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First published online 9 March 2004
doi: 10.1242/jcs.00995

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The flagella connector of Trypanosoma brucei: an unusual mobile transmembrane junction

Laura J. Briggs¹, Paul G. McKean², Andrea Baines¹, Flavia Moreira-Leite^1,*, Jacqueline Davidge¹, Sue Vaughan¹ and Keith Gull^1,

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

View larger version (112K): [in a new window]   Fig. 1. Visualization of the flagella connector at the distal tip of the new flagellum. (A) The cell cycle of procyclic trypanosomes. At G1, trypanosomes posses a single kinetoplast and a single nucleus (1K1N) with a single attached flagellum. During the cell cycle, new flagellum elongation coincides with kinetoplast duplication and segregation (2K1N). The new flagellum is physically attached to the old flagellum via the flagella connector. Following mitosis (2K2N) and the initiation of cytokinesis, this connection is released. (B-D) Negatively stained whole-mount cytoskeletons of procyclic trypanosomes. (B) The flagella connector (asterisk) is positioned at the tip of the new flagellum (nf) and along the side of the axoneme (ax) of the old flagellum (of) and not the paraflagellar rod (pfr). (C) The flagella connector consists of a trilaminar core structure composed of three distinct layers of electron density (layers 1,2,3) and filamentous extensions (marked with a bar) extending to the tip of the new flagellum. This gives roughly an overall triangular appearance. (D) No flagella connector structure is evident at the tip of the old flagellum. Scale bars, 200 nm (B), 100 nm (C,D).

View larger version (131K): [in a new window]   Fig. 2. Ultrastructure of the flagella connector viewed by thin-section EM. (A) Cross section though the flagella connector (asterisk). The new flagellum (nf) is always observed to the left of the old flagellum (of) when viewed from the posterior end of the cell. The flagella connector is composed of three discrete connection zones between two opposing flagellar membranes. These membranes display a more electron dense, linear profile, which is exemplified in (B), in which the section is cut through the extreme distal tip of the new flagellum. (C) A glance section through the flagella pocket reveals the presence of the flagella connector just distal to the transition zone (tz) at the end of the basal body, revealing its early appearance during flagellum elongation [axoneme (ax) paraflagella rod (pfr)]. Scale bars, 200 nm.

View larger version (40K): [in a new window]   Fig. 3. Model illustrating the structure of the flagella connector. (A) Procyclic T. brucei cell. The arrow indicates a hypothetical plane of section passing through the region of the flagella connector. (B) Model based on thin-section TEM data. The axonemal outer doublets that are linked to the flagella connector are numbered. (C) Model of the flagella connector structure compatible with both negative staining TEM and thin-section TEM data. (D) Cross-section of the putative organization of protein components in the interacting membrane domains of the flagella.

View larger version (131K): [in a new window]   Fig. 4. Immunogold EM with the AB1 monoclonal antibody. Cytoskeletons fixed with methanol (A,B) and unfixed cytoskeletons (C) both show 10-nm gold staining of the flagella connector (*), which is defined by the presence of electron-dense material between the tip of the new flagellum (nf) and the side of the old flagellum (of). Scale bars, 200 nm

View larger version (48K): [in a new window]   Fig. 5. T. brucei procyclic cells at various phases of the cell duplication cycle. Phase-contrast images and corresponding immunofluorescence images; DNA is labelled with DAPI (blue), the flagellum is identified by L8C4, anti-PFR antibody (green) and the flagellar connector is labelled by AB1 antibody (red). (A) An AB1 signal is not seen in most 1K1N cells. (B) AB1 is observed in a proportion of cells just above the basal body in phase contrast. (C,D) The AB1 signal is seen in cells that are elongating a new flagellum at the correct position for the flagella connector and throughout the cell cycle. (E) The AB1 signal is not seen in cells that have entered cytokinesis. Scale bars, 2 µm

View larger version (94K): [in a new window]   Fig. 6. Lack of apparent connector in other trypanosome cell types. (A) SEM of a procyclic T. brucei cell. The tip of the new flagellum is associated with the lateral aspect of the old flagellum, via the flagella connector, indicated by the white arrow. (B,C) SEM of bloodstream T. brucei forms. The tip of the new flagellum (nf) is close to the old flagellum (of) but a physical connection is not observed. (D) Negatively stained whole-mount cytoskeleton of a T. brucei bloodstream form. Although the tip of the new flagellum is close to the old flagellum, there is no visible flagella connector. In addition, the new axoneme lies adjacent to the old PFR. (E) Immunofluorescence images of T. brucei bloodstream forms. Phase-contrast and corresponding merged immunofluorescence image; DAPI is in blue, PFR is labelled with anti-PFR antibody (L8C4) in green and the flagella connector is labelled by AB1 in red. (i,ii) 1K1N cells. (iii) 2K1N cell. (iv) 2K2N cell. In no case was an AB1 signal observed at the tip of the new axoneme. (F,G) Negatively stained whole-mount cytoskeletons of T. cuzi epimastigote (F) and Leishmania promastigote (G). Both cells are in cell division and extending a new flagellum, but a flagella connector structure is not observed. Scale bars, 2 µm.

View larger version (18K): [in a new window]   Fig. 7. Cartoon of the cell cycle in T. brucei trypomastigotes. (A-D) Procyclic form. (E-H) Bloodstream forms. Apart from differences in cell shape between the two forms, the segregation of the kinetoplast is more limited in the bloodstream form (G) than in the procyclic form (C). (D) In the procyclic form, the new kinetoplast and nucleus are located towards the posterior end of the cell, but the orientation of the nuclei in a post-mitotic bloodstream cell are not in the same configuration (H). The two kinetoplasts are located at the posterior end of the cell and the nuclei are both located anterior to the kinetoplast.

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