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Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The “9+2” axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked if there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana, have an extensive RSP complement including two divergent RSP9 orthologs, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither ortholog is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 ortholog. deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked with the different mode of assembly of trypanosomatid versus Plasmodium flagella.

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.

The movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green algaChlamydomonashas adapted this process for gliding motility, using IFT to move adhesive glycoproteins (FMG-1B) in the flagella membrane. Although Ca2+signalling contributes directly to the gliding process, uncertainty remains over the mechanisms through which Ca2+acts to influence the movement of IFT trains. Here we show that flagella Ca2+elevations regulate IFT primarily by initiating the movement of paused retrograde IFT trains. Flagella Ca2+elevations exhibit complex spatial and temporal properties, including high frequency repetitive Ca2+elevations that prevent the accumulation of paused retrograde IFT trains. We show that flagella Ca2+elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane. The results suggest that flagella Ca2+elevations directly disrupt the interaction between retrograde IFT particles and flagella membrane glycoproteins to modulate gliding motility and the adhesion of the flagellum to a surface.

Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22-74/81 interactions. Structure-based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP-loading is not strictly required.

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.

Cilium Conundrum The cilium has emerged as the antenna of eukaryotic cells, having numerous functions in sensory reception and developmental signaling. Several disorders, such as polycystic kidney disease, are the result of compromised cilia structure. Bhogaraju et al. (p. 1009 ) elucidate how the intraflagellar transport machinery recognizes tubulin, a ciliary cargo that is integral to cilium maintenance and formation and is constantly turned over at the cilium tip.

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.

Objective The role of Intraflagellar transport (IFT) in the construction of cilia and flagella is well established in numerous species. In contrast, its involvement in flagellum maintenance has only been shown in Chlamydomonas. Here, the protist Trypanosoma brucei was used as a model to investigate the role of IFT in flagellum maintenance. This organism has the advantage to maintain the existing flagellum whilst assembling the new one during the cell cycle. Knocking down the expression of any IFT protein inhibits flagellum formation in trypanosomes. Nevertheless, 25% of the cells still possess a flagellum of variable length. It was initially proposed that this was a consequence of RNAi that targets mRNA and not protein, hence IFT would still be active in these flagella.

Trypanosoma brucei is a flagellated eukaryotic pathogen responsible for sleeping sickness in central Africa. Because of the presence of a long motile flagellum (>20 μm) and its amenity to genetic manipulation, it is becoming an attractive model to study the assembly and the functions of cilia and flagella. In recent years, several aspects have been investigated, especially intraflagellar transport (IFT) that has been exhaustively characterized at the light microscopy level. In this manuscript, we review various methods to express fluorescent fusion proteins and to record IFT in living trypanosomes in normal or mutant contexts. We present an approach for separating anterograde and retrograde IFT, hence facilitating quantification of train speed, frequency, and size. A statistical analysis to discriminate different subpopulations of IFT trains is also summarized. These methods have proven their efficiency for the study of IFT in trypanosomes and could be applied to any other organism.

Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the α2 and α3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo . In E. coli , the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilus argR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.

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.