A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis. Since Rab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation. Expression of GFP-Rab7 resulted in labeling of both early and late phagosomes containing yeast, but not forming phagocytic cups. In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal system and maturing phagosomes, latex bead containing (LBC) phagosomes were purified from wild-type cells at various times after internalization. Glycosidases, cysteine proteinases, Rab7 and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. LBC phagosomes isolated from cells overexpressing dominant negative (DN) Rab7 contained very low levels of LmpA (lysosomal integral membrane protein) and α-mannosidase was not detectable. Interestingly, cysteine proteinases were delivered to phagosomes as apparent pro-forms in cells overexpressing DN Rab7. Despite these defects, phagosomes in cells overexpressing DN Rab7 matured to form multi-particle spacious phagosomes, except that these phagosomes remained significantly more acidic than control phagosomes. These results suggested that Rab7 regulates both an early and late steps of phagosomal maturation, similar to its role in the endo/lysosomal system.
The Rab small molecular mass GTPases have taken center stage as master regulators of vesicle trafficking events (Somsel and Wandinger-Ness, 2000). These proteins are thought to regulate formation of transport vesicles from donor compartments, transport of membrane vesicles on cytoskeletal structures, tethering of vesicles to acceptor compartments and fusion of lipid bilayers. Rab proteins are activated in the GTP-bound state and interact with effectors until GTP hydrolysis has occurred (Bourne, 1988).
As the hypothesis that Rab proteins regulate the specificity of fusion events would predict (though it is now clear that other factors such as membrane tethers act in concert with Rabs to provide specificity), individual Rab proteins have been localized to specific compartments in the endo/lysosomal pathway. For instance, Rab4 and Rab5 have been localized to early endosomes. Rab5 regulates fusion of clathrin-coated vesicles with early endosomes and homotypic fusion of early endosomes (Bucci et al., 1992; Barbieri et al., 1994), while Rab4 regulates recycling of membrane receptors to the plasma membrane (van der Sluijs et al., 1991; van der Sluijs et al., 1992; Daro et al., 1996). Rab7 and Rab9 have been localized to late endosomes (Chavrier et al., 1990; Soldati et al., 1995), where Rab9 regulates recycling of membranes back to the Golgi complex (Lombardi et al., 1993).
Reports have suggested that Rab7 plays a role in vesicular transport from early endosomes to late endosomes (Feng et al., 1995; Mukhopadhyay et al., 1997; Vitelli et al., 1997), and in yeast (Rab7 homolog Ypt7), from late endosomes to the vacuole (Wichmann et al., 1992; Schimmoller and Riezman, 1993). While most data suggest that Rab7 regulates trafficking from early endosomes to late endosomes, two papers propose that GTPase inactive forms of Rab7 localize predominantly to lysosomes and, in one case, that expression of a dominant negative (DN) form of Rab7 causes lysosomes to disperse from their perinuclear location in Hela cells (Bucci et al., 2000; Meresse et al., 1995). Interactions between late endosomes and lysosomes have been suggested to occur through formation of a hybrid organelle, which can then undergo fission events to result in reformation of late endosomes and lysosomes (Pryor et al., 2000; Mullock et al., 1998). Though evidence points to the involvement of a Rab protein, it is not clear whether Rab7 or some other Rab protein may be involved.
A cDNA with 85% identity to human Rab7 has been cloned from Dictyostelium and indirect immunofluorescence has shown that Rab7 associates with endosomes and post-lysosomes (Buczynski et al., 1997). The endo/lysosomal system of Dictyostelium consists of a linear pathway including endosomes, lysosomes and a terminal secretory compartment known as a post-lysosome (Padh et al., 1993). Rates of phagocytosis and endocytosis are lower in a cell line overexpressing DN Rab7 (T22N mutation) (Buczynski et al., 1997). In addition, the rate of fluid-phase efflux is slower, while paradoxically lysosomal enzymes are over-secreted (Buczynski et al., 1997), and these cells also contain vacuoles the size of post-lysosomes that are acidic (post-lysosomes are normally close to neutral pH). It was hypothesized that Rab7 regulates membrane and hydrolase recycling from post-lysosomes to an earlier endosomal compartment since cells overexpressing wild-type Rab7 retain lysosomal enzymes more efficiently, while rates of fluid-phase efflux are increased. This recycling event may be necessary for proper maturation of post-lysosomes; thus, expression of DN Rab7 slows efflux, and results in a membrane ‘traffic jam’, which in turn slows the internalization pathways. Consistent with these observations is a report demonstrating that Rab7 regulates homotypic fusion events between early endosomes (Laurent et al., 1998). Thus, Rab7 might direct recycling vesicles to early endosomes and may also regulate homotypic interactions between early endosomes.
We were interested in pursuing the role of Rab7 in phagocytosis and phagosomal maturation, since expression of DN Rab7 inhibited phagocytosis of latex beads, and subcellular fractionation experiments demonstrated that Rab7 was enriched in early phagosomes (Buczynski et al., 1997). A role for Rab7 in phagosomal maturation in mammalian cells has not been described, though Rab7 does localize to phagosomes (Rabinowitz et al., 1992; Desjardins et al., 1994). Rab5 has been the key protein described to regulate interactions of early endosomes and late endosomes with phagosomes, though it is unclear which Rab might regulate fusion of lysosomes with phagosomes (Alvarez-Dominguez et al., 1996; Funato et al., 1997; Jahraus et al., 1998). Furthermore, pathogens such as Listeria and Salmonella have mechanisms to recruit Rab5 to the phagosomal membrane and somehow delay subsequent maturation steps (Hashim et al., 2000; Alvarez-Dominguez et al., 1996; Alvarez-Dominguez et al., 1997). Thus, the role of Rab proteins during phagosomal maturation is important and much remains to be discovered.
