Polo-like kinase phosphorylation of bilobe-resident TbCentrin2 facilitates flagellar inheritance in Trypanosoma brucei

In the protist parasite Trypanosoma brucei, the single Polo-like kinase (TbPLK) controls the inheritance of a suite of organelles that help position the parasite''s single flagellum. These include the basal bodies, the bilobe, and the flagellar attachment zone (FAZ). TbCentrin2 was previously shown to be a target for TbPLK in vitro, and this is extended in this study to in vivo studies, highlighting a crucial role for serine 54 in the N-terminal domain. Duplication of the bilobe correlates with the presence of TbPLK and phospho-TbCentrin2, identified using phosphospecific antiserum. Mutation of S54 leads to slow growth (S54A) or no growth (S54D), the latter suggesting that dephosphorylation is needed to complete bilobe duplication and subsequent downstream events necessary for flagellum inheritance.     

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.     

An interplay between Centrin2 and Centrin4 on the bi-lobed structure in Trypanosoma brucei

Centrins are conserved calcium-binding proteins important for various regulatory functions. In procyclic Trypanosoma brucei, TbCentrin2 and TbCentrin4 have distinct effects on cell division but both localize to the basal bodies that seed the flagellum, and a bi-lobed structure important for organelle duplication and cell division. Here we show that TbCentrin2 and TbCentrin4 both bind to the basal bodies and bi-lobed structure through the conserved C-terminal domain. Molecular genetic manipulation of TbCentrin4 levels greatly affects TbCentrin2 association with the bi-lobed structure. Using established synchronization methods, TbCentrin2 expression level is shown to be relatively constant throughout the cell cycle while TbCentrin4 level fluctuates, decreasing most during early S-phase when the bi-lobe undergoes duplication. These results thus suggest a co-ordinated action between these two centrin proteins, where the cell cycle-dependent TbCentrin4 expression could regulate the abundance of TbCentrin2 on the bi-lobed structure.     

Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.     

Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.     

A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation

The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell–cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQ-associated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei.     

Proteomics and the Trypanosoma brucei cytoskeleton: advances and opportunities

Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.     

Functional analyses of an axonemal inner‐arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation

The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility‐generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner‐arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner‐arm dynein heavy chain TbIAD5‐1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5‐1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5‐1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner‐arm dynein complex in cell division, and provided molecular insights into the flagellum motility‐mediated cytokinesis initiation in the bloodstream form of T. brucei.     

A kinetoplastid-specific kinesin is required for cytokinesis and for maintenance of cell morphology in Trypanosoma brucei

Kinesins are motor‐based transport proteins that play diverse roles in various cellular processes. The trypanosome genome lacks the homologues of many conserved mitotic kinesins, but encodes a number of trypanosome‐specific kinesins with unknown function. Here, we report the biochemical and functional characterization of TbKIN‐C, a trypanosome‐specific kinesin, which was initially identified through an RNAi screen for cytokinesis genes in T. brucei. TbKIN‐C possesses in vitro ATPase activity and associates with cytoskeletal tubulin microtubules in vivo. It is distributed throughout the cytoskeleton with a focal enrichment at the posterior end of the cell during early cell cycle stages. RNAi of TbKIN‐C resulted in distorted cell shape with an elongated posterior filled with tyrosinated tubulin microtubules. Silencing of TbKIN‐C impaired the segregation of organelles and cytoskeletal structures and led to detachment of the new flagellum and a small portion of the cytoplasm. We also show that RNAi of TbKIN‐C compromised cytokinesis and abolished the trans‐localization of TbCPC1, a subunit of the chromosomal passenger complex, from the central spindle to the initiation site of cytokinesis. Our results suggest an essential role of TbKIN‐C in maintaining cell morphology, likely through regulating microtubule dynamics at the posterior end of the cell.     

Trypanin, a component of the flagellar Dynein regulatory complex, is essential in bloodstream form African trypanosomes

The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

Proteomic identification of novel cytoskeletal proteins associated with TbPLK,an essential regulator of cell morphogenesis in Trypanosoma brucei

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.     

Biochemical analysis of PIFTC3, the Trypanosoma brucei orthologue of nematode DYF-13, reveals interactions with established and putative intraflagellar transport components

DYF-13, originally identified in Caenorhabditis elegans within a collection of dye-filling chemosensory mutants, is one of several proteins that have been classified as putatively involved in intraflagellar transport (IFT), the bidirectional movement of protein complexes along cilia and flagella and specifically in anterograde IFT. Although genetic studies have highlighted a fundamental role of DYF-13 in nematode sensory cilium and trypanosome flagellum biogenesis, biochemical studies on DYF-13 have lagged behind. Here, we show that in Trypanosoma brucei the orthologue to DYF-13, PIFTC3, participates in a macromolecular complex of approximately 660 kDa. Mass spectroscopy of affinity-purified PIFTC3 revealed several components of IFT complex B as well as orthologues of putative IFT factors DYF-1, DYF-3, DYF-11/Elipsa and IFTA-2. DYF-11 was further analysed and shown to be concentrated near the basal bodies and in the flagellum, and to be required for flagellum elongation. In addition, by coimmunoprecipitation we detected an interaction between DYF-13 and IFT122, a component of IFT complex A, which is required for retrograde transport. Thus, our biochemical analysis supports the model, proposed by genetic analysis in C. elegans, that the trypanosome orthologue of DYF-13 plays a central role in the IFT mechanism.     

Independent analysis of the flagellum surface and matrix proteomes provides insight into flagellum signaling in mammalian-infectious Trypanosoma brucei

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.     

The Trypanosoma brucei cytoskeleton: Ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy

We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule-membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quickfreezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction with the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules.     

The Fine Structure of the Flagellum and Kinetoplast-Chondriome of Trypanosoma (Schizotrypanum) cruzi in Tissue Culture*

SYNOPSIS. Trypanosoma cruzi in tissue cultures was studied with the electron microscope after double fixation in glutaraldehyde and osmium tetroxide, and embedding in Epon. Previous findings on its fine structure were confirmed, and some new structures were found in the flagellum and kinetoplast-chondriome. In the flagellum, an intraflagellar body was found, similar to that observed in other trypanosomes, beginning at the base of the flagellum and running along the axial fibre bundle thruout its length. The axial fibre bundle is formed by interconnecting tubules, the outer ones apparently smooth, the inner ones with a helical substructure. Lateral extensions from the outer tubules in the flagellar bundle seem to enter the intraflagellar body. The kinetopiast in the leishmania bodies has the same electrondense structure described before. In the trypanosome form it has assumed a large spherical shape, in which the formerly short, compressed fibres have grown in length, are more dispersed and have an irregular shape. They are oriented in the direction of the body's length in parallel array. The whole formation is continuous with a long mitochondrion which begins in the region of the nucleus and extends up almost to the tip of the trypanosome. The matrix of the kinetoplast in these forms is electron-transparent; the matrix of the mitochondria is rather dense. In a few extracellular trypanosomes, a special structure was found in which the kinetoplast is composed of electron-transparent formations, arranged in orderly horizontal lines quite similar to the mitochondrial cristae of the parasite. The significance of this structure is uncertain.     

Cell-to-flagellum attachment and surface architecture in kinetoplastids

A key morphological feature of kinetoplastid parasites is the position and length of flagellum attachment to the cell body. This lateral attachment is mediated by the flagellum attachment zone (FAZ), a large complex cytoskeletal structure, which is essential for parasite morphogenesis and pathogenicity. Despite the complexity of the FAZ only two transmembrane proteins, FLA1 and FLA1BP, are known to interact and connect the flagellum to the cell body. Across the different kinetoplastid species, each only has a single FLA/FLABP pair, except in Trypanosoma brucei and Trypanosoma congolense where there has been an expansion of these genes. Here, we focus on the selection pressure behind the evolution of the FLA/FLABP proteins and the likely impact this will have on host–parasite interactions.     

Form and function in the trypanosomal secretory pathway

Recent advances in secretory biology of African trypanosomes reveal both similarities and striking differences with other model eukaryotic organisms. Secretion is streamlined for rapid and selective transport of the major cargo, VSG. Selectivity in the early and post-Golgi compartments is dependent on glycosylphosphatidyl inositol anchors. Streamlining includes reduced organellar abundance, and close association of ER exit sites with Golgi and with unique flagellar cytoskeletal elements that govern organellar replication and segregation. These elements include a novel centrin containing bilobe structure. Innate signals for post-Golgi sorting of biosynthetic lysosomal cargo trafficking have been defined, as have pathways for both biosynthetic and endocytic trafficking to the lysosome. Less well-defined secretory organelles such as the multivesicular body and acidocalcisomes are receiving closer scrutiny.     

Flagellar Membrane and Paraxial Rod Proteins of Leishmania: Characterization Employing Monoclonal Antibodies

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family ( Endotrypanum, Trypanosoma, Leishmania ) suggesting that these molecules may be evolutionarily conserved.     

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo- and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket.