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doi: 10.1242/10.1242/jcs.00050

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RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion

Department of Microbiology and Immunology and Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA 71130, USA

View larger version (36K): [in a new window]   Fig. 1. RabD has a higher homology to Rab 14 than to Rab4. The amino-acid sequence of human Rab 14, Dictyostelium RabD and human Rab4 were compared using the BLAST program available on the NCBI database. Vertical lines represent identity and dotted lines represent similarity.

View larger version (45K): [in a new window]   Fig. 2. RabDQ67L expression results in the formation of large vacuolar structures. Ax4 cells were transformed with a plasmid containing the HA-tagged rabD constitutively active mutant cDNA driven by an inducible promoter that is turned off in the presence of folate or turned on in the absence of folate. The level of RabD expression was analyzed by western blot using anti-RabD antibodies (inset). The top panel indicates that the growth rate for wild-type and mutant cells are identical. Bar, 3 µm.

View larger version (100K): [in a new window]   Fig. 3. The enlarged endocytic vesicles in cells expressing RabQ67L are primarily lysosomes. Both wild-type cells (A-F) and RabDQ67L-expressing cells (G-L) were pulsed with FITC-dextran for 5 minutes and chased for 0 minutes to view macropinosomes (A,B,G,H); chased for 15 minutes to view lysosomes (C,D,I,J); and 60 minutes to view post-lysosomes (E,F,K,L). Cells were gently fixed and viewed using a fluorescence microscope. Bar, 2 µm. The arrows reveal structures described in the text.

View larger version (56K): [in a new window]   Fig. 4. Most of the enlarged vesicles are acidic lysosomes in RabDQ67L-expressing cells. In A, both wild-type () and RabDQ67L () cells were pulsed with FITC-dextran, washed and chased in fresh HL-5. At the indicated time points, cells were harvested, washed and resuspended in cold 50 mM MES buffer, pH 6.5. The ratio of fluorescence of FITC-dextran at 525 nm emission upon excitation at 495 nm/450 nm were measured against an in vitro standard curve from which the pH was extrapolated (n=3). Normal size lysosomes in the wild-type Ax4 cells (B,C) and the enlarged lysosomes in the RabDQ67L cells (D,E) were observed when live cells were incubated with the acidophilic dye LysoSensor DND-189 (Molecular Probes). Bar, 2 µm. Arrows reveal enlarged vesicles in D and E.

View larger version (70K): [in a new window]   Fig. 5. GFP-RabDQ67L rings primarily enlarged lysosomes but not post-lysosomes. Both wild-type cells expressing GFP-RabDwt and cells expressing GFP-RabDQ67L (A-F) and RabDQ67L (G-L) cells were pulsed with Texas Red dextran for 5 minutes and chased for 0 minutes to view macropinosomes (A,B,G,H); chased for 15 minutes to view lysosomes (C,D,I,J); and 60 minutes to view post-lysosomes (E,F,K,L). Cells were gently fixed and viewed using a fluorescence microscope using GFP and rhodamine filter sets. GFP-RabD(WT) is normally predominately localized to the contractile vacuole (B,D,F), whereas GFP-RabDQ67L rings large vesicles (H,J,L) that accumulate fluid with the kinetics of lysosomes. Bar, 2 µm. The arrows in G and H reveals a macropinosome, whereas the arrows in I and J reveal enlarged lysosomes.

View larger version (76K): [in a new window]   Fig. 6. The enlarged vesicles in the RabDQ67L-expressing cells are the result of an increase in the rate of fusion between newly formed endo-lysosomes. Wild-type (A,C,E,G) and RabDQ67L-expressing (B,D,F,H) cells were first pulsed with RITC-dextran for 5 minutes (C,D), washed, pulsed with FITC-dextran for 5 minutes (E,F), washed, chased for 5 minutes and gently fixed with formaldehyde. Captured images of cells were viewed in the fluorescein, rhodamine and merged channels. The extent of fusion between endo-lysosomes containing both fluors was measured in the wild-type (G) and RabDQ67L-expressing (H) cells by detecting colocalization of both fluors in the same vesicle. Bar, 2.5 µm. The colored arrows indicate FITC-dextran-positive, RITC-dextran-positive and FITC/RITC-dextran-positive vesicles.

View larger version (14K): [in a new window]   Fig. 7. RabD regulates phagocytosis in Dictyostelium Ax4 cells. In A, the rate of phagocytosis was measured using fluorescent latex beads (F-8816) as a phagocytic marker. Cells were shaken in suspension, and for each time point, 3x106 cells were harvested, washed and lysed. Bead fluorescence was measured at an excitation of 625 nm and emission of 645 nm. B depicts the number of beads that were internalized at the 30 minute time point of the phagocytosis assay. Both mutants had statistically significant differences in the number of beads internalized compared with the control. (RabDQ67L: P<0.0001; RabDN121I: P<0.001).

View larger version (59K): [in a new window]   Fig. 8. Phagosome-phagosome fusion rates are increased in cells expressing RabDQ67L. Control (left panels), RabDQ67L (middle panels) and RabDQ67L treated with 20 µM LY294002 (right panels) cells were incubated with FITC bacteria for 10 minutes, washed and chased for 60 minutes. Cells were gently fixed and phase contrast and fluorescent images were taken using a fluorescence microscope. Bar, 3 µm.

View larger version (85K): [in a new window]   Fig. 9. RabDQ67L promotes fusion of separate phagosomes containing bacteria and beads. Both Ax4 and RabDQ67L-expressing cells were pulsed with FITC-bacteria for 10 minutes, washed and pulsed with deep blue latex beads (L-1398) for 10 minutes in shaking suspension. After the sequential pulse periods, the cells were washed and allowed to chase in fresh HL-5 while settling on coverslips for 30 minutes. The cells were gently fixed and images were taken of control cells (A,B) and RabDQ67L cells (C,D) in the GFP and rhodamine channels of a fluorescent microscope. The scale bar represents 3 µm. The red and green arrows reveal separate latex bead and bacterial phagosomes. The yellow arrow marks a phagosome that has beads and bacteria.

View larger version (14K): [in a new window]   Fig. 10. Phagosomes fuse at a higher rate in cells expressing RabDQ67L, and this is dependent on PI 3-kinase activity. In A, control and RabDQ67L cells incubated with or without the PI 3-kinase inhibitor, 20 µM LY294002 (Calbiochem), were pulsed for 10 minutes with FITC-bacteria in shaking suspension, washed and allowed to chase for 60 minutes in fresh HL-5 on coverslips. After a gentle fixation, phase contrast and fluorescent images were taken at 1000x magnification. The number of phagosomes within each cell and the number of bacteria within each phagosome were counted in a pool of at least 20 cells. A fusion event is defined as FE=n-1, where FE is a fusion event and n equals the number of bacteria within the phagosome. Differences in FEs between control and RabDQ67L cells were very significant (P<0.0001). FEs between control and RabDQ67L cells + LY294002 were not significantly different (P=0.2623). B depicts the comparison of fusion events between control and RabDN121I-expressing cells in the same conditions described above. The difference in the number of fusion events between the two strains is significant (P=0.0232).

View larger version (65K): [in a new window]   Fig. 11. GFP-RabDQ67L rings multiparticle phagosomes. Cells expressing GFP-RabDQ67L were incubated with RITC-labeled bacteria for 10 minutes in shaking suspension, washed and chased in fresh HL-5 while settling on coverslips. Live cells were examined using a fluorescent microscope in which images were taken in the rhodamine channel (A,C) and GFP channel (B,D). Bar, 3 µm. The arrows mark multiparticle phagosomes ringed with GFP-RabD.

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