RESEARCH ARTICLE 2449 Rab7 regulates phagosome maturation in Dictyostelium Adam Rupper1, Bryon Grove2 and James Cardelli1,* 1Department of Microbiology and Immunology and The Feist/Weiller Cancer Center, LSUHSC, Shreveport, LA 71130, USA 2Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, North Dakota 58202, USA *Author for correspondence (e-mail: [email protected]) Accepted 6 April 2001 Journal of Cell Science 114, 2449-2460 © The Company of Biologists Ltd SUMMARY A Dictyostelium Rab7 homolog has been demonstrated to LBC phagosomes isolated from cells overexpressing regulate fluid-phase influx, efflux, retention of lysosomal dominant negative (DN) Rab7 contained very low levels of hydrolases and phagocytosis. Since Rab7 function LmpA (lysosomal integral membrane protein) and α- appeared to be required for efficient phagocytosis, we mannosidase was not detectable. Interestingly, cysteine sought to further characterize the role of Rab7 in proteinases were delivered to phagosomes as apparent pro- phagosomal maturation. Expression of GFP-Rab7 resulted forms in cells overexpressing DN Rab7. Despite these in labeling of both early and late phagosomes containing defects, phagosomes in cells overexpressing DN Rab7 yeast, but not forming phagocytic cups. In order to matured to form multi-particle spacious phagosomes, determine if Rab7 played a role in regulating membrane except that these phagosomes remained significantly more traffic between the endo/lysosomal system and maturing acidic than control phagosomes. These results suggested phagosomes, latex bead containing (LBC) phagosomes that Rab7 regulates both an early and late steps of were purified from wild-type cells at various times after phagosomal maturation, similar to its role in the internalization. Glycosidases, cysteine proteinases, Rab7 endo/lysosomal system. and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. Key words: Rab7, Phagocytosis, Phagosome maturation INTRODUCTION vacuole (Wichmann et al., 1992; Schimmoller and Riezman, 1993). While most data suggest that Rab7 regulates trafficking The Rab small molecular mass GTPases have taken center from early endosomes to late endosomes, two papers propose stage as master regulators of vesicle trafficking events (Somsel that GTPase inactive forms of Rab7 localize predominantly to and Wandinger-Ness, 2000). These proteins are thought to lysosomes and, in one case, that expression of a dominant regulate formation of transport vesicles from donor negative (DN) form of Rab7 causes lysosomes to disperse compartments, transport of membrane vesicles on cytoskeletal from their perinuclear location in Hela cells (Bucci et al., structures, tethering of vesicles to acceptor compartments and 2000; Meresse et al., 1995). Interactions between late fusion of lipid bilayers. Rab proteins are activated in the GTP- endosomes and lysosomes have been suggested to occur bound state and interact with effectors until GTP hydrolysis through formation of a hybrid organelle, which can then has occurred (Bourne, 1988). undergo fission events to result in reformation of late As the hypothesis that Rab proteins regulate the specificity endosomes and lysosomes (Pryor et al., 2000; Mullock et al., of fusion events would predict (though it is now clear that other 1998). Though evidence points to the involvement of a Rab factors such as membrane tethers act in concert with Rabs to protein, it is not clear whether Rab7 or some other Rab protein provide specificity), individual Rab proteins have been may be involved. localized to specific compartments in the endo/lysosomal A cDNA with 85% identity to human Rab7 has been cloned pathway. For instance, Rab4 and Rab5 have been localized to from Dictyostelium and indirect immunofluorescence has early endosomes. Rab5 regulates fusion of clathrin-coated shown that Rab7 associates with endosomes and post- vesicles with early endosomes and homotypic fusion of early lysosomes (Buczynski et al., 1997). The endo/lysosomal endosomes (Bucci et al., 1992; Barbieri et al., 1994), while system of Dictyostelium consists of a linear pathway including Rab4 regulates recycling of membrane receptors to the plasma endosomes, lysosomes and a terminal secretory compartment membrane (van der Sluijs et al., 1991; van der Sluijs et al., known as a post-lysosome (Padh et al., 1993). Rates of 1992; Daro et al., 1996). Rab7 and Rab9 have been localized phagocytosis and endocytosis are lower in a cell line to late endosomes (Chavrier et al., 1990; Soldati et al., 1995), overexpressing DN Rab7 (T22N mutation) (Buczynski et al., where Rab9 regulates recycling of membranes back to the 1997). In addition, the rate of fluid-phase efflux is slower, Golgi complex (Lombardi et al., 1993). while paradoxically lysosomal enzymes are over-secreted Reports have suggested that Rab7 plays a role in vesicular (Buczynski et al., 1997), and these cells also contain vacuoles transport from early endosomes to late endosomes (Feng et the size of post-lysosomes that are acidic (post-lysosomes are al., 1995; Mukhopadhyay et al., 1997; Vitelli et