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First published online 22 July 2003
doi: 10.1242/jcs.00676

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An essential role of Rab5 in uniformity of synaptic vesicle size

Hideyuki Shimizu1, Satoru Kawamura1,2 and Koichi Ozaki1,2,*

1 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
2 Graduate School of Frontier Biosciences, Osaka University, Toyonaka, Osaka 560-0043, Japan



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  		Fig. 1. Localization of Rab5 and its functional inhibition with Rab5N142I. (A) EGFP fluorescence was observed in longitudinal section of the eye from Rh1-GAL4/UAS-EGFP Rab5 fly. R, retina; L, lamina. (B-D) Immunoelectron microscopy using an anti-GFP antibody identified subcellular localization of EGFP-Rab5 in the cell body (B,C) and the nerve terminal (D) of photoreceptor cells. Scale bars, 100 µm (A), 200 nm (B,C), 400 nm (D). (E) Antigen specificity of anti-Rab5 antiserum. (Top) CBB staining of Drosophila Rab2, Rab3, Rab4, Rab5, Rab6 expressed in E. coli. (Bottom) Immunoblot analysis using the anti-Rab5 antiserum against the above Rab proteins. (F) Heads homogenate was fractionated by velocity sedimentation. Fractions containing synaptotagmin (Syt), syntaxin (Syx) or Rab5 were identified by immunoblotting. (G) The numbers of MVBs and Golgi bodies were counted on electron micrographs of photoreceptor cells from wild-type (open bars) or hs-GAL4/UASRab5N142I mutant (shaded bars) flies.

 


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  		Fig. 2. Enlargement of synaptic vesicles caused by dysfunction of Rab5. (A-C) Nerve terminals of the photoreceptor cells from wild-type (A) and Rh1-GAL4/UAS-Rab5N142I mutant (B,C) flies. (D) A nerve terminal of the photoreceptor cell expressing antisense Rab5 RNA (GMR-GAL4/UAS-antisenseRab5). Inset, immunoblot analysis of the expression of Rab5 in the eyes of the wild-type (WT) and the GMR-GAL4/UAS-antisenseRab5 (AS) flies. (E) A typical nerve terminal of the Rh1-GAL4/UASRab5Q88L mutant. (F) A nerve terminal of Rh1-GAL4/UAS-EGFP-Rab5. The arrowheads and arrows indicate enlarged and tubular synaptic vesicles, respectively. Scale bar, 250 nm.

 


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  		Fig. 3. Enlargement of the synaptic vesicles in the dark incubation. Histograms indicate the size distributions of synaptic vesicles in the wild-type flies under light illumination (open bars) or dark condition (hatched bars), and in the Rh1-GAL4/UAS-Rab5N142I flies under light illumination (shaded bars) or in the dark (closed bars).

 


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  		Fig. 4. Reconstitution of homotypic fusion of synaptic vesicles in vitro. (A,B) Synaptic vesicles prepared from the hs-GAL4/UASRab5N142I mutant flies were electron microscopically observed before (A) and after (B) fusion reaction. Scale bar, 200 nm. (C) The averaged diameters of synaptic vesicles prepared from the wild-type and mutant flies were measured on electron micrographs before (open bars) and after (shaded bars) fusion reaction. (D) Synaptic vesicles prepared from the wild-type and mutant flies were subjected to velocity sedimentation before (0 min) and after (15 min) the fusion reaction, and detected by immunoblotting using anti-synaptotagmin antibodies. (E) Synaptic vesicles from fractions (3+4) and (5+6) of the mutant flies were separately collected and their averaged diameters were measured on the electron micrographs.

 


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  		Fig. 5. Membrane cycle of enlarged synaptic vesicles induced by Rab5N142I. (A-C) Cross sections of photoreceptor nerve terminals indicate the exo- and endocycle of synaptic vesicles in the shi1;;Rh1-GAL4 UAS-Rab5N142I/+ double mutant. After keeping the mutant flies at 20°C (A), they were put at 30°C for 1 minute under light illumination (B) followed by the incubation at 20°C for 30 minutes (C). Scale bar, 500 nm. (D) Changes in the diameter of the R1-6 photoreceptor nerve terminals of shi1 (open bars) and shi1;;Rh1-GAL4 UAS-Rab5N142I/+ (shaded bars) mutants caused by the above temperature shift.

 


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  		Fig. 6. Impaired synaptic transmission in Rab5N142I-expressing fly. (A) ERG recordings from the light-adapted (L) and dark-adapted (D) wild-type and Rh1-GAL4/UAS-Rab5N142I flies. Arrowheads indicate the off-transient responses. (B) The proportion of wild-type and Rh1-GAL4/UASRab5N142I flies exhibiting stop-walk responses against light-off stimuli.

 




© The Company of Biologists Ltd 2003