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.

A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes.

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, TbFOPL and TbOFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei, showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, TbFOPL, affects neither axoneme nor flagellum elongation. Instead, TbFOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar TbFOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, TbFOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for TbFOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of TbFOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication.

We report an optimised centrifugal counter-flow elutriation protocol for the rapid and direct isolation of G1 cell cycle synchronised populations of both the procyclic and bloodstream form stages of Trypanosoma brucei that yields viable and proliferative cells. The high quality of the synchronisation achieved can be judged by the uniform DNA content, narrow size distribution, synchronous division, and the maintenance of synchronicity into subsequent cell cycles. We show that early-eluting fractions represent different G1 subpopulations that progress through the cell cycle with distinct temporal profiles post-elutriation, as exemplified by the observation of the maturation of a second flagellar basal body in late G1 phase, DNA replication in S phase, and dimethylation of histone H3 in mitosis/cytokinesis. We use our temporal observations to construct a revised model of the relative timing and duration of the nuclear and kinetoplast cell cycle that differs from the current model.

Variation in Basal Body Localisation and Targeting of Trypanosome RP2 and FOR20 Proteins.

TOF-LisH-PLL motifs define FOP family proteins; some members are involved in flagellum assembly. The critical role of FOP family protein FOR20 is poorly understood. Here, we report relative localisations of the four FOP family proteins in parasitic Trypanosoma brucei: TbRP2, TbOFD1 and TbFOP/FOP1-like are mature basal body proteins whereas TbFOR20 is present on pro- and mature basal bodies - on the latter it localises distal to TbRP2. We discuss how the data, together with published work for another protist Giardia intestinalis, informs on likely FOR20 function. Moreover, our localisation study provides convincing evidence that the antigen recognised by monoclonal antibody YL1/2 at trypanosome mature basal bodies is FOP family protein TbRP2, not tyrosinated α-tubulin as widely stated in the literature. Curiously, FOR20 proteins from T. brucei and closely related African trypanosomes possess short, negatively-charged N-terminal extensions absent from FOR20 in other trypanosomatids and other eukaryotes. The extension is necessary for protein targeting, but insufficient to re-direct TbRP2 to probasal bodies. Yet, FOR20 from the American trypanosome T. cruzi, which lacks any extension, localises to pro- and mature basal bodies when expressed in T. brucei. This identifies unexpected variation in FOR20 architecture that is presently unique to one clade of trypanosomatids.

Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions.

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

An alternative model for the role of RP2 protein in flagellum assembly in the African trypanosome.

The tubulin cofactor C domain-containing protein TbRP2 is a basal body (centriolar) protein essential for axoneme formation in the flagellate protist Trypanosoma brucei, the causal agent of African sleeping sickness. Here, we show how TbRP2 is targeted and tethered at mature basal bodies and provide novel insight into TbRP2 function. Regarding targeting, understanding how several hundred proteins combine to build a microtubule axoneme is a fundamental challenge in eukaryotic cell biology. We show that basal body localization of TbRP2 is mediated by twinned, N-terminal TOF (TON1, OFD1, and FOP) and LisH motifs, motifs that otherwise facilitate localization of only a few conserved proteins at microtubule-organizing centers in animals, plants, and flagellate protists. Regarding TbRP2 function, there is a debate as to whether the flagellar assembly function of specialized, centriolar tubulin cofactor C domain-containing proteins is processing tubulin, the major component of axonemes, or general vesicular trafficking in a flagellum assembly context. Here we report that TbRP2 is required for the recruitment of T. brucei orthologs of MKS1 and MKS6, proteins that, in animal cells, are part of a complex that assembles at the base of the flagellum to regulate protein composition and cilium function. We also identify that TbRP2 is detected by YL1/2, an antibody classically used to detect α-tubulin. Together, these data suggest a general processing role for TbRP2 in trypanosome flagellum assembly and challenge the notion that TbRP2 functions solely in assessing tubulin "quality" prior to tubulin incorporation into the elongating axoneme.

The tubulin cofactor C family member TBCCD1 orchestrates cytoskeletal filament formation.

TBCCD1 is an enigmatic member of the tubulin-binding cofactor C (TBCC) family of proteins required for mother-daughter centriole linkage in the green alga Chlamydomonas reinhardtii and nucleus-centrosome-Golgi linkage in mammalian cells. Loss of these linkages has severe morphogenetic consequences, but the mechanism(s) through which TBCCD1 contributes to cell organisation is unknown. In the African sleeping sickness parasite Trypanosoma brucei a microtubule-dominant cytoskeleton dictates cell shape, influencing strongly the positioning and inheritance patterns of key intracellular organelles. Here, we show the trypanosome orthologue of TBCCD1 is found at multiple locations: centrioles, the centriole-associated Golgi 'bi-lobe', and the anterior end of the cell body. Loss of Trypanosoma brucei TBCCD1 results in disorganisation of the structurally complex bi-lobe architecture and loss of centriole linkage to the single unit-copy mitochondrial genome (or kinetoplast) of the parasite. We therefore identify TBCCD1 as an essential protein associated with at least two filament-based structures in the trypanosome cytoskeleton. The last common ancestor of trypanosomes, animals and green algae was arguably the last common ancestor of all eukaryotes. On the basis of our observations, and interpretation of published data, we argue for an unexpected co-option of the TBCC domain for an essential non-tubulin-related function at an early point during evolution of the eukaryotic cytoskeleton.

Flagellum assembly and function during the Leishmania life cycle.

