The Trypanosoma brucei flagellum is unusual as it is attached along the cell body and contains, in addition to an apparently conventional axoneme, a structure called the paraflagellar rod, which is essential for cell motility. Here, we investigated flagellum behaviour in normal and mutant trypanosome cell lines where expression of genes encoding various axoneme proteins (PF16, PF20, DNAI1, LC2) had been silenced by RNAi. First, we show that the propulsive wave (normally used for forward motility) is abolished in the absence of outer dynein arms, whereas the reverse wave (normally used for changing direction) still occurs. Second, in contrast to Chlamydomonas - but like metazoa, the central pair adopts a fixed orientation during flagellum beating. This orientation becomes highly variable in central-pair- and outer-dynein-arm-mutants. Third, the paraflagellar rod contributes to motility by facilitating three-dimensional wave propagation and controlling cell shape. Fourth, motility is required to complete the last stage of cell division in both insect and bloodstream stages of the parasite. Finally, our study also reveals the conservation of molecular components of the trypanosome flagellum. Coupled to the ease of reverse genetics, it raises the interest of trypanosomes as model organisms to study cilia and flagella.

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 microm/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.

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.

The atypical small G protein Rab-like 5 has been shown to traffic in sensory cilia of Caenorhabditis elegans, where it participates in signalling processes but not in cilia construction. In this report, we demonstrate that RABL5 colocalises with intraflagellar transport (IFT) proteins at the basal body and in the flagellum matrix of the protist Trypanosoma brucei. RABL5 fused to GFP exhibits anterograde movement in the flagellum of live trypanosomes, suggesting it could be associated with IFT. Accordingly, RABL5 accumulates in the short flagella of the retrograde IFT140RNAi mutant and is restricted to the basal body region in the IFT88RNAi anterograde mutant, a behaviour that is identical to other IFT proteins. Strikingly, RNAi silencing reveals an essential role for RABL5 in trypanosome flagellum construction. RNAi knock-down produces a phenotype similar to inactivation of retrograde IFT with formation of short flagella that are filled with a high amount of IFT proteins. These data reveal for the first time a functional difference for a conserved flagellar matrix protein between two different ciliated species and raise questions related to cilia diversity.

KIF9B localizes to the axoneme and basal body and is needed for flagella assembly, whereas KIF9A localizes only to the axoneme and controls flagella motility without affecting their structure.

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 IFT88RNAi or IFT140RNAi 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.

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes.

The distribution of glucocorticoid receptor-expressing cells was studied in the forebrain of the rainbow trout by means of antibodies produced against a fusion protein made of the NH2-terminal fragment of the rainbow trout glucocorticoid receptor fused in frame with glutathione-S-transferase. The results indicate that glucocorticoid receptor-expressing cells are located in many brain regions from the telencephalon to the spinal cord, with the highest density in the neuroendocrine component of the brain, the preoptic region and the mediobasal hypothalamus, and in the periventricular zone of the optic tectum. In virtually all cases, the labeling was located in the nucleus of the cells, although on very rare occasions, a slight labeling of the cytoplasm was detected. Concerning the preoptic region, the most striking feature was the high density of glucocorticoid receptors in the magnocellular preoptic nucleus, known to contain corticotrophin-releasing factor (CRF)-, vasotocin-, and isotocin-expressing cells. Colocalization experiments showed that 100% of the CRF-immunoreactive neurons in the preoptic nucleus express glucocorticoid receptors. In the mediobasal hypothalamus, the highest expression was found in the nucleus lateralis tuberis and parts of the nucleus recessus lateralis. Concerning the pituitary, the glucocorticoid receptor was consistently found in the rostral pars distalis, with the exception of the prolactin cells, and in the proximal pars distalis, which in trout contains thyrotrophs, gonadotrophs, and somatotrophs. In the hindbrain, expression of glucocorticoid receptors were localized mainly in the periventricular regions.