Brooke Morriswood

Novel Bilobe Components in Trypanosoma brucei Identified Using Proximity-Dependent Biotinylation

ABSTRACT 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.

The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein.

The Golgi of the kinetoplastid parasite Trypanosoma brucei is closely apposed to a bilobe structure containing TbCentrin2 and TbCentrin4 in procyclic cells. However, both are additionally localized to the basal bodies. Here we report the characterization of a membrane occupation and recognition nexus (MORN)-repeat protein, TbMORN1, present at the bilobe but not at the basal body. The anterior part of the TbMORN1 structure partially overlapped with the flagellar attachment zone while the posterior part overlapped with the flagellar pocket. Depletion studies using RNAi showed that there was a modest growth inhibition in procyclic cells but lethality in bloodstream cells, showing that it is an essential protein in the bloodstream form of the organism. TbMORN1 appears to be a useful marker for the bilobe in T. brucei.

Morphology of the Trypanosome Bilobe, a Novel Cytoskeletal Structure

ABSTRACT 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.

A MORN-repeat protein facilitates protein entry into the flagellar pocket of Trypanosoma brucei

ABSTRACT 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.

Form, Fabric, and Function of a Flagellum-Associated Cytoskeletal Structure

Trypanosoma brucei is a uniflagellated protist and the causative agent of African trypanosomiasis, a neglected tropical disease. The single flagellum of T. brucei is essential to a number of cellular processes such as motility, and has been a longstanding focus of scientific enquiry. A number of cytoskeletal structures are associated with the flagellum in T. brucei, and one such structure—a multiprotein complex containing the repeat motif protein TbMORN1—is the focus of this review. The TbMORN1-containing complex, which was discovered less than ten years ago, is essential for the viability of the mammalian-infective form of T. brucei. The complex has an unusual asymmetric morphology, and is coiled around the flagellum to form a hook shape. Proteomic analysis using the proximity-dependent biotin identification (BioID) technique has elucidated a number of its components. Recent work has uncovered a role for TbMORN1 in facilitating protein entry into the cell, thus providing a link between the cytoskeleton and the endomembrane system. This review summarises the extant data on the complex, highlights the outstanding questions for future enquiry, and provides speculation as to its possible role in a size-exclusion mechanism for regulating protein entry. The review additionally clarifies the nomenclature associated with this topic, and proposes the adoption of the term “hook complex” to replace the former name “bilobe” to describe the complex.

Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

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. TbBILBO1 is the only known component of the flagellar pocket collar, a cytoskeletal barrier element found in trypanosomes. The N-terminal domain (NTD) of TbBILBO1 was found to be dispensable for targeting of the protein in vivo. However, overexpression of constructs lacking the NTD caused complete growth inhibition, implying an essential requirement for this domain. A high resolution structure of the NTD of TbBILBO1 showed that it forms a ubiquitin-like fold with a conserved surface patch. Mutagenesis of this patch recapitulated the phenotypic effects of deleting the entire domain and was found to cause cell death. The surface patch on the NTD of TbBILBO1 is therefore a potential drug target.

Stalemate in the Golgi Battle

A similar approach to analyze protein trafficking arrives at two opposing models of movement through the cells secretory pathway. The Golgi—a series of flattened membrane sacs (cisternae)—lies at the hub of a eukaryotic cells endomembrane system. The routes taken by transiting cargo and the Golgis resident enzymes have been a matter of long-standing controversy. The two most popular models—stable cisternae versus cisternal maturation—have attracted support and criticism in almost equal measure (1). Two recent papers, aimed at providing a decisive answer for one of the models, instead offer conflicting interpretations of similar data.

Assembly mechanism of Trypanosoma brucei BILBO1, a multidomain cytoskeletal protein

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. Trypanosoma brucei BILBO1 (TbBILBO1) is an essential component of the flagellar pocket collar of trypanosomes. We recently reported the high resolution structure of the N-terminal domain of TbBILBO1. Here, we provide further structural dissections of its other three constituent domains: EF-hand, coiled coil, and leucine zipper. We found that the EF-hand changes its conformation upon calcium binding, the central coiled coil forms an antiparallel dimer, and the C-terminal leucine zipper appears to contain targeting information. Furthermore, interdimer interactions between adjacent leucine zippers allow TbBILBO1 to form extended filaments in vitro. These filaments were additionally found to condense into fibers through lateral interactions. Based on these experimental data, we propose a mechanism for TbBILBO1 assembly at the flagellar pocket collar.

Nobody at the top

EMBO Reports (2018) e46329 Science is a global endeavour, a unifying mission to obtain an objective understanding of the natural world. Most of its practitioners will never meet each other or interact directly or even indirectly in any way, yet they all retain and share a sense of kinship in this open‐ended enterprise. They dwell in different societies or geopolitical entities, represent all nations, but remain bound together by a common search for objective truth (and occasionally, as here, by a propensity to indulge in pompous sermons on how wonderful and Enlightened they are). In this sense, science—or more accurately the scientific method—bears some of the hallmarks of an organised religion. It encourages adherence to particular creeds, displays a distrust of unorthodoxy (particularly when it comes to allocation of research funds) and requires its acolytes to undergo gruelling trials to test their knowledge and worthiness for inclusion. But there is one way in which science differs fundamentally from …

Two flagellar BAR domain proteins in Trypanosoma brucei with stage-specific regulation.

Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod.