Restriction of intraflagellar transport to some microtubule doublets: An opportunity for cilia diversification?

Cilia are unique eukaryotic organelles and exhibit remarkable conservation across evolution. Nevertheless, very different types of configurations are encountered, raising the question of their evolution. Cilia are constructed by intraflagellar transport (IFT), the movement of large protein complexes or trains that deliver cilia components to the distal tip for assembly. Recent data revealed that IFT trains are restricted to some but not all nine doublet microtubules in the protist Trypanosoma brucei. Here, we propose that restricted positioning of IFT trains could offer potent options for cilia to evolve towards more complex (addition of new structural elements like in spermatozoa) or simpler configuration (loss of some elements like in primary cilia), and therefore be a driver of cilia diversification. We present two hypotheses to explain how IFT trains could be restricted to some doublets, either by a triage process taking place at the basal body level or by the development of molecular differences between ciliary microtubules.

Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly.

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.

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands.

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

Intraflagellar transport during assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies.

Multicellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. Trypanosoma brucei assembles flagella of 3 to 30 µm during its development in the tsetse fly. This provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3D electron microscopy and live-cell imaging revealed that IFT was present in all T. brucei life cycle stages. IFT proteins are concentrated at the base, and IFT trains are located along doublets 3-4 and 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of flagellar IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.

Redistribution of FLAgellar Member 8 during the trypanosome life cycle: Consequences for cell fate prediction.

The single flagellum of African trypanosomes is essential in multiple aspects of the parasites' development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms of Trypanosoma brucei, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other parasite cycle stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian-infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating the remodelling of an existing flagellum. In the tsetse cardia, FLAM8 further localises to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum were seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered a meta-marker of the flagellum stage and maturation state in trypanosomes.

Biallelic Mutations in LRRC56, Encoding a Protein Associated with Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects.

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

IFT25 is required for the construction of the trypanosome flagellum.

Intraflagellar transport (IFT), the movement of protein complexes responsible for the assembly of cilia and flagella, is remarkably conserved from protists to humans. However, two IFT components (IFT25 and IFT27) are missing from multiple unrelated eukaryotic species. In mouse, IFT25 (also known as HSPB11) and IFT27 are not required for assembly of several cilia with the noticeable exception of the flagellum of spermatozoa. Here, we show that the Trypanosoma brucei IFT25 protein is a proper component of the IFT-B complex and displays typical IFT trafficking. By performing bimolecular fluorescence complementation assays, we reveal that IFT25 and IFT27 interact within the flagellum in live cells during the IFT process. IFT25-depleted cells construct tiny disorganised flagella that accumulate IFT-B proteins (with the exception of IFT27, the binding partner of IFT25) but not IFT-A proteins. This phenotype is comparable to the one following depletion of IFT27 and shows that IFT25 and IFT27 constitute a specific module that is necessary for proper IFT and flagellum construction in trypanosomes. Possible reasons why IFT25 and IFT27 would be required for only some types of cilia are discussed.

Dealing with several flagella in the same cell.

Flagella are sophisticated organelles found in many eukaryotic microbes where they perform functions related to motility, signal detection, or cell morphogenesis. In many cases, several flagella are present per cell, and these can have a different composition, length, age, or function, raising the question of how this is managed. When the flagella are equivalent and constructed simultaneously such as in Chlamydomonas or Naegleria, we propose an equal access model where molecular components have free access to each organelle. By contrast, Trypanosoma and Leishmania contain temporally distinct organelles and elongate a new flagellum whilst maintaining the existing one. The equal access model could function providing that the mature flagellum is "locked" so that it can no longer be elongated or shortened. Alternatively, access of flagellar components could be restricted at the level of the basal body, the transition zone, or the loading on intraflagellar transport trains. In organisms that contains flagella of different age and composition such as Giardia, a temporal dimension is necessary, with the production of protein components of flagella spreading over one or more cell cycles. In the future, deciphering the molecular mechanisms involved in these processes should reveal new insights in flagellum assembly and function.

Ultrastructural Changes of the Mitochondrion During the Life Cycle of Trypanosoma brucei.

The mitochondrion is crucial for ATP generation by oxidative phosphorylation, among other processes. Cristae are invaginations of the mitochondrial inner membrane that house nearly all the macromolecular complexes that perform oxidative phosphorylation. The unicellular parasite Trypanosoma brucei undergoes during its life cycle extensive remodeling of its single mitochondrion, which reflects major changes in its energy metabolism. While the bloodstream form (BSF) generates ATP exclusively by substrate-level phosphorylation and has a morphologically highly reduced mitochondrion, the insect-dwelling procyclic form (PCF) performs oxidative phosphorylation and has an expanded and reticulated organelle. Here, we have performed high-resolution 3D reconstruction of BSF and PCF mitochondria, with a particular focus on their cristae. By measuring the volumes and surface areas of these structures in complete or nearly complete cells, we have found that mitochondrial cristae are more prominent in BSF than previously thought and their biogenesis seems to be maintained during the cell cycle. Furthermore, PCF cristae exhibit a surprising range of volumes in situ, implying that each crista is acting as an independent bioenergetic unit. Cristae appear to be particularly enriched in the region of the organelle between the nucleus and kinetoplast, the mitochondrial genome, suggesting this part has distinctive properties.

Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth.

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 µM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3'-oxime bearing an extra bulky substituent on the 3' oxime [(6-BIO-3'-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3'-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3'-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

Flagellar incorporation of proteins follows at least two different routes in trypanosomes.

