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INTRAFLAGELLAR TRANSPORT IN TRYPANOSOMES

INTRAFLAGELLAR TRANSPORT IN TRYPANOSOMES. Filippo RUSCONI Muséum National d’Histoire Naturelle INSERM U565 & CNRS UMR 5153 & MNHN 0503 rusconi@mnhn.fr. Radial spoke. IntraFlagellar Transport (IFT). IFT particle. FLA10 KinesinII. Cytoplasmic Dynein. Raft.

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INTRAFLAGELLAR TRANSPORT IN TRYPANOSOMES

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  1. INTRAFLAGELLAR TRANSPORT IN TRYPANOSOMES Filippo RUSCONI Muséum National d’Histoire Naturelle INSERM U565 & CNRS UMR 5153 & MNHN 0503 rusconi@mnhn.fr

  2. Radial spoke IntraFlagellar Transport (IFT) IFT particle FLA10 KinesinII Cytoplasmic Dynein Raft Anterograde IFT: FLA10 Kinesin II (towards flagellum tip, + end MT) Retrograde IFT: Cytoplasmic Dynein DHC1b (towards cell body) Cole et al., J Cell Biol. 141, 993-1008, 1998

  3. Trypanosoma brucei (protozoan) • Flagellated organism easy to grow in vitro • Genome sequenced • Straight forward molecular biology • Reverse genetics (RNAi, inducible expression) • Responsible for sleeping sickness • Flagellum attached to the cell body nucleus Phase Anti-flagellum + DAPI

  4. IFT in trypanosomes Gene Characteristics IFT in T. b. Motors DHC1b dynein heavy chain + DHC1bis dynein heavy chain +LC8 dynein light chain -KIF3A kinesin motor -KIF3B kinesin motor NDKAP cargo binding ND Raft components IFT20 coiled-coil +IFT27 G protein ?IFT52 coiled-coil +IFT55 coiled-coil +IFT74 coiled-coil NDIFT88 TPR +IFT122 WD40 +IFT172 WD40 + Regulators LF4 kinase +/- Vector for RNAi silencing T7 tet-inducible promoters drug resistance PFRA T7 transcription terminators intermolecular Wang et al., JBC (2000) 275, 40174 Durand-Dubief, Kohl & Bastin, MBP (2003) 129, 11-21

  5. The trypanosome cell cycle Green: Flagellum, Red lines: FAZ, Red dots: basal body, Blue: DAPI

  6. Generation of non-flagellated cells induced Green: Flagellum, Red lines: FAZ, Red dots: basal body, Blue: DAPI Defects: No new flagellum, but FAZ is still there but ... FAZ is too short non- induced

  7. Shorter flagella, shorter cells ? DIC (TbDHC1b cells, RNAi silencing induced for 48 hrs) Green: Flagellum, Red lines: FAZ, Red dots: basal body, Blue: DAPI

  8. 40 30 20 20 10 10 0 0 0 10 20 30 Flagellum and cell size are linked cell size (µm) Flagellum length (µm)

  9. Characterisation of IFT RNAi mutants Induced cells don’t have a flagellum % Flagellated cells IFT88 DHC1b

  10. FAZ FORMATION AND CYTOKINESIS Multinucleated cells Non-flagellated sibling no new flagellum no new FAZ No cytokinesis Nuclear division OK no new flagellum new FAZ Flagellated sibling no new flagellum no new FAZ Blue: DAPI Red lines: FAZ Red spots: basal bodies

  11. Summary • IFT controls flagellum assembly • IFT controls flagellum length • Flagellum length dictates FAZ elongation and defines cytokinesis initiation • Flagellum and FAZ are required for cell morphogenesis (shape, polarity) Kohl, Robinson & Bastin, EMBO J. (2003) 22,5336-5346 See also comments in Hurtley S. (2003) A dead-end short cut. Science 302, 535 Jones S. (2003) Trypanosome flagellum-motility and more. Nature Rev. Microbiol. 1, 172-173.

  12. Non-flagellated cells show dispersion of their endocytic network Normal Dispersion of : -rab5 (early endosomes) -rab11 (recycling pathway) -p67 (lysosomes) Non-flagellated Red: anti-clathrin Blue: DAPI

  13. Group Philippe BASTIN (Muséum National d’Histoire Naturelle, Paris, Dir. C. GIOVANNANGELI & J.S. SUN) Linda KOHL Filippo RUSCONI Sabrina BENGHANEM Géraldine TOUTIRAIS Mickaël DURAND-DUBIEF Carole BRANCHE Gwénola DORE Derrick ROBINSON(Université Bordeaux II) Mark FIELD(Imperial College, UK) Thanks to K. GULL, P. ENGLUND, C. CLAYTON, G. CROSS

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