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First published online 21 October 2008
doi: 10.1242/jcs.035626

Journal of Cell Science 121, 3704-3716 (2008)
Published by The Company of Biologists 2008
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Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei

Sabrina Absalon1,2,*, Thierry Blisnick1,*, Mélanie Bonhivers3, Linda Kohl4, Nadège Cayet5, Géraldine Toutirais2, Johanna Buisson1, Derrick Robinson3 and Philippe Bastin1,2,{ddagger}

1 Trypanosome Cell Biology Unit, Pasteur Institute and CNRS, 25 rue du Docteur Roux, 75015 Paris, France
2 Dynamique et Régulation des Génomes, Muséum National d'Histoire Naturelle, INSERM and CNRS, 43 rue Cuvier, 75005 Paris, France
3 Microbiologie Cellulaire et Moléculaire et Pathogénicité, Université Bordeaux 2 and CNRS, 146 Rue Léo Saignat, 33076 Bordeaux, France
4 Biologie Fonctionnelle des Protozoaires, Muséum National d'Histoire Naturelle, 61 rue Buffon, 75005 Paris, France
5 Plateforme de Microscopie Electronique, Pasteur Institute, 25 rue du Docteur Roux, 75015 Paris, France


Figure 1
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  		Fig. 1. Basal body replication takes place in the absence of a flagellum. (A-C) Merged images of double immunofluorescence staining with mAb22 (marker of the proximal end of the mature and the pro-basal body, green) and the anti-TBBC antibody (marker of the mature basal body, red) of detergent-extracted cytoskeleton from non-induced (A, –TET) or 48-hour-induced IFT140RNAi (B, +TET) cells stained with DAPI (blue). Insets show magnification of the basal body region. BB, basal body. (C) Non-flagellated cells can duplicate their basal body (a) but fail to divide, hence becoming multinucleated cells (b) that accumulate several pairs of basal bodies. a' and b' show magnification of the boxed basal body regions in a and b. (D) Transmission electron micrograph of a section through an IFT172RNAi cell induced for 72 hours, which has four basal bodies (arrows). TET, tetracycline.

 

Figure 2
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  		Fig. 2. The flagellar pocket exhibits a modified shape and accumulates vesicles in IFTRNAi mutant cells with a short or no flagellum. (A-F) Sections through the flagellar pocket region of wild-type (WT) trypanosomes (A), of the indicated IFTRNAi mutant cell lines (B-E) or of PF20RNAi cells (F) that were induced for 72 hours. The typical flask-shape of the flagellar pocket is altered and vesicles frequently accumulate in the lumen (D,E). A, anterior side of the flagellar pocket; P, posterior side; K, kinetoplast; BB, basal body; TR, transition region. Black arrows indicate the tight contact between the top of the flagellar pocket and the flagellum, the white arrow indicates a vesicle in the flagellar compartment and the arrowheads indicate excessive IFT material typical of retrograde transport defects. (G) Quantification of sections with the indicated phenotypes in wild-type (WT) and various IFTRNAi cell lines. WT, n=73; DHC1bRNAi, n=51; IFT140RNAi, n=36; IFT88RNAi, n=28; IFT172RNAi, n=31.

 

Figure 3
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  		Fig. 3. The defined orientation of the flagellar pocket collar is randomised in IFTRNAi mutants. (A) Scanning electron micrograph of a wild-type cell extracted with cold Triton X-100. The white rectangle indicates the position of the magnified area shown on the right. The FPC and the FAZ filament are indicated by white arrows and arrowheads, respectively. (B) An intact wild-type cell examined by scanning electron microscopy. The magnified area shows the region where the flagellum emerges from the cell body. The shape of the FPC is visible underneath the membrane (white arrows). (C) Images of IFT140RNAi cells (a-c) induced for 72 hours and treated with cold Triton X-100 as in A. The FPC associated with the short flagellum appears less elaborate and its orientation relative to the main axis of the cell is variable. (D) A non-extracted IFT140RNAi cell after induction of silencing for 72 hours. The shape of the FPC is clearly recognisable but its orientation is not perpendicular to the main axis of the cell.

 

Figure 4
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  		Fig. 4. The FPC is disorientated as soon as cells fail to assemble a normal flagellum. (A-B) Scanning electron microscopy of untreated non-induced (A) and 48-hour-induced IFT140RNAi cells (B). In controls (A), the two flagella emerge from separate FPCs that are parallel. In cells that possess an old flagellum and an aberrantly short new flagellum (B), the orientation of the FPC associated with the new flagellum is not fixed relative to the long axis of the cell nor to the orientation of the FPC of the old flagellum. Arrows show the FPC, and arrowheads indicate the flagellar sleeve present in IFT mutants.

