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First published online 1 August 2006
doi: 10.1242/jcs.03078

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Conserved and specific functions of axoneme components in trypanosome motility

Carole Branche^1,*, Linda Kohl², Géraldine Toutirais¹, Johanna Buisson¹, Jacky Cosson³ and Philippe Bastin^1,*,

¹ INSERM U565 and CNRS UMR5153 and MNHN USM0503, Muséum National d'Histoire Naturelle, 43 rue Cuvier, 75231 Paris cedex 05, France
² USM0504, Muséum National d'Histoire Naturelle, 61 rue Buffon, 75231 Paris cedex 05, France
³ CNRS UMR7009, Station zoologique, BP28, 06230 Villefranche sur Mer, France

View larger version (58K): [in a new window]   Fig. 1. Structural and molecular aspects of the trypanosome flagellum. (A) Drawing of cross-section through the T. brucei flagellum, indicating predicted location of proteins studied in this work. Microtubules are shown in black, nexin links in green, radial spokes in magenta, dynein arms in orange and central-pair projections in yellow. The PFR is shown in blue, with its proximal, intermediate and distal domain (relative to the axoneme). The inset shows a magnified central pair and its projections. To measure orientation of the central pair relative to the PFR, lines were drawn on the axis passing through the two microtubules or through the proximal domains of the PFR (dashed grey lines) and angles measured. (B) Cross-sections of wild-type flagellum from whole cells. (Left) The flagellum appears to lie on its side, with the PFR in proximity of the FAZ filament (dark spot between subpellicular microtubules) and the axoneme closer to the four specialised microtubules associated to the smooth endoplasmic reticulum (left side of the image). (Right) The PFR is positioned between the cell body and the axoneme. (C) Cross-section of demembranated flagellum where dynein arms, radial spokes and central-pair projections are more visible. (D) Longitudinal section of the flagellum showing the axoneme (Axo) with the central pair (CP) microtubules and some projections and the paraflagellar rod (PFR) confined between the axoneme and the cell body. (E) Representation of proteins analysed in this study with their main particular domains (from Pfam-domain searches). (F) Percentage of identity between flagellum proteins from T. brucei and two other kinetoplastids, T. cruzi and L. major, and between human, mouse and Chlamydomonas reinhardtii. Alignment was performed using the Jotun-Hein method, except for TbDNAH where Clustal W was used.

View larger version (24K): [in a new window]   Fig. 2. Sedimentation shows reduction of motility. Trypanosomes were incubated in cuvettes and optical density was measured before and after mixing. The recipient cell line 29-13 was used as a negative control and the PFR2 RNAi-mutant snl-2 as positive control. RNAi mutant cell lines were induced for 2 days and grown with (full lines) or without (dotted lines) shaking. Reduction in cell motility was severe in both conditions.

View larger version (74K): [in a new window]   Fig. 3. Central-pair-presence and -orientation in normal and mutant cell lines. (A) Cross-sections of flagella from (TbPF16)RNAi and (TbPF20)RNAi cells induced for 3 days. The central pair can be positioned at very different angles relative to the PFR axis. In several sections, one or both microtubules of the central pair can be absent. (B) Loss of some outer dynein arms in (TbPF20)RNAi- and (TbPF16)RNA mutants, and of most of them (eight) in (TbDNAI1)RNAi mutants. Yellow arrows indicate the position of the missing arm. (C) Longitudinal section of a (TbPF16)RNAi mutant where the central pair is visible in a bent region, in this case it appears parallel to the bend plane. (D) Central-pair orientation relative to the PFR proximal axis. A negative angle indicates that lines drawn from the axis of the central pair and of the PFR would cross in direction of doublet 4 of the axoneme (right side of images in B). Sections were classified as belonging to any 15° interval. Owing to the difficulty in unambiguously distinguishing the C1 from the C2 microtubule, angles were limited to 180° but could be more important. As no significant differences could be seen between sections of whole cells or de-membranated samples, data from both sets were pooled. From top to bottom: wild-type (n=82), (TbPF20)RNAi (n=65), (TbPF16)RNAi (n=116) and (TbDNAI1)RNAi (n=44). Whereas a determined orientation is found in wild-type cells, it appeared highly variable in axoneme mutants.

View larger version (24K): [in a new window]   Fig. 4. Cumulative growth curves of the axoneme or PFR mutants under non-induced conditions (full line), induced conditions (dashed line) and induced conditions while shaking the culture (dotted line).

View larger version (67K): [in a new window]   Fig. 5. Cell-body separation is impaired when axoneme mobility is strongly affected. (A-C) Still images of movies of (TbPF16)RNAi mutants (A) non-induced or induced for (B) 48 hours or (C) 72 hours. (A) Both flagella are motile and the cell is actively moving and twisting, but no net movement is observed. (B) The left flagellum is motile but the right one is not. The active flagellum drags the couple towards its anterior end. (C) Both flagella are paralysed and division is slowed down. (D) Field of (TbPF20)RNAi-mutant cells induced for 72 hours, grown (left) without shaking or (right) with shaking. Large cell aggregates are visible when cultures are not shaken; most of them disappear upon shaking. (E) Bloodstream form trypanosomes electroporated with (left) GFP or (right) PFR2 dsRNA. After 5 hours, flagella were labelled with the anti-PFR2 antibody (green, bottom images) and DNA-stained with DAPI (blue); superimposed phase-contrast image (top images). Cells with two flagella that received GFP dsRNA showed complete PFR2 staining in both flagella, whereas those with PFR2 dsRNA did not stain for PFR2 at the distal part of the flagellum, the normal site of PFR construction. (F) After 18 hours cells that had failed to divide (left images) were abundant only in the PFR2-transfected samples. The cells could re-enter the cell cycle (middle images) but rapidly degenerated (right images).

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