In Dictyostelium, phagosomal maturation consists of an early acidic phase followed by a late non-acidic phase, similar to that seen in the endo/lysosomal system (Rupper et al., 2001). Early phagosomes are acidified to a pH of approximately 5, while late phagosomes have a pH of over 6. Concomitant with the rise in pH, mature phagosomes fuse to give rise to late, spacious phagosomes. We have found that an increase in phagosomal pH and fusion of mature phagosomes requires phosphatidylinositol 3-kinase activity, as cells with a disruption in both ddpik1 and ddpik2 (two homologs of mammalian class I PI 3-kinases) and cells inhibited with PI 3-kinase inhibitors were blocked in these functions (Rupper et al., 2001). Furthermore, a cell line with a genetic disruption of PKB (a Dictyostelium protein kinase B homolog) was also blocked in these steps, suggesting that PI 3-kinases act through a PKB dependent pathway (Rupper et al., 2001).
Other work has demonstrated that proteins from the endo/lysosomal system are rapidly delivered to nascent phagosomes in Dictyostelium (Rezabek et al., 1997) and that two types of lysosomes may sequentially fuse with nascent phagosomes containing bacteria (Souza et al., 1997). These two types of lysosomes contain hydrolases with distinct sugar modifications (see below).
In the present study, we sought to answer the following questions. Does Rab7 direct internal membrane to the phagosomal cup in Dictyostelium, accounting for its role in phagocytosis? Does Rab7 regulate phagosomal pH changes or delivery of endo-lysosomal proteins to maturing phagosomes?
MATERIALS AND METHODS
Cells and culture conditions
D. discoideum, strain Ax4, was grown axenically at 18°C in HL5 growth medium (1% Oxoid proteose peptone, 1% glucose, 0.5% yeast extract, 2.4 mM Na2HPO4, and 8.8 mM KH2PO4, pH 6.5) either in shaking suspension or in tissue culture flasks. Construction of the Ax4 strain overexpressing DN Rab7 was described in a previous publication (Buczynski et al., 1997). In brief, site-directed mutagenesis was performed to generate a Thr 22 to Asn mutation in the Rab7 cDNA. The resulting cDNA was cloned into the pDA80-HA vector behind and in-frame with the flu hemagglutinin epitope, transformed into Dictyostelium and G418 resistant colonies were selected and screened for overexpression by western blot analysis. A cell line expressing green fluorescent protein (GFP) fused in-frame to the N terminus of Rab7 was generated as follows. A cDNA encoding GFP with the following mutations, F64L and S65T, was cloned into the KpnI site of pVEIIΔATG (Rebstein et al., 1993). The full-length cDNA encoding wt Rab7 was cloned into the SacI site of the resulting plasmid. The resulting plasmid was purified using (Qiagen Inc., Valencia, CA, USA) Qiafilter Maxi Prep columns and was transformed into D. discoideum, Ax3 strain, by the calcium phosphate precipitation method (Sadeghi et al., 1988). Geneticin (Sigma, St Louis, MO, USA) resistant colonies were screened for expression of GFP fluorescence by observation with a fluorescent microscope. Construction of the GFP-ABD expressing cell line was described in Pang et al. (Pang et al., 1998) and was a kind gift of Dr David Knecht.
Magnetic purification of the vesicles from the endo/lysosomal system
Ax3 cells were harvested during log-phase growth between 3-5×106 cells/ml, centrifuged at 1000 g for 5 minutes and resuspended at 2.0×108 cells/ml in HL5. The cells were then fed dextran-coated colloidal iron at 3 mg/ml for 60 minutes to load vesicles of the endo/lysosomal system. Iron-containing vesicles were purified as described elsewhere (Rodriguez-Paris et al., 1993).
Purification of latex bead containing phagosomes
Control cells or cells overexpressing DN Rab7 were harvested during log-phase growth between 3-5×106 cells/ml, centrifuged at 1000 g for 5 minutes and resuspended at 2.5×107 cells/ml in HL5. The cells were allowed to recover for 10 minutes prior to addition of latex beads. Latex beads (0.8 μm diameter, blue-dyed; Sigma, St Louis, MO, USA) were added at a concentration of 100 beads/cell and incubated with the cells for various periods. The cells were then washed 4 times with cold HL5 by centrifugation at 1000 g for 5 minutes. The cells were then resuspended at 2.5×107 cells/ml in HL5 and chased to various time points in shaking suspension. At each time point, 5.6×108 cells were harvested, washed once in cold homogenization buffer (100 mM sucrose, 5 mM glycine, pH 8.5) and the pellet was kept on ice. The cell pellets were resuspended in 2.8 ml of cold homogenization buffer supplemented with protease inhibitors (0.1 mM TLCK, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 5 μg/ml aprotinin and 0.5 mM phenyl methyl sulphonyl fluoride, final concentrations; Sigma, St Louis, MO, USA) and the cells were broken by passage through doubled, 5 μm pore size, polycarbonate filters (Osmonics, Inc.). Protease inhibitors were not included in preparations where protease activity was to be measured. Phagosomes were prepared from cell homogenates as described.