 

Figure 5
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  		Fig. 5. Organisation of the FPC protein BILBO1 is modified in IFTRNAi cells. (A-C) Immunofluorescence staining of the indicated cell lines with an antibody recognising BILBO1 (green), stained with DAPI (blue). Cells were either non-induced (–TET) or induced for 72 hours (+TET). BILBO1 staining is less defined in cells that lack flagella (C). Arrows indicate cells with modified BILBO1 staining and arrowheads indicate short flagella.

 

Figure 6
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  		Fig. 6. The flagellar pocket protein CRAM is mislocalised in IFT140RNAi cells. (A-C) Cells from the indicated cell lines were fixed and permeabilised with Nonidet before immunofluorescence with the anti-CRAM antiserum and stained with DAPI. Bottom panels show anti-CRAM signal only (white) and top panels show merged phase contrast, anti-CRAM (green) and DAPI (blue) images. (D-G) Cells from non-induced (D) and induced (E-G) IFT140RNAi cells were fixed and incubated with the anti-CRAM antiserum without permeabilisation, followed by incubation with Protein AG coupled to gold particles. Arrows indicate short dilated flagella typical of retrograde transport inhibition; arrowheads indicate flagellar sleeves. Scale bars: 1 µm.

 

Figure 7
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  		Fig. 7. Various vesicles are present in short flagella and in the flagellar pocket area of IFTRNAi cell lines. (A-C) Wild-type cells. FP, flagellar pocket; BB, basal body; E, endosomes, G, Golgi complex, K, kinetoplast. The white arrow indicates the flat compartment visible close to the flagellar pocket membrane. (D) Section through the flagellar pocket of an IFT140RNAi cell induced for 3 days. Notice the accumulation of IFT-like material in the short flagellum (IFT excess), the flagellum attachment zone (FAZ) and the presence of `double' (black arrowheads) and bent (black arrow) vesicles close to the flagellar pocket/FAZ region. (E,F) Sections through the flagellum of DHC1bRNAi cells induced for 72 hours. Notice the presence of round or flat vesicles in the flagellar compartment (some are indicated by short white arrows). (G,H) IFT88RNAi cells induced for 72 hours showing the flagellar pocket region where vesicles are also seen in a flagellar sleeve compartment (G) and the Golgi complex, close to which some double membrane (black arrowheads) or bent (black arrows) vesicles are frequently identified (H). (I) Quantification of sections that show a flat compartment close to the flagellar pocket membrane (black bars), at least one double membrane vesicle (`double', light grey) or at least one `cup-like' vesicle (dark grey) in the indicated cell lines. WT, n=63; DHC1bRNAi, n=42; IFT140RNAi, n=36; IFT88RNAi, n=23.

 

Figure 8
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  		Fig. 8. The function of the flagellar pocket is altered in the absence of a normal flagellum in IFTRNAi cells. (A-D) Capture and accumulation of the lipophilic tracer FM4-64 (red) in the indicated cell lines induced for 48 hours. Cells were fixed and stained with DAPI (blue) immediately after washes. White arrows indicate non-flagellated cells. (C) Quantification of the amount of FM4-64 signal in the indicated cells. For IFT172RNAi induced samples, counts for flagellated and non-flagellated cells from the same slide are presented. FP+++, bright signal at the posterior end of the cell; FP+/–, weak signal at the posterior end; diffuse+++, bright signal through a large zone of the cell (always the posterior end for flagellated cells, more variable for non-flagellated ones); diffuse+/–, weak signal through a large zone of the cell. n=50-120.

 

Figure 9
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  		Fig. 9. Diagram summarising the role of the flagellum in flagellar pocket formation. (A) General view of the monoflagellated trypanosome cell where the flagellar pocket area has been made transparent to visualise flagellar pocket associated components. For simplicity, only the main organelles studied in this work have been represented. (B-D) Situation in wild-type cells with one flagellum (B), one old and one new flagellum prior to microtubule assembly (C) and after flagellar pocket duplication (D). (E) Situation in a nonflagellated cell where anterograde IFT was inhibited. (F) Situation in a cell where a short flagellum has been produced upon blockage of retrograde transport. The internal region of the emerging flagellum is also shown. See text for details.

 

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© The Company of Biologists Ltd 2008