Professor Sue Vaughan

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

Leishmania are flagellated eukaryotic parasites that cause leishmaniasis and are closely related to the other kinetoplastid parasites such as Trypanosoma brucei. In these parasites there is a cell membrane invagination at the base of the flagellum called the flagellar pocket, which is tightly associated with and sculpted by cytoskeletal structures including the flagellum attachment zone (FAZ). The FAZ is a complex interconnected structure linking the flagellum to the cell body and has critical roles in cell morphogenesis, function and pathogenicity. However, this structure varies dramatically in size and organisation between these different parasites, suggesting changes in protein localisation and function. Here, we screened the localisation and function of the Leishmania orthologs of T. brucei FAZ proteins identified in the genome-wide protein tagging project TrypTag. We identified 27 FAZ proteins and our deletion analysis showed that deletion of two FAZ proteins in the flagellum, FAZ27 and FAZ34 resulted in a reduction in cell body size, and flagellum loss in some cells. Furthermore, after null mutant generation, we observed distinct and reproducible changes to cell shape, demonstrating the ability of the parasite to adapt to morphological perturbations resulting from gene deletion. This process of adaptation has important implications for the study of Leishmania mutants.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown function. Using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localisation of 89% of the T. brucei proteome. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultra-conserved cellular architecture of eukaryotes, including the first comprehensive ‘cartographic’ analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

The closely-related parasites Trypanosoma brucei, T. congolense, and T. vivax cause neglected tropical diseases collectively known as African Trypanosomiasis. A characteristic feature of bloodstream form T. brucei is the flagellum that is laterally attached to the side of the cell body. During the cell cycle, the new flagellum is formed alongside the old flagellum, with the new flagellum tip embedded within a mobile transmembrane junction called the groove. The molecular composition of the groove is currently unknown, which limits the analysis of this junction and assessment of its conservation in related trypanosomatids. Here, we identified 13 proteins that localise to the flagellar groove through a small-scale tagging screen. Functional analysis of a subset of these proteins by RNAi and gene deletion revealed three proteins, FCP4/TbKin15, FCP7, and FAZ45, that are involved in new flagellum tip attachment to the groove. Despite possessing orthologues of all 13 groove proteins, T. congolense and T. vivax did not assemble a canonical groove around the new flagellum tip according to 3D electron microscopy. This diversity in new flagellum tip attachment points to the rapid evolution of membrane-cytoskeleton structures that can occur without large changes in gene complement and likely reflects the niche specialisation of each species.

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm

The centriole/basal body (CBB) is an evolutionarily conserved organelle acting as a microtubule organising centre (MTOC) to nucleate cilia, flagella, and the centrosome. SAS4/CPAP is a conserved component associated with BB biogenesis in many model flagellated cells. Plasmodium, a divergent unicellular
eukaryote and causative agent of malaria, displays an atypical, closed mitosis with an MTOC (or centriolar plaque), reminiscent of an acentriolar MTOC, embedded in the nuclear membrane. Mitosis during male gamete formation is accompanied by flagella formation. There are two MTOCs in male gametocytes: the acentriolar nuclear envelope MTOC for the mitotic spindle and an outer centriolar MTOC (the basal body) that organises flagella assembly in the cytoplasm. We show the coordinated location, association and assembly of SAS4 with the BB component, kinesin8B, but no association with the kinetochore protein, NDC80, indicating that SAS4 is part of the BB and outer centriolar MTOC in the cytoplasm. Deletion of the SAS4 gene produced no phenotype, indicating that it is not essential for either male gamete formation or parasite transmission.

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time livecell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are flagellate eukaryotic parasites that cause serious diseases in humans and animals. These parasites have cell shapes defined by a subpellicular microtubule array and all share a number of important cellular features. One of these is the flagellar pocket, an invagination of the cell membrane around the proximal end of the flagellum, which is an important organelle for endo/exocytosis. The flagellar pocket plays a crucial role in parasite pathogenicity and persistence in the host and has a great influence on cell morphogenesis and cell division. Here, we compare the morphology and function of the flagellar pockets between different trypanosomatids, with their life cycles and ecological niches likely influencing these differences.