Western blot and protease activity analysis
Equivalent amounts of PNS and phagosome protein samples were loaded onto gels, and separated by SDS-PAGE using the discontinuous buffer system of Laemmli (Laemmli, 1970). In the case of silver stains, we used the Silver Stain Plus kit from Bio-Rad and followed the manufacturer’s recommended protocol. For western blots, proteins were electroblotted to nitrocellulose membranes in Towbin buffer at 100 V for 1 hour (Towbin et al., 1979). The membranes were blocked overnight with TBSTG buffer (10 mM Tris base, 150 mM NaCl, 0.05% (w/v) Tween-20, 0.1% (v/v) gelatin). All antibodies were diluted with TBSTG buffer. The following antibodies were used in this study: B30.2 (recognizes 70 kDa subunit of the V-ATPase specifically) (Fok et al., 1993), 1:100; B832 (recognizes 100 kDa subunit of the V-ATPase specifically) (Fok et al., 1993), 1:100; LSU 27-2 (recognizes α-mannosidase specifically) (J. Cardelli, unpublished results), 1:5000; LSU 18-2 (recognizes 41 kDa subunit of V-ATPase specifically) (Temesvari et al., 1996), 1:2000; 7-2-4 (recognizes Rab7 specifically) (Buczynski et al., 1997), 1:200; anti-Cathepsin D (recognizes cathepsin D specifically) (Journet et al., 1999), 1:200; anti-CPp36 (recognizes cysteine proteinase p36 specifically, a kind gift of J. Garin), 1:1000; anti-LmpA (a CD36, LIMP II homologue) (recognizes LmpA specifically) (Karakesisoglou et al., 1999), 1:2000 and anti-Glc-Nac-1-phosphate (Souza et al., 1997), 1:1000. After incubation with the antibody of interest and extensive washing, blots were incubated with goat anti-mouse (BioRad, 1:3000 dilution in TBSTG) or goat anti-rabbit (Sigma, 1:30,000 dilution), both alkaline phosphatase-conjugated secondary antibodies. The blots were developed in NBT buffer (100 mM Tris base, 100 mM NaCl, 5 mM Mg/Cl2) containing the alkaline phosphatase substrates BCIP (1.2 mM, Amresco) and Nitro Blue Tetrazolium (0.6 mM, Amresco).
Cysteine proteinase activity was detected following electrophoretic separation of phagosome protein samples as described above except that the polyacrylamide gels contained 0.2% gelatin (w/v) as a substrate for the proteinases and SDS-PAGE was performed at 4°C to prevent proteinase activity during migration. As a control, some PNS and purified phagosomal fractions were incubated with trans-epoxysuccinyl-L-leucylamido-(4-guanadino) butane (E-64, 10 μM) at room temperature for 30 minutes prior to being loaded on the polyacrylamide gel. Following electrophoresis, the gel was incubated in 2.5% Triton X-100 (v/v) with mild agitation for 30 minutes at room temperature, and then in 0.1 M sodium acetate/acetic acid buffer (pH 4.0) with 1 mM dithiothreitol for 16 hours. The gels were then stained with Coomassie Brilliant Blue (0.25% (w/v) in 50% (v/v) methanol and 10% (v/v) acetic acid) and destained with a solution of 50% (v/v) methanol and 10% (v/v) acetic acid in order to visualize proteolyzed bands. Incubation of the samples with E64 inhibited protease activity completely except in the top two bands visible in the gel, suggesting that a majority of the visible activity was due to cysteine proteinases.
Immunofluorescence (IF) microscopy
1×106 cells were allowed to attach to glass coverslips for 10 minutes and then fixed with 2% formaldehyde in phosphate-buffered saline (PBS) containing one-third strength HL5 and 0.1% DMSO for 5 minutes at room temperature, followed by permeabilization at –20°C in 100% methanol containing 1% formaldehyde. LmpA was detected by staining with a 1:500 dilution of a rabbit polyclonal antibody to LmpA (Karakesisoglou et al., 1999) and α-mannosidase was detected by staining with a mouse monoclonal antibody 2H9 at a dilution of 1:50 (Bush and Cardelli, 1989). The primary antibodies were detected, after washing, with Texas Red-labeled donkey anti-mouse or FITC-labeled donkey anti-rabbit at a 1:100 dilution. Antibodies were diluted in PBS containing 2.5 mg/ml bovine serum albumin and 0.1% saponin. The cells were imaged with an Olympus BX50 fluorescence microscope using a CCD camera.
Cells were allowed to internalize a mixture of FITC-labeled E. coli and Crimson Red latex beads (Molecular Probes, Eugene, OR, USA) for 10 minutes in shaking suspension. The cells were then washed twice with growth medium and chased for 120 minutes in shaking suspension. 10 minutes prior to the end point cells were harvested and allowed to attach to glass coverslips. Images were captured immediately with a BioRad MRC-1000 confocal microscope using a 60× oil objective. Laser lines of 488 nm and 568 nm of the Krypton/Argon laser were used at 3% laser power to simultaneously excite FITC and Crimson Red, respectively. FITC fluorescence was imaged after passing through a 522±35 nm filter and Crimson Red fluorescence was imaged after passing through a 605±32 nm filter. The images were analyzed with IPLAB software (Scanalytics, Inc., Fairfax, VA, USA) and scored for the total number of phagosomes labeled by both FITC-bacteria and Crimson Red latex bead fluorescence per cell. Since FITC was cleaved from the bacterial surface by the end point and thus filled the phagosome with fluorescence, phagosomes were scored based on colocalization of FITC and Crimson Red fluorescence.
