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Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium

¹ Department of Microbiology and Immunology, LSU Health Sciences Center, Shreveport, LA 71130, USA
² Department of Botany, Kyoto University, Kyoto 606-8502 Japan
³ Department of Biology, University of Arkansas at Little Rock, Little Rock, AR 72204, USA
^* Author for correspondence (e-mail: jmbush{at}ualr.edu )

View larger version (52K): [in a new window]   Fig. 1. Sequence alignments and Southern and northern blots of DdRab11. The nucleotide sequence of the DdRab11 gene was analyzed using MacVector software of IBI and the putative protein sequence is shown. The amino acid sequence for the Dictyostelium Rab11b, the human Rab11, human Rab25, and the yeast protein, YPT3, are also shown. Optimum amino acid alignment is shown in the figure.

View larger version (34K): [in a new window]   Fig. 2. Dictyostelium DdRab11 is associated with contractile vacuole membranes.(A,B) Post-nuclear extracts prepared from homogenized cells were fractionated on Percoll gradients as described in Materials and Methods. After fractionation, samples were subjected to SDS-PAGE followed by western blot analysis using antibodies to DdRab11 and RabD. DdRab11 protein was confined to the top fractions of the gradient that corresponded to non-lysosomal membranes vesicles. Bottom fractions represent lysosomal membrane fraction that contained lysosomal marker enzymes. (C,D) Protein samples (20 µg) from post-nuclear supernatant (PNS), purified contractile vacuole (light membranes) and purified lysosomes were subjected to SDS-PAGE and western blot analysis. DdRab11 and RabD were both enriched in contractile vacuole membranes, but only RabD was enriched in purified lysosomes.

View larger version (160K): [in a new window]   Fig. 3. DdRab11 co-localizes with the 100-kDa vacuolar ATPase in the contractile vacuole. Indirect immunofluorescence techniques were used to stain and visualize fixed cells for DdRab11 (A), ATPase (B,D) and RabD (C). Arrows denote reticular elements that contain DdRab11, RabD, and the 100 kDa proton pump subunit. Scale bars: 5 µm.

View larger version (74K): [in a new window]   Fig. 4. GFP-DdRab11 co-localizes with the 100 kDa proton pump subunit in the contractile vacuole. Cells expressing CV-localized GFP-DdRab11 were observed and photographed (A,B). Co-localization experiments in cells expressing GFP-DdRab11 are shown in C-E. (C) GFP-DdRab11; (D) Texas Red-tagged antibody staining of the 100 kDa ATPase proton pump protein. (E) A dual fluorescent image of both C,D that demonstrates complete co-localization of GFP-DdRab11 and the 100 kDa ATPase proton pump protein. Scale bar: 5 µm.

View larger version (132K): [in a new window]   Fig. 5. GFP-DdRab11 associates with expulsing contractile vacuoles. Images 1-12 represent a photographic sequence from a 12 second movie of unfixed cells expressing GFP-DdRab11. The sponguimal network and bladders are easily visualized by the GFP-tagged DdRab11. The CV at the six o'clock position is shown in the process of water expulsion.

View larger version (115K): [in a new window]   Fig. 6. GFP-DdRab11 is associated with newly forming CV bladders and CV bladders undergoing homotypic fusions. Images 1-12 represent a photographic sequence from a 12 second movie of unfixed cells expressing GFP-DdRab11. The dashed circle in images 1, 4 and 9 show the progression of a newly forming CV. The arrowheads in images 3, 4 and 8 show a homotypic fusion event between two CV, while in image 11, the fusion enlarged CV is shown to have split back into two smaller CVs. In addition, the double arrows in images 2 and 7 show two CVs undergoing water expulsion and network disengagement.

View larger version (105K): [in a new window]   Fig. 7. DdRab11 co-localizes with the calmodulin and RabD, but not the 100 kDa proton pump subunit in cells exposed to hyper-osmotic conditions. Cells growing on coverslips in HL5 were exposed to sucrose (100 mM) in HL5 for 1 hour. Cells were fixed with formaldehyde and decorated with antibodies to DdRab11 (A,E), RabD (C), calmodulin (B,D) and the proton pump subunit (F). Cells were visualized using a fluorescence microscope. White arrows denote a contractile membrane structure that contains DdRab11 and calmodulin proteins (A,B) and RabD and calmodulin (C,D). DdRab11 protein was found in reticular contractile vacuolar membranes when compared with the ATPase-positive vesicular structures accumulating in cells exposed to hyper-osmotic conditions. The white arrow shows the ATPase-positive DdRab11-negative region (F). Scale bar: 5 µm.

View larger version (114K): [in a new window]   Fig. 8. DdRab11 no longer co-localizes with RabD or the proton pump in cells exposed to hypo-osmotic conditions. Cells attached to coverslips were placed in distilled water for 20 minutes. Cells were fixed and decorated with antibodies to RabD (A), DdRab11 (C) and the proton pump subunit (B,D). Cells were viewed in a fluorescence microscope. White arrows (A,B) show co-localization of RabD and the proton pump. Arrowheads (C,D) show DdRab11 and the proton pump, respectively, not co-localized. The arrow in D indicates a swollen vesicle in a cell stained for the proton pump. The bright field image of this same cell is shown in E. Scale bar: 5 µm.

View larger version (119K): [in a new window]   Fig. 9. Both DdRab11 and RabD no longer associates with the proton pump following treatment of cells with the ATPase inhibitor concanamycin A. Cells growing in HL5 medium we exposed to concanamycin A (2 µM final concentration) for 1 hour, then fixed and prepared for fluorescence microscopy using antibodies to DdRab11 (A), RabD (D) and the 100 kDa proton pump subunit (B,E). Arrows indicate proton pump positive vacuoles that show little staining for DdRab11 or RabD. The cell stained for DdRab11 (A) and 100 kDa proton pump subunit (B) was also photographed under bright field conditions (C). Scale bar: 5 µm.

View larger version (88K): [in a new window]   Fig. 10. DdRab11N125I-expressing cells are defective in contractile vacuolar function. Cells expressing a DdRab11N125I dominant negative protein were generated and contractile vacuolar function was tested. Wild-type (A) and mutant cells (B) grown in growth media were placed in distilled water and photographed. Photomicrographs of wild-type cells in water (A) show cells with amoeboid morphology and slightly enlarged contractile vacuoles (black arrowhead). However, mutant cells in water (B) were detached from the plastic and contained rounded spherical shapes with distinct enlarged contractile vacuoles (black arrowheads).

View larger version (88K): [in a new window]   Fig. 11. The reticular network containing the 100 kDa ATPase subunit is altered in cells expressing DdRab11N125I. Mutant and wild-type cells were fixed and stained for the 100 kDa ATPase protein. The distribution of the 100 kDa subunit was altered in mutant cells (A) when compared with the ATPase distribution in wild-type cells (B). Scale bar: 5 µm.

View larger version (16K): [in a new window]   Fig. 12. Cells expressing DdRab11N125I are normal in endocytic uptake and release rates but are enhanced in the rate of phagocytosis. DdRab11N125I (+) cells were incubated in HL5 medium containing FITC-dextran (1 mg/ml). At the indicated times (top), cells were recovered and washed by centrifugation and accumulated FITC-dextran was measured using a spectrophotometer. In addition (middle), after 3 hours of accumulation, cells were washed and resuspended in growth medium. At the indicated times, cells were washed and the FITC-dextran remaining intracellular was measured. Finally (bottom), cells in growth medium were incubated with fluorescent latex beads (at the indicated times), the cells were washed, and accumulated beads were measured using a spectrophotometer.

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