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to “lock” at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei . CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

Eukaryotic flagella are complex microtubule based organelles and in many organisms there are extra axonemal structures present, including the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition fibre-transition zone, and the flagellum. It begins with translation of protein components, followed by their sorting and trafficking into the flagellum, transport to the assembly site and then incorporation. Flagella are formed from over 500 proteins; the principles governing axonemal component assembly are relatively clear. However, the coordination and sites of extra-axonemal structure assembly processes are less clear. We have discovered two cytoplasmic proteins in T. brucei that are required for PFR formation, PFR assembly factors 1 and 2. Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The presence of cytoplasmic factors required for PFR formation aligns with the concept of processes occurring across multiple compartments to facilitate axoneme assembly and this is likely a common theme for extra-axonemal structure assembly.

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B–GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum, and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence. 

Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts.\nWe report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids.\nOur data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

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.

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.

Flagella are highly conserved organelles present in a wide variety of species. In Trypanosoma brucei the single flagellum is necessary for morphogenesis, cell motility and pathogenesis, and is attached along the cell body. A new flagellum is formed alongside the old during the cell division cycle. In the (insect) procyclic form, the flagella connector (FC) attaches the tip of the new flagellum to the side of the old flagellum, ensuring faithful replication of cell architecture. The FC is not present in the bloodstream form of the parasite. We show here, using new imaging techniques including serial block-face scanning electron microscopy (SBF-SEM), that the distal tip of the new flagellum in the bloodstream form is embedded within an invagination in the cell body plasma membrane, named the groove. We suggest that the groove has a similar function to the flagella connector. The groove is a mobile junction located alongside the microtubule quartet (MtQ) and occurred within a gap in the subpellicular microtubule corset, causing significant modification of microtubules during elongation of the new flagellum. It appears likely that this novel form of morphogenetic structure has evolved to withstand the hostile immune response in the mammalian blood.

Trypanosomes use a microtubule-focused mechanism for cell morphogenesis and cytokinesis. We used scanning electron and video microscopy of living cells to provide the first detailed description of cell morphogenesis and cytokinesis in the early-branching eukaryote Trypanosoma brucei. We outline four distinct stages of cytokinesis and show that an asymmetric division fold bisects the two daughter cells, with a cytoplasmic bridge-like structure connecting the two daughters immediately prior to abscission. Using detection of tyrosinated α-tubulin as a marker for new or growing microtubules and expression of XMAP215, a plus end binding protein, as a marker for microtubule plus ends we demonstrate spatial asymmetry in the underlying microtubule cytoskeleton throughout the cell division cycle. This leads to inheritance of different microtubule cytoskeletal patterns and demonstrates the major role of microtubules in achieving cytokinesis. RNA interference techniques have led to a large set of mutants, often with variations in phenotype between procyclic and bloodstream life cycle forms. Here, we show morphogenetic differences between these two life cycle forms of this parasite during new flagellum growth and cytokinesis. These discoveries are important tools to explain differences between bloodstream and procyclic form RNAi phenotypes involving organelle mis-positioning during cell division and cytokinesis defects.

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

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.

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.

Trypanosomes and Leishmanias are important human parasites whose cellular architecture is centred on the single flagellum. In trypanosomes, this flagellum is attached to the cell along a complex flagellum attachment zone (FAZ), comprising flagellar and cytoplasmic components, the integrity of which is required for correct cell morphogenesis and division. The cytoplasmic FAZ cytoskeleton is conspicuously associated with a sheet of endoplasmic reticulum termed the ‘FAZ ER’. In the present work, 3D electron tomography of bloodstream form trypanosomes was used to clarify the nature of the FAZ ER. We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket. TbVAP is an orthologue of VAMP-associated proteins (VAPs), integral ER membrane proteins whose mutation in humans has been linked to familial motor neuron disease. The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket. Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.

Centrioles are found in nearly all eukaryotic cells and are required for growth and maintenance of the radial array of microtubules, the mitotic spindle, and cilia and flagella. Different types of microtubule structures are often required at different places in a given cell; centrioles must move around to nucleate these varied structures. Here, we draw together recent data on diverse centriole movements to decipher common themes in how centrioles move. Par proteins establish and maintain the required cellular asymmetry. The actin cytoskeleton facilitates movement of multiple basal bodies. Microtubule forces acting on the cell cortex, and nuclear cytoskeletal links, are important for positioning individual centrosomes, and during cell division. Knowledge of these common mechanisms can inform the study of centriole movements across biology.

Eukaryotic flagella are microtubule-based structures required for a variety of functions including cell motility and sensory perception. Most eukaryotic flagella grow out from a cell into the surrounding medium, but when the flagellum of the protozoan parasite Trypanosoma brucei exits the cell via the flagellar pocket, it is attached along the length of the cell body by a cytoskeletal structure called the flagellum attachment zone (FAZ). The exact reasons for flagellum attachment have remained elusive, but evidence is emerging that the attached flagellum plays a major role in cell morphogenesis in this organism. In this review we discuss evidence published in the past four years that is unravelling the role of the flagellum in organelle segregation, inheritance of cell shape and cytokinesis.

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1-S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1-S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

Trypanosoma brucei is a unicellular parasite causing African sleeping sickness in cattle and humans. Due to the ease with which these cells can be cultured and genetically manipulated, it has emerged as a model organism for the kinetoplastids.In this chapter we describe the preparation of T. brucei for transmission electron microscopy. A thorough explanation of conventional sample preparation through chemical fixation of whole cells and detergent extracted cytoskeletons followed by dehydration and Epon embedding is given. We also introduce a novel high-pressure freezing protocol, which followed by rapid freeze substitution and HM20 embedding generates T. brucei samples displaying good cell morphology, which are suitable for immunocytochemistry.

Proteins from the calpain super-family are involved in developmentally- and environmentally regulated re-modelling of the eukaryotic cytoskeleton and the dynamic organisation of signal transduction cascades. In trypanosomatid parasites, calpain-related gene families are unusually large, but we have little insight in to the functional roles played by these molecules during trypanosomatid lifecycles. Here we report that CAP5.5, a cytoskeletal calpain-related protein subject to strict stage-specific expression in the sleeping sickness parasite Trypanosoma brucei, is essential and required for correct cell morphogenesis of procyclic (tsetse mid-gut stage) T. brucei. Striking consequences of CAP5.5 RNA interference are the loss of protein from the posterior cell-end, organelle mis-positioning giving rise to aberrant cytokinesis, and disorganisation of the sub-pellicular microtubules that define trypanosome cell shape. We further report that the stage-specificity of CAP5.5 expression can be explained by the presence of a paralogue, CAP5.5V, which is required for cell morphogenesis in bloodstream T. brucei; RNAi against this paralogous protein results in a qualitatively similar phenotype to that described for procyclic CAP5.5 RNAi mutants. By comparison to recently described phenotypes for other procyclic trypanosome RNAi mutants, likely functions for CAP5.5 and CAP5.5V are discussed.

Abstract: This study uses electron tomography linked to a variety of other EM methods to provide an integrated view of the flagellar pocket and basal body area of the African trypanosome procyclic trypomastigote. We reveal the pocket as an asymmetric membranous 'balloon' with two boundary structures. One of these - the collar - defines the flagellum exit point. The other defines the entry point of the flagellum into the pocket and consists of both an internal transitional fibre array and an external membrane collarette. A novel set of nine radial fibres is described in the basal body proximal zone. The pocket asymmetry is invariably correlated with the position of the probasal body and Golgi. The neck region, just distal to the flagellum exit site, is a specialised area of membrane associated with the start of the flagellum attachment zone and signifies the point where a special set of four microtubules, nucleated close to the basal bodies, joins the subpellicular array. The neck region is also associated with the single Golgi apparatus of the cell. The flagellar exit point interrupts the subpellicular microtubule array with discrete endings of microtubules at the posterior side. Overall, our studies reveal a highly organised, yet dynamic, area of cytoplasm and will be informative in understanding its function.

The flagellum is attached along the length of the cell body in the protozoan parasite Trypanosoma brucei and is a defining morphological feature of this parasite. The flagellum attachment zone ( FAZ) is a complex structure and has been characterised morphologically as comprising a FAZ filament structure and the specialised microtubule quartet (MtQ) plus the specialised areas of flagellum: plasma membrane attachment. Unfortunately, we have no information as to the molecular identity of the FAZ filament components. Here, by screening an expression library with the monoclonal antibody L3B2 which identifies the FAZ filament we identify a novel repeat containing protein FAZ1. It is kinetoplastid-specific and provides the first molecular component of the FAZ filament. Knockdown of FAZ1 by RNA interference (RNAi) results in the assembly of a compromised FAZ and defects in flagellum attachment and cytokinesis in procyclic trypanosomes. The complexity of FAZ structure and assembly is revealed by the use of other monoclonal antibody markers illustrating that FAZ1 is only one protein of a complex structure. The cytokinesis defects provide further evidence for the role of an attached flagellum in cellular morphogenesis in these trypanosomes.

Undoubtedly, there are fundamental processes driving the structural mechanics of cell division in eukaryotic organisms that have been conserved throughout evolution and are being revealed by studies on organisms such as yeast and mammalian cells. Precision of structural mechanics of cytokinesis is however probably no better illustrated than in the protozoa. A dramatic example of this is the protozoan parasite Trypanosoma brucei, a unicellular flagellated parasite that causes a devastating disease (African sleeping sickness) across Sub-Saharan Africa in both man and animals. As trypanosomes migrate between and within a mammalian host and the tsetse vector, there are periods of cell proliferation and cell differentiation involving at least five morphologically distinct cell types. Much of the existing cytoskeleton remains intact during these processes, necessitating a very precise temporal and spatial duplication and segregation of the many single-copy organelles. This structural precision is aiding progress in understanding these processes as we apply the excellent reverse genetics and post-genomic technologies available in this system. Here we outline our current understanding of some of the structural aspects of cell division in this fascinating organism.

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.

The most common form of the motile eukaryotic flagellum contains a 9 + 2 axoneme, consisting of nine outer doublet microtubules and a central pair of single microtubules. The flagellum of the African trypanosome Trypanosoma brucei fits this pattern, but in addition possesses a paraflagellar rod (PFR) connected to the axoneme at doublets 4 through 7. The PFR acts as a structural platform for metabolic enzymes and is essential for motility (for review, see [1]). Here we describe a novel intra-lumenal microtubule structure, which we term the ponticulus, that bridges the B-tubule lumen in all 9 outer doublet microtubules of the 9 + 2 axoneme. We show that ponticuli are not incorporated into the axoneme during new flagellum assembly, but instead are a post- assembly modification.

By tilting many axoneme sections in the transmission electron microscope, we confirmed that a bridge-like structure is present in the B-tubule of all nine outer doublets but is never present in the central pair microtubules (Figure 1A and inset). We have termed this structure a ponticulus (little bridge). Ponticuli are present in both tsetse-form and bloodstream trypanosomes and in axonemes of Leishmania and Crithidia.

Background

Tubulin isotypes and expression patterns are highly regulated in diverse organisms. The genome sequence of the protozoan parasite Leishmania major contains three distinct β-tubulin loci. To investigate the diversity of β-tubulin genes, we have compared the published genome sequence to draft genome sequences of two further species L. infantum and L. braziliensis. Untranscribed regions and coding sequences for each isoform were compared within and between species in relation to the known diversity of β-tubulin transcripts in Leishmania spp.

Results

All three β-tubulin loci were present in L. infantum and L. braziliensis, showing conserved synteny with the L. major sequence, hence confirming that these loci are paralogous. Flanking regions suggested that the chromosome 21 locus is an amastigote-specific isoform and more closely related (either structurally or functionally) to the chromosome 33 'array' locus than the chromosome 8 locus. A phylogenetic network of all isoforms indicated that paralogs from L. braziliensis and L. mexicana were monophyletic, rather than clustering by locus.

Conclusion

L. braziliensis and L. mexicana sequences appeared more similar to each other than each did to its closest relative in another species; this indicates that these sequences have evolved convergently in each species, perhaps through ectopic gene conversion; a process not yet evident among the more recently derived L. major and L. infantum isoforms. The distinctive non-coding regions of each β-tubulin locus showed that it is the regulatory regions of these loci that have evolved most during the diversification of these genes in Leishmania, while the coding regions have been conserved and concerted. The various loci in Leishmania satisfy a need for innovative expression of β-tubulin, rather than elaboration of its structural role.

BACKGROUND:

alpha- and beta-tubulin are fundamental components of the eukaryotic cytoskeleton and cell division machinery. While overall tubulin expression is carefully controlled, most eukaryotes express multiple tubulin genes in specific regulatory or developmental contexts. The genomes of the human parasites Trypanosoma brucei and Leishmania major reveal that these unicellular kinetoplastids possess arrays of tandem-duplicated tubulin genes, but with differences in organisation. While L. major possesses monotypic alpha and beta arrays in trans, an array of alternating alpha- and beta tubulin genes occurs in T. brucei. Polycistronic transcription in these organisms makes the chromosomal arrangement of tubulin genes important with respect to gene expression.

RESULTS:

We investigated the genomic architecture of tubulin tandem arrays among these parasites, establishing which character state is derived, and the timing of character transition. Tubulin loci in T. brucei and L. major were compared to examine the relationship between the two character states. Intergenic regions between tubulin genes were sequenced from several trypanosomatids and related, non-parasitic bodonids to identify the ancestral state. Evidence of alternating arrays was found among non-parasitic kinetoplastids and all Trypanosoma spp.; monotypic arrays were confirmed in all Leishmania spp. and close relatives.

CONCLUSION:

Alternating and monotypic tubulin arrays were found to be mutually exclusive through comparison of genome sequences. The presence of alternating gene arrays in non-parasitic kinetoplastids confirmed that separate, monotypic arrays are the derived state and evolved through genomic restructuring in the lineage leading to Leishmania. This fundamental reorganisation accounted for the dissimilar genomic architectures of T. brucei and L. major tubulin repertoires.

Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.

Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. T. brucei multiplies extracellularly in the bloodstream, relying on antigenic variation of a dense variant surface glycoprotein (VSG) coat to escape antibody-mediated lysis. We investigated the role of VSG in proliferation and pathogenicity by using inducible RNA interference to ablate VSG transcript down to 1-2% normal levels. Inhibiting VSG synthesis in vitro triggers a rapid and specific cell cycle checkpoint blocking cell division. Parasites arrest at a discrete precytokinesis stage with two full-length flagella and opposing flagellar pockets, without undergoing additional rounds of S phase and mitosis. A subset (<10%) of the stalled cells have internal flagella, indicating that the progenitors of these cells were already committed to cytokinesis when VSG restriction was sensed. Although there was no obvious VSG depletion in vitro after 24-h induction of VSG RNA interference, there was rapid clearance of these cells in vivo. We propose that a stringent block in VSG synthesis produces stalled trypanosomes with a minimally compromised VSG coat, which can be targeted by the immune system. Our data indicate that VSG protein or transcript is monitored during cell cycle progression in bloodstream-form T. brucei and describes precise precytokinesis cell cycle arrest. This checkpoint before cell division provides a link between the protective VSG coat and cell cycle progression and could function as a novel parasite safety mechanism, preventing extensive dilution of the protective VSG coat in the absence of VSG synthesis.

The FtsZ protein is a polymer-forming GTPase which drives bacterial cell division and is structurally and functionally related to eukaryotic tubulins. We have searched for FtsZ-related sequences in all freely accessible databases, then used strict criteria based on the tertiary structure of FtsZ and its well-characterized in vitro and in vivo properties to determine which sequences represent genuine homologues of FtsZ. We have identified 225 full-length FtsZ homologues, which we have used to document, phylum by phylum, the primary sequence characteristics of FtsZ homologues from the Bacteria, Archaea, and Eukaryota. We provide evidence for at least five independent ftsZ gene-duplication events in the bacterial kingdom and suggest the existence of three ancestoral euryarchaeal FtsZ paralogues. In addition, we identify “FtsZ-like” sequences from Bacteria and Archaea that, while showing significant sequence similarity to FtsZs, are unlikely to bind and hydrolyze GTP.

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.

