessin, 2001). Fig.…”

CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum

“…6B; localization indicated by arrows). The Dictyostelium ER has been studied using various markers, all of which produce a similar localization surrounding the nucleus and throughout the cytoplasm (Müller-Taubenberger et al, 2001;Somesh et al, 2006). In order to confirm the ER localization, we used an antibody against calnexin, an integral transmembrane protein of the ER.…”

RacG Regulates Morphology, Phagocytosis, and Chemotaxis

“…They are involved in chemotaxis, cell motility, endocytosis, cytokinesis, and development (13,31,39). More recently, RacH has been implicated specifically in the regulation of endocytic-vesicle trafficking (53). However, with few exceptions, the exact signaling pathways regulated by these proteins remain largely unknown.…”