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A developmentally regulated Rab11 homologue in Trypanosoma brucei is involved in recycling processes

Wellcome Trust Laboratories for Molecular Parasitology, Imperial College of Science, Technology and Medicine, Department of Biochemistry, Exhibition Rd, London, SW7 2AY, UK
^* Author for correspondence (e-mail: mfield{at}ic.ac.uk )

View larger version (109K): [in a new window]   Fig. 1. TbRAB11 is a Rab11 / Ypt31 homolog. ClustalX alignment of the predicted translation of the ORF of TbRAB11 with its closest kinetoplastid, mammalian, yeast and plant orthologues identified by BLASTp analysis at GenBank. Tc, T. cruzi; At, A. thaliana; Mm, M. musculus; Ypt, S. cerevisiae. Boxes indicate identity and shading similarity.

View larger version (52K): [in a new window]   Fig. 2. TbRAB11 is a developmentally regulated GTPase. (A) Northern blot analysis demonstrating that BSF parasites express TbRAB11 mRNA at a higher level than PCF. Total RNA was isolated from 107 cells and resolved on a formaldehyde gel. After transfer to a nylon membrane the blot was probed with the full length TbRAB11 ORF. Positions of RNA standards in kb are shown to the left and ethidium bromide stain of the rRNA bands shown as a control for loading equivalence at the base of the figure. (B) Western blot analysis with affinity purified anti-TbRAB11 generated in rabbits using full length recombinant protein as antigen. Fresh parasite cultures were resuspended in protein sample buffer, 107 parasite equivalents loaded in each lane and resolved on a 12% SDS-polyacrylamide gel. After electrophoretic transfer to nitrocellulose, equivalence of loading was checked by Ponceau S staining. Molecular weight standards are indicated to the left in kDa. (C) GTPase assay using recombinant GST-TbRAB2 (lanes 1,3) and GST-TbRAB11 (lanes 2,4). Recombinant protein loaded with [-32P]GTP was incubated at 37°C for 0 minutes (lanes 1,2) or for 60 minutes (lanes 3,4). After incubation [-32P]GDP was separated from [-32P]GTP by thin layer chromatography. (D) Graphical representation of GTPase activity of recombinant GST-TbRAB2 () and GST-TbRAB11 (). Recombinant protein loaded with [-32P]GTP was incubated at 37°C for 0, 5, 10, 20, 40 or 60 minutes. After incubation [-32P]GDP was separated from [-32P]GTP by thin layer chromatography and the percent hydrolysis at each time point determined using phosphorimager analysis.

View larger version (38K): [in a new window]   Fig. 3. TbRAB11 localisation in 427 BSF and PCF T. brucei. Immunofluorescence analysis of PCF (A-C) and BSF parasites (D-F). Parasites were stained with rabbit anti-TbRAB11 followed by an anti-rabbit Cy3 conjugate (red) and with DAPI for DNA (blue). Phase-contrast images merged with DNA stain are shown to the right of each fluorescence image.

View larger version (28K): [in a new window]   Fig. 4. TbRAB11 migrates coordinately with the basal body during mitosis. PCF parasites were treated for microscopy and stained with mouse anti-basal body (BB4) followed by an anti-mouse FITC conjugate (green), rabbit anti-TbRAB11 followed by an anti-rabbit Cy3 conjugate (red) and with DAPI for DNA (blue). For each image the basal body stain is shown to the left, the central figure corresponds to TbRAB11 and the right image is the merge. (A-C) Cells prior to entering mitosis with one kinetoplast and one nucleus. (D-F) Cells with one nucleus and an elongate duplicating kinetoplast and basal body. (G-I) Cells in which the kinetoplast and basal body have both duplicated while there is still a single nucleus.

View larger version (39K): [in a new window]   Fig. 5. TbRAB11 is absent from lysosomal and Golgi elements. (A-D) BSF parasites were treated for fluorescent microscopy and stained with mouse anti-p67 followed by an anti-mouse FITC conjugate (green), rabbit anti-TbRAB11 followed by an anti-rabbit Cy3 conjugate (red) and DAPI for DNA (blue). (E-H) BSF parasites were exposed to biotinylated ConA at 4°C for 10 minutes prior to chase for 30 minutes at 37°C to permit lysosomal ConA accumulation. Cells were then treated for microscopy and stained with FITC-avidin (green), rabbit anti-TbRAB11 followed by an anti-rabbit Cy3 conjugate (red) and DAPI for DNA (blue). (I-L) BSF parasites were treated with BODIPY-TR ceramide (red) to stain the Golgi (Field et al., 2000). Cells were subsequently processed for microscopy and counterstained with rabbit anti-TbRAB11 followed by an anti-rabbit Oregon Green conjugate (green) and DAPI for DNA (blue).

View larger version (19K): [in a new window]   Fig. 6. The TbRAB11 compartment is juxtaposed to, but distinct from, early endosomes and partially colocalises with clathrin-coated structures. (A-C), Confocal microscopy was used to examine the relationship of TbRAB11 with TbRAB5A. Cells were stained with mouse anti-TbRAB11 followed by an anti-mouse FITC conjugate (green) and with rabbit anti-TbRAB5A followed by an anti-rabbit Cy3 conjugate (red). (D-F) Confocal fluorescent microscopy was used to examine the relationship of TbRAB11 with trypanosomal clathrin heavy chain. Cells were stained with mouse anti-TbRAB11 followed by an anti-mouse FITC conjugate (green) and with rabbit anti-TbCLH followed by an anti-rabbit Cy3 conjugate (red). C and F represent the merge in each case. Bars, 0.5 µm. * indicates the location of the nucleus; arrowheads indicate regions of colocalisation.

View larger version (20K): [in a new window]   Fig. 7. TbRAB11 is not associated with endocytic events. BSF parasites were exposed to FITC-ConA (green) for 15 seconds (A-C,G-I) or two minutes (D-F,J-L) prior to treatment for microscopy. Cells were counterstained with either rabbit anti-TbRAB11 (red in A-F) or rabbit anti-TbCLH (red in G-L) followed by an anti-rabbit Cy3 conjugate. Arrowheads in panel J-L represent regions of colocalisation. Bars, 0.5 µm. * indicates the location of the nucleus.

View larger version (25K): [in a new window]   Fig. 8. The TbRAB11 compartment is juxtaposed to the collecting tubules. BSF parasites were exposed to FITC-ConA for 10 minutes at 4°C and chased in FITC-ConA free media equilibrated either at 4°C (A-C,G-I) or 12°C (D-F,J-L) for 30 minutes. Cells were subsequently processed for microscopy and stained for either TbRAB11 (red in A-F) or TbRAB5A (red in G-L). C,F,I,L are merged images. * indicates the location of the nucleus, except in G-I where the nucleus is just to the right-hand side of the field of view. Bars, 0.5 µm.

View larger version (21K): [in a new window]   Fig. 9. The TbRAB11 compartment contains the trypanosomal transferrin receptor (ESAG6/7). BSF parasites were stained with mouse anti-TbRAB11 followed by an anti-mouse FITC conjugate (green), rabbit-ESAG6/7 followed by an anti-rabbit Cy3 conjugate (red) and DAPI for DNA (blue). A-D represent standard epifluorescent images, whereas E-G are confocal images; * represents the nucleus. D,G are merged images.

View larger version (16K): [in a new window]   Fig. 10. Anti-VSG antibody traffics through the TbRAB11 compartment subsequent to internalisation. BSF parasites were incubated with anti-VSG antibody for 1 hour and subsequently processed for fluorescent microscopy. Rabbit anti-VSG was detected using an anti-rabbit Cy3 conjugate (red). TbRAB11 was detected using mouse anti-TbRAB11 followed by an anti-mouse FITC conjugate (green). (A-C) Epifluorescent images; blue, DAPI for DNA. (D-F) Confocal images; * represents the position of the nucleus. C,F are merged images.

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