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Construction of cilia and flagella relies on Intraflagellar Transport (IFT). Although IFT proteins can be found in multiple locations in the cell, transport has only been reported along the axoneme. Here, we reveal that IFT concentration at the base of the flagellum of Trypanosoma brucei is required for proper assembly of IFT trains. Using live cell imaging at high resolution and direct optical nanoscopy with axially localized detection (DONALD) of fixed trypanosomes, we demonstrate that IFT proteins are localised around the 9 doublet microtubules of the proximal portion of the transition zone, just on top of the transition fibres. Super-resolution microscopy and photobleaching studies reveal that knockdown of the RP2 transition fibre protein results in reduced IFT protein concentration and turnover at the base of the flagellum. This in turn is accompanied by a 4- to 8-fold drop in the frequency of IFT train injection. We propose that the flagellum base provides a unique environment to assemble IFT trains.

The movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using retrograde IFT motors to move adhesive glycoproteins in the flagella membrane. Ca2+ signalling contributes directly to the gliding process, although uncertainty remains over the mechanism through which it acts. Here, we show that flagella Ca2+ elevations initiate the movement of paused retrograde IFT trains, which accumulate at the distal end of adherent flagella, but do not influence other IFT processes. On highly adherent surfaces, flagella exhibit high-frequency Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. Flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane, suggesting that Ca2+ acts by directly disrupting an interaction between retrograde IFT trains and flagella membrane glycoproteins. By regulating the extent to which glycoproteins on the flagella surface interact with IFT motor proteins on the axoneme, this signalling mechanism allows precise control of traction force and gliding motility in adherent flagella.

Intraflagellar transport (IFT) is the movement of large protein complexes responsible for the construction of cilia and flagella. Using a combination of three-dimensional electron microscopy and high-resolution live imaging, Bertiaux et al. show that IFT takes place on only four microtubule doublets out of the nine available in the trypanosome flagellum., Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3–4 and 7–8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3–4 s while remaining on the same side of the axoneme.

Les cils et flagelles sont des organites essentiels chez la plupart des eucaryotes, y compris chez l’humain. Dans cette revue, nous abordons le mode d’assemblage de ces organites complexes, qui fait intervenir un processus dynamique denomme transport intraflagellaire. Le transport intraflagellaire apporte les elements structuraux au bout distal du cil ou a lieu l’assemblage, permettant ainsi la croissance de l’organite. Nous exposons ensuite trois modeles differents de controle de la longueur du cil avant de discuter de leur alteration lors des ciliopathies, maladies genetiques associees aux defauts ciliaires.

International audience; Intraflagellar transport (IFT) is required for construction of most cilia and flagella. Here, we used electron microscopy, immunofluorescence and live video microscopy to show that IFT is absent or arrested in the mature flagellum of Trypanosoma brucei upon RNA interference (RNAi)-mediated knockdown of IFT88 and IFT140, respectively. Flagella assembled prior to RNAi did not shorten, showing that IFT is not essential for the maintenance of flagella length. Although the ultrastructure of the axoneme was not visibly affected, flagellar beating was strongly reduced and the distribution of several flagellar components was drastically modified. The R subunit of the protein kinase A was no longer concentrated in the flagellum but was largely found in the cell body whereas the kinesin 9B motor was accumulating at the distal tip of the flagellum. In contrast, the distal tip protein FLAM8 was dispersed along the flagellum. This reveals that IFT also functions in maintaining the distribution of some flagellar proteins after construction of the organelle is completed.

International audience; Cilia and flagella are essential organelles in most eukaryotes including human beings. In this review, we will discuss the mode of assembly of these complex organelles that depends on a dynamic process called intraflagellar transport or IFT. IFT delivers structural elements at the distal end of the cilium where assembly takes place, thereby allowing the growth of the organelle. We next discuss the different models for control of cilium length and their alterations in ciliopathies, genetic diseases associated to ciliary defects.; Les cils et flagelles sont des organites essentiels chez la plupart des eucaryotes, y compris chez l’humain. Dans cette revue, nous abordons le mode d’assemblage de ces organites complexes, qui fait intervenir un processus dynamique dénommé transport intraflagellaire. Le transport intraflagellaire apporte les éléments structuraux au bout distal du cil où a lieu l’assemblage, permettant ainsi la croissance de l’organite. Nous exposons ensuite trois modèles différents de contrôle de la longueur du cil avant de discuter de leur altération lors des ciliopathies, maladies génétiques associées aux défauts ciliaires.