γ-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of γ-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented 1, 2 and 3, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of γ-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 γ-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 γ-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.

African Trypanosomes are flagellated protozoan parasites that cause sleeping sickness in humans and Nagana in cattle. During its life cycle, Trypanosoma brucei alternates between an insect vector (tsetse fly) and a mammalian host. Within each of these, the parasite proliferates and undergoes separate periods of differentiation in preparation for each new host/vector environment. The differentiated cell types of the trypanosome life cycle are defined morphologically by the position of the single flagellum, nucleus and kinetoplast (the single mass of mitochondrial DNA). The flagellum is key to these morphological events and hence much attention has focused recently on understanding its role in trypanosome morphogenesis and pathogenicity. However, the tractable cell biology, reverse genetics and advanced genome project mean that the trypanosome is also emerging as an ideal model organism for the studies of eukaryotic flagella and cilia in general.

Although most eukaryotic cells can express multiple isotypes of alphabeta-tubulin, the significance of this diversity has not always been apparent. Recent data indicate that particular alphabeta-tubulin isotypes, both genome encoded and those derived by post-translational modification, can directly influence microtubule structure and function--thus validating ideas originally proposed in the multitubulin hypothesis over 25 years ago. It has also become increasingly evident over the past year that some (but intriguingly not all) eukaryotes encode several other tubulin proteins, and to date five further members of the tubulin superfamily, gamma, delta, epsilon, zeta and eta, have been identified. Although the role of gamma-tubulin in the nucleation of microtubule assembly is now well established, far less is known about the functions of delta-, epsilon-, zeta- and eta-tubulin. Recent work has expanded our knowledge of the functions and localisation of these newer members of the tubulin superfamily, and the emerging data suggesting a restricted evolutionary distribution of these 'new' tubulin proteins, conforms to established knowledge of microtubule cell biology. On the basis of current evidence, we predict that delta-, epsilon-, zeta- and eta-tubulin all have functions associated with the centriole or basal body of eukaryotic cells and organisms.

More than 20 years ago, biochemical analysis of the eukaryotic cell cytoskeleton revealed the major component proteins. The heterodimeric (α/β) protein tubulin was defined as the building block of microtubules, assembled in a polar manner into specifically arranged protofilaments in the microtubule wall [1].

The next two members of the tubulin protein superfamily were both discovered by genetic means — γ tubulin in Aspergillus [2] and δ tubulin in Chlamydomonas [3]. The γ tubulin is essential for microtubule function and is located in centroso mes and other microtubule-organising centres [4]. The δ tubulin is encoded by the UNI3 gene in Chlamydomonas and a uni3-1 mutation resulted in flagellar basal bodies that possess doublet rather than triplet microtubules [3]. These four members of the tubulin superfamily can be characterised by their distinct intracellular locations and expression patterns, which are reflected in unique sequence characteristics.

The large number of tubulin sequences available in current databases, coupled with the considerable divergence of those sequences, complicates the task of reliable identification and characterisation of tubulin family members. During the Saccharomyces cerevisiae genome project, sequencing revealed the presence of a tubulin gene that was only around 30% identical to the yeast α and β tubulins. This Tub4 protein was conjectured to be a novel tubulin rather than an α, β or γ tubulin [5]. However, subsequent analysis of the completed S. cerevisiae genome and molecular and biochemical studies have led to an accepted view that Tub4 is the budding yeast γ tubulin [4]. Consequently, it has been suggested that caution is required in using certain types of sequence analysis methods to classify novel tubulin sequences [6].

Within the genome of the protozoan parasite Trypanosoma brucei, the genes for α and β tubulin exist as a cluster of repeated α/β pairs [7]. Recently, we identified the single γ tubulin gene in T. brucei [8]. We then conducted a search by PCR and other means for the presence of the T. brucei homologue of δ tubulin. To our surprise, after we cloned the T. brucei δ tubulin homologue, we also identified two new divergent tubulin-like sequences. The general features can be readily visualised in the automatically generated alignment illustrated in Figure 1. Both of these new sequences are also present within the T. brucei genome project databases at the Sanger Centre and TIGR as partial or complete sequences (see Figure 1).