Fluorescent labeling of bacteria and yeast
E. coli from a stationary-phase culture were washed once with 0.1 M NaHCO3, pH 9.0, and resuspended in 0.1 M NaHCO3, pH 9.0, with 1 mg/ml FITC Isomer 1 (Sigma) for 1 hour at 25°C with gentle stirring. The labeled E. coli were then washed with PBS until the supernatant was free of visible FITC. The bacteria were resuspended in a suitable volume of PBS and stored at 4°C until needed. Yeast from a stationary-phase culture were washed once with PBS and then heat-killed by boiling for 15 minutes. The yeast were then washed with 0.1 M NaHCO3, pH 9.0, and resuspended in 0.1 M NaHCO3, pH 9.0, with 1 mg/ml Rhodamine B isothiocyanate (Sigma) for 1 hour at 25°C with gentle stirring. The labeled yeast were then washed with PBS until the supernatant was free of visible Rhodamine.
Measurement of phagosomal pH
Control cells or cells overexpressing DN Rab7 were harvested in log-phase growth in shaking suspension and resuspended at 5×106 cells/ml in HL5 and allowed to recover 10 minutes. Control cells and cells overexpressing DN Rab7 were pulsed with FITC-labeled E. coli for 10 minutes in shaking suspension. The cells were then diluted with ice-cold HL5 and centrifuged at 1000 g at 4°C; this was repeated 3 times, followed by resuspension at 5×106 cells/ml in room temperature HL5. At each time point 5×106 cells were harvested and washed twice with ice-cold HL5 and once with ice-cold 50 mM MES (pH 6.5) buffer. The cells were resuspended in 2 ml of MES buffer and fluorescence was measured with a Hitachi fluorimeter (Model F-4010) at 520 nm following excitation at 450 nm and 495 nm. The ratio of fluorescence emission at 450 nm and 495 nm was calculated and used to determine the intraphagosomal pH by extrapolation from an in vitro standard curve determined by measuring the fluorescence ratio of FITC-labeled E. coli in buffers from pH 4-7.
GFP-Rab7 localizes rapidly to nascent phagosomes as actin is removed
It has been reported that Rab GTPases might regulate exocytosis of internal membrane to the phagosomal cup and thus play a direct role in regulating phagocytosis (Bajno et al., 2000; Hackam et al., 1998; Cox et al., 2000). Since expression of DN Rab7 in Dictyostelium causes a decrease in phagocytosis (30% of control rate) and Rab7 was found enriched in purified phagosomal membranes after a short pulse (2 minutes) with latex beads, we hypothesized that Rab7 might regulate delivery of internal membrane to the phagosomal cup (Buczynski et al., 1997). To test this hypothesis, we generated a Dictyostelium cell line that expresses a green fluorescent protein N-terminal fusion with Rab7 (see Materials and Methods). This cell line was allowed to internalize live yeast for 10 minutes, then fixed with 1% formaldehyde, or the cells were washed free of uninternalized yeast and incubated in yeast-free growth medium for 120 minutes before fixation. Images taken by fluorescence microscopy clearly demonstrated that yeast-containing phagosomes were ringed with GFP-Rab7 at both early and late time points (Fig. 1). Many other vesicular structures from the endo/lysosomal system were ringed with GFP-Rab7 and this labeling pattern agreed nicely with previous published results of indirect immunofluorescence using a specific antibody to Rab7 (Buczynski et al., 1997).
These results confirmed that Rab7 localizes to early phagosomal membranes. We next determined the kinetics of delivery of GFP-Rab7 to phagosomal membranes by performing confocal videomicroscopy with living cells. Cells expressing GFP-Rab7 were allowed to attach to glass coverslips and then challenged with heat-killed yeast labeled with rhodamine B isothiocyanate (RITC-yeast). A cell with bright GFP-Rab7 expression was then brought into focus in a plane that also contained RITC-yeast and images were captured every 5 seconds. Contrary to our expectations, no detectable GFP-Rab7 localized to the base of forming phagosomal cups, nor could any association of GFP-Rab7 with the plasma membrane be found, but GFP-Rab7 localized rapidly to phagosomes within 60 seconds following internalization (Fig. 2).
We were able to gain more insight into the timing of GFP-Rab7 association with phagosomal membranes by studying the dynamics of association of filamentous actin (F-actin) during particle internalization. Previous studies have shown that F-actin is required for phagocytosis in Dictyostelium and that F-actin-binding proteins transiently associate with early phagosomal membranes (Maniak et al., 1995; Rezabek et al., 1997). We sought to determine if the removal of F-actin coincided with the delivery of GFP-Rab7 to the phagosomal membrane. We again used confocal video microscopy to visualize the association of the actin cytoskeleton with forming phagosomes in a cell line that strongly expresses a GFP fusion with the actin-binding domain (GFP-ABD) of ABP-120 (actin binding protein 120), an actin cross-linking protein in Dictyostelium. It has been shown that the staining pattern of GFP-ABD mimics that of fluorescent phalloidin, strongly suggesting that GFP-ABD only bound to F-actin in the cell (Pang et al., 1998).
By examining images captured by confocal microscopy of GFP-ABD cells internalizing RITC-yeast, we found that GFP-ABD strongly labeled the forming phagocytic cup and completely ringed the newly internalized phagosomes (Fig. 3). Once the phagosome closed, GFP-ABD was rapidly removed from the phagosomal membrane within 30-60 seconds of phagosomal closure. In some cases it appeared that GFP-ABD was more rapidly removed from the hemisphere of the phagosome facing the cytoplasm than it was from the surface of the phagosome adjacent to the cell cortex. We cannot conclude from these images that removal of F-actin was complete or that F-actin does not reassociate with phagosomes later during maturation. The timing of removal of the actin cytoskeleton from maturing phagosomes coincides with the association of GFP-Rab7 with phagosomal membranes, as described above.
Endo-lysosomally localized proteins are rapidly delivered to maturing phagosomes
Since Rab7 did not appear to play a direct role in the internalization of phagocytic particles, we hypothesized that DN Rab7 might slow the rate of phagocytosis by blocking phagosomal maturation. Rab7 could be involved in the removal of actin from the phagosomal membrane, or more likely, involved in the delivery of components from the endo/lysosomal system to nascent phagosomes. We chose to look at delivery of components from the endo/lysosomal system to phagosomes by purifying latex bead-containing phagosomes on sucrose step gradients.
Previous studies had demonstrated that latex bead-containing phagosomes can be purified to high purity from Dictyostelium and that those membranes were minimally contaminated by enzymes from the endoplasmic reticulum and Golgi complex (Rezabek et al., 1997). Since phagosomes fuse with endosomes and lysosomes, it was difficult to demonstrate that purified phagosomes were free from contaminating endosomes and lysosomes or other membranes of the endo/lysosomal system. We performed the following experiment to determine if purified latex bead-containing phagosomes were indeed free from endosome and lysosome contamination. First, we found that latex bead-containing phagosomes could be prepared from unlabeled cells broken in the presence of 35S-methionine labeled cells (which had not ingested beads), without significant contamination from labeled proteins (Fig. 4A). This strongly suggested that other organelles from labeled cells, such as endosomes and lysosomes, which would have contained 35S-methionine labeled proteins, did not copurify with latex bead-containing phagosomes. As expected, latex bead-containing phagosomes from 35S-methionine labeled cells gave an enrichment of protein bands (analyzed by SDS-PAGE) when compared to the protein banding pattern of whole cell extracts from the same cells (Fig. 4B). The pattern of proteins observed was comparable to those in previously published gels. Finally, when added to broken 35S-labeled cells, latex beads bound many labeled proteins, but the protein banding pattern observed by SDS-PAGE was clearly different from the pattern observed from purified latex bead-containing phagosomes (Fig. 4C). These data suggested that cell breakage did not compromise the integrity of the phagosomal membrane and that latex bead-containing phagosomes could be purified with minimal contamination from other membranes. This was further substantiated by electron microscopy of sections of one of the embedded purified preparations. Fig. 5 demonstrates that the purified phagosomes were minimally contaminated with membranes that did not contain an enclosed latex bead.
We next determined the kinetics of delivery of various proteins from the endo/lysosomal system to maturing phagosomes in control cells. Western blot analysis (Fig. 6A) indicated that the following proteins were significantly enriched in lysosomes prepared from control cells by magnetic fractionation (see Materials and Methods for details): LmpA (lysosomal integral membrane protein) (Karakesisoglou et al., 1999), α-mannosidase (lysosomal glycosidase), the 41 and 100 kDa vacuolar proton pump subunits (s.u.), Rab7, cathepsin D (proteinase) and CPp36 (cysteine proteinase). Phagosomes were purified from cells pulsed for 5 minutes with latex beads, washed free of non-ingested beads and then chased for up to 2 hours in growth medium (Fig. 6B). Equal amounts of phagosomal proteins and whole cell homogenate were separated by SDS-PAGE and then visualized by western blot analysis. All of the lysosomally associated antigens tested were delivered to maturing phagosomes rapidly, all being enriched even at 0 minutes of chase. The kinetics of delivery of these proteins fell into two classes: (1) proteins that were delivered rapidly and remained at constant levels during the chase period (LmpA, proton pump subunits, α-mannosidase, Rab7 and cathepsin D) and (2) proteins that, though enriched in phagosomes initially at time zero of chase, reproducibly accumulated to higher levels during the chase (CPp36 and cysteine proteinase activity). Interestingly, the proteins belonging to the second class were all proteases (CPp36 and cysteine proteinase activity), while lysosomal membrane proteins and α-mannosidase fell into the first class. Comparable results were observed in three independent experiments (results not shown).
