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Rab7 regulates phagosome maturation in Dictyostelium

¹ Department of Microbiology and Immunology and The Feist/Weiller Cancer Center, LSUHSC, Shreveport, LA 71130, USA
² Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, North Dakota 58202, USA

View larger version (165K): [in a new window]   Fig. 1. GFP-Rab7 localizes to early and late phagosomal membranes. GFP-Rab7 expressing cells were pulsed with live yeast for 10 minutes and then fixed with 1% formaldehyde and viewed by fluorescence (A) or DIC (A') microscopy. The arrow denotes an early phagosome ringed with GFP-Rab7. GFP-Rab7 expressing cells were pulsed with live yeast for 120 minutes then fixed with 1% formaldehyde and viewed by fluorescence (B) or DIC (B') microscopy. The arrow denotes a multi-particle phagosome ringed with GFP-Rab7. Bar, 2 µm.

View larger version (90K): [in a new window]   Fig. 2. GFP-Rab7 associates with phagosomes within 60 seconds of internalization. GFP-Rab7 expressing cells were allowed to attach to glass coverslips and mounted in a chamber to facilitate imaging by confocal microscopy. RITC-labeled yeast were added to the growth media and images were captured every 5 seconds. Selected images of a single experimental series are shown. Note that the newly internalized yeast particle in the right portion of the cell does not receive GFP-Rab7 until at least 20 seconds after uptake (panel labeled 60''). The particle in the left portion of the cell appears to be surrounded by GFP-Rab7 vesicles during internalization but a closer inspection of the cup, particularly at 0 seconds and 40 seconds reveals Rab7-free areas. Bar, 5 µm.

View larger version (79K): [in a new window]   Fig. 3. GFP-ABD is rapidly removed from nascent phagosomes. GFP-ABD expressing cells were allowed to attach to glass coverslips and mounted in a chamber to facilitate imaging by confocal microscopy. RITC-labeled yeast were added to the growth media and images were captured every 5 seconds. Selected images of a single experimental series are shown. Bar, 5 µm.

View larger version (103K): [in a new window]   Fig. 4. Purified phagosomes containing latex beads are free from contamination by endosomes and lysosomes. Cells were incubated with growth medium containing 35S-methionine for 1 hour in order to label proteins. Unlabeled cells (A) were pulsed with latex beads for 20 minutes, washed, mixed with labeled cells and latex bead containing phagosomes were purified as described in Materials and Methods. Labeled cells (B) were treated as described for unlabeled cells in A. Latex beads (C) were added to labeled cell homogenate and then purified from the homogenate as in A. Equal protein loads were separated by SDS-PAGE and 35S-met labeled proteins were visualized by fluorography. H, cell homogenate; P, purified phagosomal membranes. The positions of marker proteins (kDa) are shown.

View larger version (133K): [in a new window]   Fig. 5. Electron microscopic evidence that purified latex bead-containing phagosomes are free from contamination by other organelles. Latex bead-containing phagosomes were purified as described in the Materials and Methods section and thin sections (Buczynski et al., 1997) were examined using a transmission electron microscope. Arrow points to a multiparticle phagosome; arrowhead points to a single particle phagosome.

View larger version (30K): [in a new window]   Fig. 6. Overexpression of DN Rab7 blocks delivery of -mannosidase and LmpA to phagosomes. In (A), vesicles of the endo/lysosomal system were purified by pulsing control cells with iron dextran for 60 minutes and purifying iron dextran-containing vesicles using a magnetic column (see Materials and Methods). Western blots of cell homogenate (H) and endo/lysosomal vesicles (E/L) were decorated with antibodies to LmpA (lysosomal integral membrane protein), 100 kDa and 41 kDa subunits of the V-ATPase (100 kDa S.U. and 41 kDa S.U.), -mannosidase (-mann.), cathepsin D (Cath. D), cysteine proteinase P36 (CPp36) and Rab7 as described in Materials and Methods. In (B), phagosomes were purified as described in Materials and Methods from cells pulsed with latex beads for 5 minutes and chased for the indicated periods of time. Western blots were performed as in A. Bottom strip, cysteine proteinase activity of phagosomal fractions. In (C), phagosomes were purified from control cells (x4) and cells overexpressing DN Rab7 (7TN) pulsed with latex beads for 15 minutes. Western blots were performed as in A. The positions of marker proteins (kDa) are shown.

View larger version (83K): [in a new window]   Fig. 7. The silver stain profile of proteins from phagosomes isolated from cells overexpressing DN Rab7 lacks bands found in phagosomes isolated from control cells. Phagosomes were purified as described in Materials and Methods from control cells and cells overexpressing DN Rab7 pulsed with latex beads for 30 minutes. Phagosomal proteins were separated by SDS-PAGE and silver stained as described in Materials and Methods. H, whole cell homogenate; X4, control phagosomes; DN7, phagosomes from cells overexpressing DN Rab7. Arrows denote bands missing from the DN Rab7 phagosome protein profile. The positions of marker proteins (kDa) are shown.

View larger version (66K): [in a new window]   Fig. 8. Both the banding pattern of cysteine proteinase activity from purified phagosomes and the banding pattern of a western blot decorated with an antibody to GlcNac-1-P suggest that immature cysteine proteinases are delivered to phagosomes in cells overexpressing DN Rab7. (A) Proteins from phagosomes purified from control cells and cells overexpressing DN Rab7 were separated by SDS-PAGE on gels made with 0.2% gelatin. Phagosomes were formed by pulsing cells with latex beads for 30 minutes. Cysteine proteinase activity was visualized as described in Materials and Methods. + indicates samples that were pre-incubated with E-64. H, whole cell homogenate; P, phagosomes; X4, control and 7TN, DN Rab7 overexpressors. (B) Western blots of whole cell lysates and purified phagosomes from control cells and cells overexpressing DN Rab7 were made using the GlcNac-1-P-recognizing antibody AD7.5. Phagosomes were formed by pulsing cells with latex beads for 30 minutes. Abbreviations as for A. The positions of marker proteins (kDa) are shown.

View larger version (10K): [in a new window]   Fig. 9. The formation of spacious phagosomes is not blocked by overexpression of DN Rab7. Control cells and cells overexpressing DN Rab7 were fed a mixture of FITC-labeled bacteria and Crimson Red latex beads for 10 minutes, washed and then chased for 120 minutes. During the last 10 minutes of chase, cells were allowed to attach to coverslips and then imaged by LSCM, as described in Materials and Methods. The mean number of phagosomes labeled by both crimson red beads and FITC-bacteria per cell were plotted (n=3). Values are means ± s.e.m. P<0.05 for X4 versus LY294002. X4, control cells; 7TN, cells overexpressing DN Rab7; LY294002, cells incubated in the presence of 20 µM LY294002.

View larger version (12K): [in a new window]   Fig. 10. The regulation of phagosomal pH is aberrant in cells overexpressing DN Rab7. Control cells (closed circles, X4) and cells overexpressing DN Rab7 (open circles, 7TN) were pulsed for 10 minutes with FITC-labeled E. coli, washed and then chased to various time points in growth medium without bacteria. The internalized FITC-bacteria were used as a pH-sensitive probe to determine the pH of the phagosomes in vivo (see Materials and Methods). Values are means ± s.e.m.; n=4 for X4 and n=6 for 7TN. P<0.05 for 15, 30, 45 and 90 minute time points.

View larger version (25K): [in a new window]   Fig. 11. A model proposing the role of Rab7 in regulating phagosomal maturation. The model depicts that Rab7 is responsible for transporting vesicles containing glycosidases and LmpA from post-lysosomes (the most distal endosomal compartment in Dictyostelium formed by lysosome fusion) to either early endosomes or newly formed phagosomes. Transport to phagosomes might be direct (route 2) or indirect (route 1) where the glycosidases and LmpA are transported to early endosomes that in turn fuse with newly formed phagosomes. Rab7 is proposed not to regulate delivery of cysteine proteinases that reside in compartments distinct from those containing glycosidases. Rab7 also does not appear to regulate fusion of phagosomes to form spacious phagosomes, a role played by PI 3-kinase and PKB. Endo, endocytosis of fluid; Exo, exocytosis of fluid; E/L=endo/lysosomal vesicles; PL, post-lysosomes; EP, early phagosomes; MP, mature phagosomes; LP, late spacious phagosomes; CP, cysteine proteinases; G, glycosidases; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B.