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MBC in Press, published online ahead of print December 19, 2007
Mol. Biol. Cell 10.1091/mbc.E07-08-0749

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Submitted on August 3, 2007
Revised on October 16, 2007
Accepted on December 6, 2007

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

Sabrina Absalon,*{dagger} Thierry Blisnick,* Linda Kohl,{dagger}{ddagger} Géraldine Toutirais,{dagger} Gwénola Doré,{dagger} Daria Julkowska,* Arounie Tavenet,{dagger} and Philippe Bastin*{dagger}

*Trypanosome Cell Biology Unit, Pasteur Institute and Centre National de la Recherche Scientifique (CNRS), 75015 Paris, France; {dagger}Dynamique et Régulation des Génomes, Muséum National d’Histoire Naturelle, Institut National de la Santé et de la Recherche Médicale and CNRS, 75005 Paris, France; {ddagger}Biologie Fonctionnelle des Protozoaires, Muséum National d’Histoire Naturelle, 75005 Paris, France

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 9 conventional IFT genes and 5 novel Putative IFT genes (PIFT) in Trypanosoma brucei, that maintains its existing flagellum while assembling a new one. Immunostaining against IFT172 or expression of tagged IFT20 or 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. RNAi 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, 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 3 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)




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