During a complex digenetic life cycle flagellated Leishmania parasites alternate between promastigote and amastigote forms which differ significantly in cellular morphology and flagellum length. Recent studies have provided important new insights into mechanisms by which Leishmania regulate expression of genes required for flagellum assembly, and mechanisms used to modify flagellum length. While the critical role of the promastigote flagellum in parasite biology has long been appreciated, the importance of the amastigote flagellum has often been disregarded. However, recent work suggests that the 'rudimentary' amastigote flagellum may serve indispensable roles in cellular organisation, and/or sensory perception, which are critical for intracellular survival of Leishmania within host macrophages.

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy.

The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone.

Combining RNA interference mutants and comparative proteomics to identify protein components and dependences in a eukaryotic flagellum.

Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.

Swimming with protists: perception, motility and flagellum assembly.

In unicellular and multicellular eukaryotes, fast cell motility and rapid movement of material over cell surfaces are often mediated by ciliary or flagellar beating. The conserved defining structure in most motile cilia and flagella is the '9+2' microtubule axoneme. Our general understanding of flagellum assembly and the regulation of flagellar motility has been led by results from seminal studies of flagellate protozoa and algae. Here we review recent work relating to various aspects of protist physiology and cell biology. In particular, we discuss energy metabolism in eukaryotic flagella, modifications to the canonical assembly pathway and flagellum function in parasite virulence.

An essential quality control mechanism at the eukaryotic basal body prior to intraflagellar transport.

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.

Trypanosome IFT mutants provide insight into the motor location for mobility of the flagella connector and flagellar membrane formation.

The flagella connector (FC) of procyclic trypanosomes is a mobile, transmembrane junction important in providing cytotactic morphogenetic information to the daughter cell. Quantitative analyses of FC positioning along the old flagellum, involving direct observations and use of the MPM2 anti-phosphoprotein monoclonal reveals a ;stop point' is reached on the old flagellum which correlates well with the initiation of basal body migration and kinetoplast segregation. This demonstrates further complexities of the FC and its movement in morphogenetic events in trypanosomes than have hitherto been described. We used intraflagellar transport RNAi mutants to ablate the formation of a new flagellum. Intriguingly the FC could still move, indicating that a motor function beyond the new flagellum is sufficient to move it. When such a FC moves, it drags a sleeve of new flagellar membrane out of the flagellar pocket. This axoneme-less flagellar membrane maintains appropriate developmental relationships to the cell body including following the correct helical path and being connected to the internal cytoskeleton by macula adherens junctions. Movement of the FC in the apparent absence of intraflagellar transport raises the possibility of a new form of motility within a eukaryotic flagellum.

Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes.

Productive beating of eukaryotic flagella and cilia requires a strict regulation of axonemal dynein activation. Fundamental to any description of axonemal beating is an understanding of the significance of the central pair microtubules and the degree to which central pair rotation has a role. However, for the majority of organisms, it is unclear whether the central pair actually rotates. Using an extra-axonemal structure as a fixed reference, we analysed the orientation of the central pair in African trypanosomes and other kinetoplastid protozoa. A geometric correction allowed the superposition of data from many cross-sections, demonstrating that the axis of the central pair is invariant and that there is no central pair rotation in these organisms. Analysis of mutants depleted in particular flagellar and basal body proteins [gamma-tubulin, delta-tubulin, Parkin co-regulated gene product (PACRG) or the paraflagellar rod protein PFR2] allowed a dissection of the mechanisms for central pair constraint. This demonstrated that orientation is independent of flagellum attachment and beating, but is influenced by constraints along its length and is entirely dependent on correct positioning at the basal plate.

Flagellar motility is required for the viability of the bloodstream trypanosome.

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.

The flagella connector of Trypanosoma brucei: an unusual mobile transmembrane junction.

Throughout its elongation, the new flagellum of the procyclic form of the African trypanosome Trypanosoma brucei is tethered at its tip to the lateral aspect of the old flagellum. This phenomenon provides a cytotactic mechanism for influencing inheritance of cellular pattern. Here, we show that this tethering is produced via a discrete, mobile transmembrane junction - the flagella connector. Light and electron microscopy reveal that the flagella connector links the extending microtubules at the tip of the new flagellum to the lateral aspect of three of the doublet microtubules in the old flagellar axoneme. Two sets of filaments connect the microtubules to three plates on the inner faces of the old and new flagellar membranes. Three differentiated areas of old and new flagellar membranes are then juxtaposed and connected by a central interstitial core of electron-dense material. The flagella connector is formed early in flagellum extension and is removed at the end of cytokinesis, but the exact timing of the latter event is slightly variable. The flagella connector represents a novel form of cellular junction that is both dynamic and mobile.

Coordination of cell cycle and cytokinesis in Trypanosoma brucei.

In common with all eukaryotic cells, trypanosomes must coordinate a complex series of morphogenetic events both temporally and spatially during the cell cycle. The structural and molecular cues that synchronise these events in trypanosomes have started to be elucidated, and intriguingly although similarities to cell cycle events in other eukaryotes can be identified, trypanosomes have also evolved novel solutions to the common challenges faced by dividing eukaryotic cells. Although cellular morphology is clearly pivotal for successful progression through the trypanosome cell cycle, most cytological studies to date have focused exclusively on procyclic form trypanosomes. These studies provide an excellent framework for understanding cell cycle events in trypanosomes, however recent data indicates that profound differences might exist between different life cycle stages in relation to the regulation of cell cycle and cytokinesis.

Gamma-tubulin functions in the nucleation of a discrete subset of microtubules in the eukaryotic flagellum.

gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.

Myristoyl-CoA:protein N-myristoyltransferase, an essential enzyme and potential drug target in kinetoplastid parasites.

Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions. The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei. In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions. Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli. Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania. In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death. This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents.