The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies-troublesome proteins and technical limitations-are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.

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The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.

Summary Myosin VI has been implicated in many cellular processes including endocytosis, secretion, membrane ruffling and cell motility. We carried out a yeast two-hybrid screen and identified TRAF6-binding protein (T6BP) and nuclear dot protein 52 (NDP52) as myosin VI binding partners. Myosin VI interaction with T6BP and NDP52 was confirmed in vitro and in vivo and the binding sites on each protein were accurately mapped. Immunofluorescence and electron microscopy showed that T6BP, NDP52 and myosin VI are present at the trans side of the Golgi complex, and on vesicles in the perinuclear region. Although the SKICH domain in T6BP and NDP52 does not mediate recruitment into membrane ruffles, loss of T6BP and NDP52 in RNAi knockdown cells results in reduced membrane ruffling activity and increased stress fibre and focal adhesion formation. Furthermore, we observed in these knockdown cells an upregulation of constitutive secretion of alkaline phosphatase, implying that both proteins act as negative regulators of secretory traffic at the Golgi complex. T6BP was also found to inhibit NF-κB activation, implicating it in the regulation of TRAF6-mediated cytokine signalling. Thus myosin VI-T6BP interactions may link membrane trafficking pathways with cell adhesion and cytokine-dependent cell signalling.

Background: TbBILBO1 is the only known component of the flagellar pocket collar, a cytoskeletal structure in the parasite Trypanosoma brucei. Results: The TbBILBO1 N-terminal domain has a ubiquitin-like fold with a conserved surface patch; overexpression of constructs with a mutagenized patch is lethal. Conclusion:The conserved surface patch is essential for TbBILBO1 function. Significance: The surface patch is a potential therapeutic target.

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.T rypanosoma brucei is an important parasite of humans and domestic animals in sub-Saharan Africa, as the causative agent of sleeping sickness and nagana, respectively. Its complex life cycle involves transitions between tsetse fly vectors (its definitive hosts) and mammalian intermediate hosts. This life cycle involves a number of different cell stages, of which the procyclic form (found in the tsetse fly) and the slender bloodstream form (BSF) (found in the mammalian bloodstream) are the best studied in a laboratory setting. The procyclic form and the BSF of T. brucei share similar cytoskeletal architectures (1, 2).The principal feature of this cytoskeleton is a corset of microtubules that lie directly underneath the plasma membrane and impart to the cell its distinctive shape (3). A single invagination of the plasma membrane, termed the flagellar pocket (FP), constitutes a distinct subdomain and is found at the posterior end of the cell (4). The FP is the site of all endo-and exocytic traffic (5, 6). Abutting the FP membrane is a basal body that nucleates the single flagellum of the trypanosome cell. The flagellum exits the FP and is adhered longitudinally to the cell body along a left-handed helical path (7). Once outside the FP, the axoneme of the flagellum is paralleled by an associated intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of a paracrystalline lattice and is associated with cellular motility (8). Nucleated adjacent to the basal body is a specialized microtubule quartet that traces around the FP and then underlies the flagellum as far as the anterior end of the cell (4).The small cylinder of membrane that connects the FP to the rest of the plasma membrane constitutes a third subdomain and is called the flagellar pocket neck (FPN) (4). A number of discrete cytoskeletal structures cluster around the FPN membrane on its cytoplasmic face. Of these, the best characterized is an electrondense horseshoe-shaped structure named th...

Background: TbBILBO1 is the only known protein component of the flagellar pocket collar, but its assembly remains unknown. Results: Structural dissections of the three different domains of TbBILBO1 revealed their roles in protein assembly. Conclusion: TbBILBO1 forms a linear filament that interacts laterally to form a fibrous bundle. Significance: The data show how two types of coiled coil act together to assemble TbBILBO1 into long filaments.

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