Rab7 function is required for delivery of proteins from the endo/lysosomal system to early phagosomes
In order to test the hypothesis that Rab7 might be required for trafficking of proteins from the endo/lysosomal system to early phagosomes, we purified latex bead-containing phagosomes from cells expressing DN Rab7 and compared the protein content of those phagosomes to phagosomes purified from control cells. Cells were allowed to ingest latex beads for 15 minutes and then latex bead-containing phagosomes were purified on sucrose step gradients. Equal protein loads were separated by SDS-PAGE and analyzed by silver staining. A comparison of banding patterns revealed that many bands present in control phagosomes were missing from DN Rab7 phagosomes (i.e. bands marked by arrows in Fig. 7). In light of this result, we tried to identify proteins that were not delivered to phagosomes in cells expressing DN Rab7 by performing western blots on purified phagosomes with antibodies to known proteins enriched in the endo/lysosomal system. We discovered that delivery of both LmpA and α-mannosidase was greatly reduced in cells expressing DN Rab7 (Fig. 6C). Interestingly, two subunits of the V-ATPase, the 100 kDa and 41 kDa subunits, were delivered to phagosomes in DN Rab7-overexpressing cells, though the amount of 41 kDa subunit delivered to phagosomes purified from mutant cells was 68.5±5.3% (mean ± s.d., n=2) the amount delivered to control phagosomes. Delivery of the 100 kDa subunit was only slightly decreased by less than 10%. The higher molecular mass band recognized by the Rab7 antibody was the overexpressed hemagglutinin-tagged DN Rab7. Identical results were also observed with longer chase points, suggesting that delivery of proteins was not simply delayed (results not shown).
Though cells expressing DN Rab7 oversecrete α-mannosidase (Buczynski et al., 1997), this did not appear to explain the complete lack of α-mannosidase delivery to phagosomes in these cells. Immunofluorescence microscopy of cells expressing DN Rab7 revealed that no mislocalization or detectable reduction of levels of α-mannosidase or LmpA was observed as compared to control cells (results not shown). Interestingly, LmpA and α-mannosidase did not colocalize to a large extent, but this is in agreement with previous published results where LmpA did not colocalize with another lysosomal hydrolase, α-fucosidase (Karakesisoglou et al., 1999).
Apparent immature forms of cysteine proteinases and proteins with GlcNac-1-P linked to serine residues are delivered to phagosomes in DN Rab7 expressing cells
The majority of cysteine proteinases (CPs) in Dictyostelium contain N-acetylglucosamine-1-phosphate (GlcNac-1-P) linked to serine residues (Mehta et al., 1996). These proteins lack mannose-6-phosphate (Man-6-P) modifications that are normally found on lysosomal enzymes such as α-mannosidase, and while CPs are enriched in a lysosomal compartment, proteins with Man-6-P oligosaccharides or GlcNac-1-P are found in distinct lysosomal compartments (Souza et al., 1997). We were, therefore, interested to determine if the delivery of CPs to phagosomes was also disrupted by expression of DN Rab7.
We first looked at proteinase activity in cellular lysates from control cells and cells expressing DN Rab7. Protease activity was visualized using protease gels, also known as zymograms (see Materials and Methods). Lysates from cells expressing DN Rab7 contained a number of protease activity bands, which were not present in control cell lysates (Fig. 8A). The majority of protease activity less than 35 kDa in molecular weight in these lysates was inhibited by trans-epoxysuccinyl-L-leucylamido-(4-guanadino)butane (E-64), a specific inhibitor of cysteine proteinases. The fact that this activity is present at pH 4.0 and can be inhibited by E-64 strongly suggests that this activity is due to cysteine proteinases. These proteases were enriched in phagosomes from control cells and cells overexpressing DN Rab7, and the differences in bands mimicked the differences seen in the whole cell lysates. DN Rab7 phagosomes yielded protease activity bands at higher molecular masses, such as the top band at the 50.8 kDa marker (Fig. 8A, lane marked 7TNP), which were absent in control phagosomes, while some lower molecular mass bands were absent in DN Rab7 phagosomes or decreased in activity compared to bands from control phagosomes (note bands close to the 28.1 kDa marker). This type of pattern suggests to us that these higher molecular mass bands may be due to unprocessed proteases and the missing lower molecular mass bands would correspond to the proteolytically processed mature forms of these enzymes that are absent. Note that incubation of immature proteinases at acidic pH in renaturing conditions could result in activation of these enzymes (see Discussion).
We also performed western blots with a monoclonal antibody specific for GlcNac-1-P (AD7.5), a modification found almost exclusively on cysteine proteinases, with similar results to those described above. There were a number of higher molecular mass bands present in whole cell lysates of DN Rab7 overexpressing cells that were absent in control cell lysates. DN Rab7 phagosomes contained an AD7.5 reactive band at 65-70 kDa that was absent from control phagosomes and DN Rab7 phagosomes lacked a doublet close to 45 kDa that was present in control phagosomes. Again, we believe this may be due to a defect in processing of proteins that contain GlcNac-1-P glycosylation, and not mistrafficking of proteins from the trans-Golgi to phagosomes.
Since DN Rab7 phagosomes appeared to be deficient in interactions with the endo/lysosomal system, we hypothesized that these phagosomes would be blocked in maturation. In Dictyostelium, phagosomal maturation consists of an early acidic phase followed by an increase in phagosomal pH and the formation of large, spacious phagosomes through homotypic fusion events (Rupper et al., 2001). Spacious phagosomes begin to appear after 90 minutes of internalization of bacteria or latex beads (Rupper et al., 2001). We tested whether phagosomes in cells expressing DN Rab7 could undergo homotypic fusion events by performing a laser scanning confocal microscope (LSCM)-based fusion assay. In this assay cells are fed a mixture of FITC-bacteria and fluorescent red beads for 10 minutes, washed extensively, and then chased for 120 minutes in medium without phagocytic probes. The cells are then imaged by LSCM and the images are scored for the number of double-labeled phagosomes/cell. We found that the rate of spacious phagosome formation in cells expressing DN Rab7 was not significantly inhibited (Fig. 9). Previous studies have demonstrated that PI 3-kinase inhibitors block spacious phagosome formation, and treatment of control cells with 20 μM LY294002 significantly inhibited phagosome fusion in this assay (Rupper et al., 2001). This suggested that adding a mixture of phagocytic probes did not result in a large number of phagosomes, which internalized phagocytic probes of both colors.
The progress of phagosomal maturation can be monitored by measuring the change in phagosomal pH over time. In control cells, nascent phagosomes are rapidly acidified and as they mature the internal pH rises, reaching a plateau after 90 minutes of maturation. We found that phagosomal pH regulation in cells overexpressing DN Rab7 was aberrant. Within the first 5 minutes, DN Rab7 phagosomes were acidified to the same level as control phagosomes, but then DN Rab7 phagosomes increased in pH more rapidly and were significantly less acidic at 15, 30 and 45 minute time points (Fig. 10). Interestingly, the phagosomal pH of DN Rab7 phagosomes reached a plateau of pH 5.9 by 20 minutes of maturation and was significantly more acidic than control phagosomes after 90 minutes of chase. This suggests that DN Rab7 phagosomes may indeed be blocked in phagosomal maturation, but apparently at a later stage than phagosome homotypic fusion. This also correlates with the fact that subunits of the V-ATPase may not be delivered to early phagosomes to the same level as is found in control phagosomes (Fig. 6C). This might be an explanation for the rapid increase in pH early on.
Though interest in the mechanisms regulating phagosomal maturation has increased, only few Rab GTPases have been studied in this process. Rab5, the best characterized GTPase, plays a pivotal role in mammalian phagosomal maturation by regulating phagosome fusion with early and late endosomes (Alvarez-Dominguez et al., 1996; Jahraus et al., 1998) and is also a target for disruption of phagosomal maturation by intracellular pathogens (Hashim et al., 2000; Alvarez-Dominguez et al., 1997). Nevertheless, other Rabs surely contribute to the process of phagosomal maturation and the destruction of internalized pathogens (Funato et al., 1997; Jahraus et al., 1998). In this paper we demonstrate that the Dictyostelium Rab7 homolog plays an important role in phagosomal maturation, probably regulating both early (select protein delivery) and late (pH regulation) steps. Rab7 associates rapidly with nascent phagosomes and regulates the delivery of a subset of lysosomal enzymes and membrane proteins to early phagosomes (the early step). Furthermore, apparently immature forms of cysteine proteinases are delivered to phagosomes in cells overexpressing DN Rab7, and although phagosomal maturation and fusion proceed, phagosomal pH regulation was aberrant in these cells (the late step).
While GFP-Rab7 did not associate with the forming phagosomal cup, thus potentially accounting for its role in regulating phagocytosis, it did rapidly associate with internalized phagosomes (Fig. 2). This confirms biochemical data that Rab7 was enriched on latex bead-containing phagosomes after a 2-minute pulse with latex beads (Buczynski et al., 1997). Interestingly, the arrival of GFP-Rab7 coincides with the removal of GFP-ABD from nascent phagosomes. We would speculate that removal of the F-actin coat facilitates or triggers the interaction of phagosomes with the endo/lysosomal system. Alternatively, the association of Rab7-containing membranes might trigger the disassociation of F-actin. We cannot conclude that removal of F-actin is complete or that F-actin does not associate with maturing phagosomes to facilitate late processing steps. In fact, others have found that F-actin associates with maturing phagosomes in a bimodal fashion (Defacque et al., 2000). The maturation of phagosomes would appear to be important in regulating internalization of bacteria since phagocytosis rates are lower in cells expressing DN Rab7.
The localization of GFP-Rab7 to early phagosomes suggested it played an important role in regulating interactions of early phagosomes with the endo/lysosomal system. As a first approach, we looked at the time course of delivery of a number of proteins from the endo/lysosomal system to phagosomes in wild-type cells, and found that all of these proteins were delivered rapidly to phagosomes, but did seem to fall into two kinetic classes (Fig. 6). Proteins such as α-mannosidase and subunits of the V-ATPase were delivered rapidly and remained enriched at the same level throughout the chase period, while cysteine proteinase activity and CP36 were enriched to higher levels at later time points in the chase. This pattern of delivery is consistent with the hypothesis that cysteine proteinases, which have GlcNac-1-P modifications and no Man-6-P modifications, are found in a separate endo/lysosomal compartment than lysosomal enzymes such as α-mannosidase, which contain Man-6-P modifications (Souza et al., 1997). Together, these data suggest that early endosomal compartments interact with phagosomes by different mechanisms. A recent report found that lysosomal proteins were also delivered with differing kinetics to phagosomes in mouse macrophages (Claus et al., 1998).