al., 1997), and normally close to neutral pH). It was hypothesized that Rab7 in yeast (Rab7 homolog Ypt7), from late endosomes to the regulates membrane and hydrolase recycling from post- 2450 JOURNAL OF CELL SCIENCE 114 (13) lysosomes to an earlier endosomal compartment since cells MATERIALS AND METHODS overexpressing wild-type Rab7 retain lysosomal enzymes more efficiently, while rates of fluid-phase efflux are increased. This Cells and culture conditions recycling event may be necessary for proper maturation of D. discoideum, strain Ax4, was grown axenically at 18°C in HL5 post-lysosomes; thus, expression of DN Rab7 slows efflux, and growth medium (1% Oxoid proteose peptone, 1% glucose, 0.5% yeast results in a membrane ‘traffic jam’, which in turn slows the extract, 2.4 mM Na2HPO4, and 8.8 mM KH2PO4, pH 6.5) either in internalization pathways. Consistent with these observations is shaking suspension or in tissue culture flasks. Construction of the Ax4 a report demonstrating that Rab7 regulates homotypic fusion strain overexpressing DN Rab7 was described in a previous events between early endosomes (Laurent et al., 1998). Thus, publication (Buczynski et al., 1997). In brief, site-directed mutagenesis was performed to generate a Thr 22 to Asn mutation in Rab7 might direct recycling vesicles to early endosomes and the Rab7 cDNA. The resulting cDNA was cloned into the pDA80-HA may also regulate homotypic interactions between early vector behind and in-frame with the flu hemagglutinin epitope, endosomes. transformed into Dictyostelium and G418 resistant colonies were We were interested in pursuing the role of Rab7 in selected and screened for overexpression by western blot analysis. A phagocytosis and phagosomal maturation, since expression of cell line expressing green fluorescent protein (GFP) fused in-frame to DN Rab7 inhibited phagocytosis of latex beads, and subcellular the N terminus of Rab7 was generated as follows. A cDNA encoding fractionation experiments demonstrated that Rab7 was GFP with the following mutations, F64L and S65T, was cloned into enriched in early phagosomes (Buczynski et al., 1997). A role the KpnI site of pVEII∆ATG (Rebstein et al., 1993). The full-length for Rab7 in phagosomal maturation in mammalian cells has not cDNA encoding wt Rab7 was cloned into the SacI site of the resulting been described, though Rab7 does localize to phagosomes plasmid. The resulting plasmid was purified using (Qiagen Inc., Valencia, CA, USA) Qiafilter Maxi Prep columns and was (Rabinowitz et al., 1992; Desjardins et al., 1994). Rab5 has transformed into D. discoideum, Ax3 strain, by the calcium phosphate been the key protein described to regulate interactions of early precipitation method (Sadeghi et al., 1988). Geneticin (Sigma, St endosomes and late endosomes with phagosomes, though it is Louis, MO, USA) resistant colonies were screened for expression of unclear which Rab might regulate fusion of lysosomes with GFP fluorescence by observation with a fluorescent microscope. phagosomes (Alvarez-Dominguez et al., 1996; Funato et al., Construction of the GFP-ABD expressing cell line was described in 1997; Jahraus et al., 1998). Furthermore, pathogens such as Pang et al. (Pang et al., 1998) and was a kind gift of Dr David Knecht. Listeria and Salmonella have mechanisms to recruit Rab5 to Magnetic purification of the vesicles from the the phagosomal membrane and somehow delay subsequent endo/lysosomal system maturation steps (Hashim et al., 2000; Alvarez-Dominguez et Ax3 cells were harvested during log-phase growth between 3-5×106 al., 1996; Alvarez-Dominguez et al., 1997). Thus, the role of cells/ml, centrifuged at 1000 g for 5 minutes and resuspended at Rab proteins during phagosomal maturation is important and 2.0×108 cells/ml in HL5. The cells were then fed dextran-coated much remains to be discovered. colloidal iron at 3 mg/ml for 60 minutes to load vesicles of the In Dictyostelium, phagosomal maturation consists of an endo/lysosomal system. Iron-containing vesicles were purified as early acidic phase followed by a late non-acidic phase, similar described elsewhere (Rodriguez-Paris et al., 1993). to that seen in the endo/lysosomal system (Rupper et al., 2001). Early phagosomes are acidified to a pH of Purification of latex bead containing phagosomes Control cells or cells overexpressing DN Rab7 were harvested during approximately 5, while late phagosomes have a pH of over 6. × 6 Concomitant with the rise in pH, mature phagosomes fuse to log-phase growth between 3-5 10 cells/ml, centrifuged at 1000 g for 5 minutes and resuspended at 2.5×107 cells/ml in HL5. The cells were give rise to late, spacious phagosomes. We have found that allowed to recover for 10 minutes prior to addition of latex beads. an increase in phagosomal pH and fusion of mature Latex beads (0.8 µm diameter, blue-dyed; Sigma, St Louis, MO, USA) phagosomes requires phosphatidylinositol 3-kinase activity, were added at a concentration of 100 beads/cell and incubated with as cells with a disruption in both ddpik1 and ddpik2 (two the cells for various periods.