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First published online 5 September 2006
doi: 10.1242/jcs.03203

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Trypanosome IFT mutants provide insight into the motor location for mobility of the flagella connector and flagellar membrane formation

Jacqueline A. Davidge^1,*, Emma Chambers^2,*, Harriet A. Dickinson¹, Katie Towers², Michael L. Ginger¹, Paul G. McKean² and Keith Gull^1,

¹ Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
² Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK

View larger version (12K): [in a new window]   Fig. 1. Graphs showing relationships between flagellar connector movement and basal body migration in procyclic form trypanosomes. Electron micrographs of whole-mount cytoskeletons were analysed and the following cytological measurements recorded: (1) distance between basal bodies; (2) length of the old flagellum (OF); (3) length of the new flagellum (NF) and (4) distance of FC migration along the old flagellum. Measurements are expressed as a ratio to old flagellum length to account for individual variation in cell length. (A) Graph showing relationship between new flagellum length and inter-basal body distance. (B) Graph showing relationship between FC migration and new flagellum length. (C) Graph showing relationship between FC migration and inter-basal body distance.

View larger version (58K): [in a new window]   Fig. 2. Visualisation of MPM2-positive phosphoproteins in procyclic trypanosomes. Immunofluorescence images of T. brucei cytoskeletons co-labelled with the anti-phospho-epitope antibody MPM2 (green) and (A) ROD1, a specific marker for the extra-axonemal PFR structure; (B) BBA4, a specific marker for the proximal pole of both basal and probasal body structures; and (C) AB1, a specific marker for the central component of the flagella connector (red). Nuclear and kinetoplast DNA are labelled with DAPI (blue). The sections identified by the white boxes are enlarged in the top right hand corner of each panel to allow closer inspection of the co-labelling pattern observed. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 3. An MPM2-positive structure is retained on the old flagellum post cytokinesis enabling distinction between procylic cells post division. (A) Immunofluorescence image of a representative 1K1N procyclic trypanosome labelled with the anti-phospho-epitope antibody MPM2 (green), showing a focus of MPM2 labelling (FC remnant) at the defined stop point of FC migration. Nuclear and kinetoplast DNA are labelled with DAPI (blue). (B) Quantitative analysis of FC migration as revealed by MPM2 labelling of procyclic cells. The graph plots FC migration (as a ratio of old flagellum length) against new flagellum length in biflagellate cells (blue diamonds). The position of the FC remnant on the old flagellum for a population of 1K1N cells (red squares) is overlaid to demonstrate that the observed focus of MPM2 on 1K1N cells precisely correlates with the defined FC stop point. The reduced overlap between the datasets is a reflection of under representation of very late division cells in the biflagellate population. (C) Cartoon summarising the correlation between the temporal and spatial positioning of the new and old flagellum, the FC and inter basal body separation as deduced from the cell measurement data shown in Fig. 1 and supplementary material Fig. S1. This cartoon also depicts the asymmetric MPM2-staining pattern predicted for 1K1N cells following cytokinesis. DIVN, dividing nucleus; K, kinetoplast; N, nucleus. Bar, 10 µm.

View larger version (119K): [in a new window]   Fig. 4. Formation and movement of the FC in the absence axonemal assembly. (A) A thin-section electron micrograph shows formation of a FC (asterisk) in the flagellar pocket of a TbCHE2 RNAi-induced cell. (B,C) Thin-section electron micrographs revealing the extrusion of flagellar membrane (arrows) beyond the flagellar pocket of TbCHE2 RNAi-induced cells and the formation of macula adherens junctions within the FAZ (white arrowheads within a square bracket). nf, new flagellum. (D-I) Exit of a flagellar stub from the flagellar pocket and the left-handed helical twist of the flagellar sleeve in TbCHE2 RNAi-induced cells as revealed by scanning electron microscopy. The boxed regions of D,F,H are shown at higher magnification in E,G, and I, respectively. of, old flagellum; arrows point to the flagellar sleeve. (J) The elongating new flagellum is positioned on the left-hand side of the old flagellum in cells with two flagella. (K) Transverse section through a TbCHE2 RNAi-induced cell reveals the presence of a flagellar sleeve (arrow) to the left-hand side of the old flagellum and is attached by a FC. Bars, 500 nm (A,B,E,G,I,J); 2 µm (D,F,H); 200 nm (C,K).

View larger version (66K): [in a new window]   Fig. 5. Detection of the FC on the old flagellum of cells not elongating a new flagellum and the flagellar sleeve is not an artefact of TbCHE2 RNAi. (A) Negatively stained whole-mount cytoskeleton showing a partial FC (asterisk in the inset) part way down the old flagellum (of) in a TbCHE2 RNAi-induced cell with two mature basal bodies (arrows). (B,C) The flagellar sleeve cannot be seen by scanning electron microscopy in a mutant that elongates a detached flagellum. The boxed region of B is shown at higher magnification in C. Bars 2 µm (A,B); 500 nm (C and inset in A).