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First published online 21 October 2008
doi: 10.1242/jcs.035626

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Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei

Sabrina Absalon^1,2,*, Thierry Blisnick^1,*, Mélanie Bonhivers³, Linda Kohl⁴, Nadège Cayet⁵, Géraldine Toutirais², Johanna Buisson¹, Derrick Robinson³ and Philippe Bastin^1,2,

¹ Trypanosome Cell Biology Unit, Pasteur Institute and CNRS, 25 rue du Docteur Roux, 75015 Paris, France
² Dynamique et Régulation des Génomes, Muséum National d'Histoire Naturelle, INSERM and CNRS, 43 rue Cuvier, 75005 Paris, France
³ Microbiologie Cellulaire et Moléculaire et Pathogénicité, Université Bordeaux 2 and CNRS, 146 Rue Léo Saignat, 33076 Bordeaux, France
⁴ Biologie Fonctionnelle des Protozoaires, Muséum National d'Histoire Naturelle, 61 rue Buffon, 75005 Paris, France
⁵ Plateforme de Microscopie Electronique, Pasteur Institute, 25 rue du Docteur Roux, 75015 Paris, France

Author for correspondence (e-mail: philippe.bastin{at}pasteur.fr)

Accepted 14 August 2008

In trypanosomes, the flagellum is rooted in the flagellar pocket, a surface micro-domain that is the sole site for endocytosis and exocytosis. By analysis of anterograde or retrograde intraflagellar transport in IFT88^RNAi or IFT140^RNAi mutant cells, we show that elongation of the new flagellum is not required for flagellar pocket formation but is essential for its organisation, orientation and function. Transmission electron microscopy revealed that the flagellar pocket exhibited a modified shape (smaller, distorted and/or deeper) in cells with abnormally short or no flagella. Scanning electron microscopy analysis of intact and detergent-extracted cells demonstrated that the orientation of the flagellar pocket collar was more variable in trypanosomes with short flagella. The structural protein BILBO1 was present but its localisation and abundance was altered. The membrane flagellar pocket protein CRAM leaked out of the pocket and reached the short flagella. CRAM also accumulated in intracellular compartments, indicating defects in routing of resident flagellar pocket proteins. Perturbations of vesicular trafficking were obvious; vesicles were observed in the lumen of the flagellar pocket or in the short flagella, and fluid-phase endocytosis was drastically diminished in non-flagellated cells. We propose a model to explain the role of flagellum elongation in correct flagellar pocket organisation and function.

Key words: Flagellum, Flagellar pocket, Endocytosis, Intraflagellar transport

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