BACKGROUND INFORMATION: Eukaryotic cilia and flagella are sophisticated organelles composed of several hundreds of proteins that need to be incorporated at the right time and the right place during assembly. RESULTS: Two methods were used to investigate this process in the model protist Trypanosoma brucei: inducible expression of epitope-tagged labelled proteins and fluorescence recovery after photobleaching of fluorescent fusion proteins. This revealed that skeletal components of the radial spokes (RSP3), the central pair (PF16) and the outer dynein arms (DNAI1) are incorporated at the distal end of the growing flagellum. They display low or even no visible turnover in mature flagella, a finding further confirmed by monitoring a heavy chain of the outer dynein arm. In contrast, the membrane-associated protein arginine kinase 3 (AK3) showed rapid turnover in both growing and mature flagella, without particular polarity and independently of intraflagellar transport. CONCLUSION: These results demonstrate different modes of incorporation for structural and membrane-associated proteins in flagella. SIGNIFICANCE: The existence of two distinct modes for incorporation of proteins in growing flagella suggests the existence of different targeting machineries. Moreover, the absence of turnover of structural elements supports the view that the length of the mature flagellum in trypanosomes is not modified after assembly.

STEM tomography analysis of the trypanosome transition zone.

The protist Trypanosoma brucei is an emerging model for the study of cilia and flagella. Here, we used scanning transmission electron microscopy (STEM) tomography to describe the structure of the trypanosome transition zone (TZ). At the base of the TZ, nine transition fibres irradiate from the B microtubule of each doublet towards the membrane. The TZ adopts a 9 + 0 structure throughout its length of ∼300 nm and its lumen contains an electron-dense structure. The proximal portion of the TZ has an invariant length of 150 nm and is characterised by a collarette surrounding the membrane and the presence of electron-dense material between the membrane and the doublets. The distal portion exhibits more length variation (from 55 to 235 nm) and contains typical Y-links. STEM analysis revealed a more complex organisation of the Y-links compared to what was reported by conventional transmission electron microscopy. Observation of the very early phase of flagellum assembly demonstrated that the proximal portion and the collarette are assembled early during construction. The presence of the flagella connector that maintains the tip of the new flagellum to the side of the old was confirmed and additional filamentous structures making contact with the membrane of the flagellar pocket were also detected. The structure and potential functions of the TZ in trypanosomes are discussed, as well as its mode of assembly.

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length.

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.

Flagellar adhesion in Trypanosoma brucei relies on interactions between different skeletal structures in the flagellum and cell body.

The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.

IFT81, encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype.

BACKGROUND: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. METHODS: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. RESULTS: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. CONCLUSIONS: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.

Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.

The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage-specific phosphorylation status.

During its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40-/- promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40-/- parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40-/- infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania-specific phosphorylation sites and their role in regulating parasite protein function.

Intraflagellar transport proteins cycle between the flagellum and its base.

Intraflagellar transport (IFT) is necessary for the construction of cilia and flagella. IFT proteins are concentrated at the base of the flagellum but little is known about the actual role of this pool of proteins. Here, IFT was investigated in Trypanosoma brucei, an attractive model for flagellum studies, using GFP fusions with IFT52 or the IFT dynein heavy chain DHC2.1. Tracking analysis by a curvelet method allowing automated separation of forward and return transport demonstrated a uniform speed for retrograde IFT (5 µm s(-1)) but two distinct populations for anterograde movement that are sensitive to temperature. When they reach the distal tip, anterograde trains are split into three and converted to retrograde trains. When a fast anterograde train catches up with a slow one, it is almost twice as likely to fuse with it rather than to overtake it, implying that these trains travel on a restricted set of microtubules. Using photobleaching experiments, we show for the first time that IFT proteins coming back from the flagellum are mixed with those present at the flagellum base and can reiterate a full IFT cycle in the flagellum. This recycling is dependent on flagellum length and IFT velocities. Mathematical modelling integrating all parameters actually reveals the existence of two pools of IFT proteins at the flagellum base, but only one is actively engaged in IFT.

A new asymmetric division contributes to the continuous production of infective trypanosomes in the tsetse fly.

African trypanosomes are flagellated protozoan parasites that cause sleeping sickness and are transmitted by the bite of the tsetse fly. To complete their life cycle in the insect, trypanosomes reach the salivary glands and transform into the metacyclic infective form. The latter are expelled with the saliva at each blood meal during the whole life of the insect. Here, we reveal a means by which the continuous production of infective parasites could be ensured. Dividing trypanosomes present in the salivary glands of infected tsetse flies were monitored by live video-microscopy and by quantitative immunofluorescence analysis using molecular markers for the cytoskeleton and for surface antigens. This revealed the existence of two distinct modes of trypanosome proliferation occurring simultaneously in the salivary glands. The first cycle produces two equivalent cells that are not competent for infection and are attached to the epithelium. This mode of proliferation is predominant at the early steps of infection, ensuring a rapid colonization of the glands. The second mode is more frequent at later stages of infection and involves an asymmetric division. It produces a daughter cell that matures into the infective metacyclic form that is released in the saliva, as demonstrated by the expression of specific molecular markers - the calflagins. The levels of these calcium-binding proteins increase exclusively in the new flagellum during the asymmetric division, showing the commitment of the future daughter cell to differentiation. The coordination of these two alternative cell cycles contributes to the continuous production of infective parasites, turning the tsetse fly into an efficient and long-lasting vector for African trypanosomes.

Forward motility is essential for trypanosome infection in the tsetse fly.

African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.