Inhibition of Rab7 function by expression of DN Rab7 blocked delivery of α-mannosidase and LmpA to early phagosomes (Fig. 6C). Interestingly, blockage of phagosome interaction with the endo/lysosomal system by overexpression of DN Rab7 was not complete, as subunits of the V-ATPase and cysteine proteinase activity were found in early phagosomal membranes from these cells. This suggests that Rab7 might regulate interaction of a subset of compartments from the endo/lysosomal system (possibly lysosomes), which interact early with phagosomes; an observation that is consistent with our data suggesting that a cysteine proteinase and cysteine proteinase activity are delivered more slowly to phagosomes than proteins such as α-mannosidase and the V-ATPase.
The complete absence of α-mannosidase in DN Rab7 phagosomes is not due to missorting of α-mannosidase, as cells overexpressing DN Rab7 process α-mannosidase normally (Buczynski et al., 1997). The cells do over-secrete the mature form of α-mannosidase, but substantial amounts of the protein remain within the cells, as observed by indirect immunofluorescence and western blotting (data not shown).
Although cysteine proteinase activity was delivered to phagosomes in cells overexpressing Rab7, the activity-banding pattern was different from that of control cells (Fig. 8). The presence of higher molecular mass bands and the absence of or decreased activity of lower molecular mass bands found in phagosomes from DN Rab7 cells as compared to phagosomes from control cells suggested that immature forms of cysteine proteases were being delivered to phagosomes in cells overexpressing DN Rab7. This pattern of cysteine proteinases was confirmed by western blots of control versus DN Rab7 phagosomes with an antibody to GlcNac-1-P. Though other proteins may have GlcNac-1-P modifications, it has been demonstrated that cysteine proteinases in Dictyostelium contain this as their sole sugar modification.
Cysteine proteinases are synthesized as pre-pro-forms, where the pre-peptide serves as a target for translocation into the ER and is cleaved during this process (Turk et al., 2000). Once these pro-enzymes have reached their targeted compartment, cellular proteases such as cathepsin D, or auto-proteolysis through an intermolecular mechanisms, results in cleavage of the pro-peptide and activation of the protease (Rowan et al., 1992; Kawabata et al., 1993; Podobnik et al., 1997). Presumably, the pro-enzyme becomes activated in the protease gel by incubation at low pH, which results in cleavage of the pro-peptide by other molecules of the same protease. For this reason we see activity in the gel where the pro-enzyme has migrated to.
There are two possible explanations for why the pro-enzymes might be unprocessed. First, in DN Rab7 overexpressing cells, the pro-enzymes might not be targeted to the proper endo/lysosomal compartment where they are normally processed. This seems unlikely because these enzymes get delivered to phagosomes. Though it is possible that pro-cysteine proteinases could be delivered directly from the trans-Golgi to phagosomes, the same machinery would also transport pro-cysteine proteinases to their endo/lysosomal compartment, thus, we would not expect transport to phagosomes without transport to the endo/lysosomal system. It is likely that the bulk of cysteine proteinases get delivered to phagosomes by interaction with the endo/lysosomal system.
The second possibility is that the conditions in the endo/lysosomal system are not correct for proper pro-enzyme processing in cells expressing DN Rab7. This could be due to one of the following reasons: (1) morphologic defects in the endo/lysosomal system, (2) the possibility that a cellular factor that catalyzes processing is over-secreted and not present at high enough concentrations in endosomes and lysosomes, or (3) defects in pH regulation of the endo/lysosomal system. The endo/lysosomal system of cells expressing DN Rab7 is morphologically different from that in control cells, consisting of large, acidic compartments the size of post-lysosomes. Control cells have both acidic endosomes and lysosomes and non-acidic post-lysosomes. Cells expressing DN Rab7 also oversecrete lysosomal enzymes. It may be that a combination of oversecretion of lysosomal enzymes and increased compartment volume causes defects in proteolytic processing of cysteine proteinases. In this situation, lysosomal enzymes would not be concentrated sufficiently for normal processing and lysosomal acidification might not be as great as in control cells. Once cysteine proteinases are delivered to phagosomes, they still do not get processed correctly. The above arguments probably also apply to DN Rab7 phagosomes because some lysosomal enzymes are not present and pH regulation of DN Rab7 phagosomes is clearly aberrant.
In conclusion, we propose that Rab7 regulates both an early and a late event during phagosomal maturation. The model shown in Fig. 11 proposes that during the early phagosomal maturation step, Rab7 regulates fusion of vesicles that primarily contain hydrolases (with Man-6-P modifications) and LmpA. These vesicles are either early endosomes (route 1) or vesicles recycling from post-lysosomes (route 2). This is consistent with the proposed role for Rab7 in endosome fusion events (Laurent et al., 1998) and endosomal recycling (Buczynski et al., 1997). Fusion of a second endosomal compartment, which primarily contains proteins with GlcNac-1-P modifications such as cysteine proteinases, and homotypic fusion of phagosomes, do not rely on Rab7 function. The regulation of phagosomal pH in cells expressing DN Rab7 is also aberrant although the delivery of the proton pump appears normal. Perhaps Rab7 regulates the delivery of a protein regulating the proton pump or ion channels that is not delivered correctly. Future experiments are being designed to further test these hypotheses.
The published research was supported by an NIH grant DK 39232 to J.C. The authors would like to thank Dr Michael Schleicher for the antibodies to LmpA, Dr Jerome Garin for the antibody to cathepsin D and the cysteine p36 protein, and Dr Hudson Freeze for the antibody to Glc-Nac-1-phosphate.