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First published online 24 February 2009
doi: 10.1242/jcs.040444

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A novel function for the atypical small G protein Rab-like 5 in the assembly of the trypanosome flagellum

¹ Trypanosome Cell Biology Unit, Pasteur Institute and CNRS, Paris, France
² Dynamique et Régulation des Génomes, Muséum National d'Histoire Naturelle, INSERM and CNRS, Paris, France

View larger version (109K): [in this window] [in a new window]   Fig. 1. Alignment of deduced amino acid sequences of RABL5 homologues. Alignment was generated using ClustalW; the most conserved residues are shown in red, the less conserved in blue. G1-G5 indicates conserved motifs implicated in nucleotide binding domains. Dashes indicate gaps introduced to optimize the alignment. Consensus indicates where the majority of residues match. Abbreviations and GenBank Accession Numbers of predicted protein sequences are as follows: Tb (Trypanosoma brucei), EAN80628/Tb11.01.8590; Tc (Trypanosoma cruzi), EAN83652; Lm (Leishmania major), CAJ08852; Ce (Caenorhabditis elegans), NP_503073; Hs (Homo sapiens), NP_073614; Mm (Mus musculus), AAH09150; Cr (Chlamydomonas reinhardtii), XP_001689669.1.

View larger version (55K): [in this window] [in a new window]   Fig. 2. RABL5 is associated with the basal body and the flagellum matrix. (A) Western blot of total protein samples showing expression of RABL5 at its expected Mr (arrow) in wild-type cells (WT) and the presence of an additional band (*) corresponding to the GFP::RABL5 in wild-type cells transfected with pPCPFReGFPRABL5 (WT+GFP::RABL5). (B) Wild-type cells fixed in methanol double-stained with the anti-axoneme marker MAb25 and the anti-RABL5 antiserum as indicated. The first panel shows the phase-contrast image merged with DAPI staining (blue) to visualise nuclear and kinetoplast DNA. Arrows indicate the flagella and arrowheads indicate basal body staining. New flagella and basal bodies are shown in yellow and old ones are shown in white. (C) GFP::RABL5 (green) -expressing trypanosomes fixed in methanol and stained with the basal body marker MAb22 (red) and with DAPI (blue). The basal body regions (arrowheads) are magnified (insets). The merged image (right) reveals that RABL5 stains the distal region of the basal body. (D) Wild-type trypanosomes were washed and treated with Nonidet P-40 1% to extract the cytoskeleton from the detergent soluble fraction. Protein samples were separated by SDS-PAGE, transferred to membranes and incubated with the indicated antibodies. The endoplasmic reticulum protein BiP was taken as marker of the soluble fraction and the paraflagellar rod (PFR) proteins were used as markers of the cytoskeleton. Most of the RABL5 protein pool is found in the soluble fraction. (E) The same treatment was applied to GFP::RABL5-expressing cells and analysed by IFA using MAb22 to reveal the fibres linked to the proximal basal body. GFP::RABL5 is still present at the basal body but cytoplasmic and flagellar staining are lost.

View larger version (34K): [in this window] [in a new window]   Fig. 3. RABL5 shows close colocalisation with IFT proteins. Wild-type cells fixed in methanol were stained with the anti-RABL5 antiserum and (A) with antibodies against IFT172, a protein required for anterograde transport, or (B) with PIFTF6/IFT144, a protein required for retrograde transport, as indicated. The left panels show the anti-IFT172 (A) or anti-PIFTD6 (B) signal, the middle panels show the RABL5 signal and the merged fluorescent images with the corresponding phase and DAPI (blue) images are shown in the right panels.

View larger version (56K): [in this window] [in a new window]   Fig. 4. RABL5 is linked to IFT. (A) GFP::RABL5 moves in the trypanosome flagellum. Still images of Movie 1 (supplementary material) are shown (elapsed time in seconds). The green arrow indicates a moving fluorescent particle and the white arrowhead shows the basal body. (B-E) Non-induced (B), IFT140RNAi (C,D) or IFT88RNAi (E) cells induced for 2 days stained with the anti-RABL5 antiserum as indicated. Arrows show flagella and arrowheads indicate basal bodies. New flagella and basal bodies are shown in yellow and old ones are shown in white. Clear accumulation of RABL5 in the short flagella of IFT140RNAi cells is detected (C,D), whereas only basal body signal is found in IFT88RNAi cells (E). B-E are at the same magnification.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Silencing of RABL5 perturbs flagellum assembly. (A) RT-PCR performed on total RNA from non-induced and RABL5RNAi cells induced for the indicated number of days shows potent silencing of the RABL5 mRNA. Aldolase was used as control. (B) Western blot showing the effect of RNAi silencing on the endogenous RABL5 protein. Total protein extract (20 µg) from non-induced and RABL5RNAi cells induced for the indicated number of days were run on a gel, transferred to a membrane and incubated with the anti-RABL5 or the anti-Bip as loading control. (C) Growth curve of non-induced (thin line) and induced (thick line) RABL5RNAi cells. (D) RABL5RNAi cells induced for 4 days stained with the anti-axoneme MAb25 (red) and with DAPI (blue), showing the presence of unusually short flagella. (E-J) Scanning electron micrographs of wild-type or RABL5RNAi cells induced for 4 days (same scale). An old (white arrow) and a new (yellow arrow) flagellum can be seen on control cells (E), whereas only a short new flagellum is constructed by the RABL5RNAi cells (F,G). (H-J) Daughter cells inheriting the short flagellum (white arrow). Arrowheads indicate membrane extensions or vesicles emerging from the flagellar pocket.

View larger version (42K): [in this window] [in a new window]   Fig. 6. RABL5RNAi assemble short flagella accumulating IFT proteins like retrograde IFT mutants. RABL5RNAi cells were non-induced (A) or induced for 4 days (B), labelled with the anti-IFT172 and counterstained with DAPI (blue). Arrow shows the accumulation of IFT proteins in the short flagellum (same scale). Arrowheads indicate the position of old (white) and new (yellow) basal bodies. (C) Cells were induced for 4 (RABL5RNAi) or 3 days (DHC1bRNAi, IFT140RNAi and IFT88RNAi), and stained with the anti-IFT172 antiserum. The proportion of short cells displaying no, weak or bright signal (n100) is shown. RABL5RNAi typically behaves like the other IFT retrograde transport mutants described above.

View larger version (76K): [in this window] [in a new window]   Fig. 7. Flagella of RABL5RNAi accumulate IFT-like material. Transmission electron micrographs showing the base of the flagellum in control cells (A) and in RABL5RNAi cells induced for 4 days (B,C) with bulbous short flagella containing IFT-like material. axo, axoneme; bb, basal body; fp, flagellar pocket; k, kinetoplast; tz, transition zone. Stars indicate the presence of vesicles within a short flagellum, and the arrow points at a short, dilated flagellum emerging from the flagellar pocket.

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