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Vol. 19, Issue 3, 929-944, March 2008

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Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes

Sabrina Absalon^*,, Thierry Blisnick^*, Linda Kohl^,, Géraldine Toutirais, Gwénola Doré, Daria Julkowska^*, Arounie Tavenet, and Philippe Bastin^*,

*Trypanosome Cell Biology Unit, Pasteur Institute and Centre National de la Recherche Scientifique, 75015 Paris, France; Dynamique et Régulation des Génomes, Muséum National d'Histoire Naturelle, Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique, 75005 Paris, France; and Biologie Fonctionnelle des Protozoaires, Muséum National d'Histoire Naturelle, 75005 Paris, France

Submitted August 3, 2007; Revised October 16, 2007; Accepted December 6, 2007
Monitoring Editor: Francis Barr

Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 µm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.

Address correspondence to: Philippe Bastin (pbastin{at}pasteur.fr)

Abbreviations used: IFA, immunofluorescence assay; IFT, intraflagellar transport; MAb, monoclonal antibody; PFA, paraformaldehyde; PFR, paraflagellar rod; PIFT, putatively involved in intraflagellar transport.