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Philippe Bastin - One of the best experts on this subject based on the ideXlab platform.
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Timing and original features of Flagellum assembly in trypanosomes during development in the tsetse fly
Parasites and Vectors, 2020Co-Authors: Moara Lemos, Brice Rotureau, Eloïse Bertiaux, Adeline Mallet, Albane Imbert, Philippe BastinAbstract:Background: Trypanosoma brucei exhibits a complex life-cycle alternating between tsetse flies and mammalian hosts. When parasites infect the fly, cells differentiate to adapt to life in various tissues, which is accompanied by drastic morphological and biochemical modifications especially in the proventriculus. This key step represents a bottleneck for salivary gland infection. Methods: Here, we monitored Flagellum assembly in trypanosomes during differentiation from the trypomastigote to the epimastigote stage, i.e. when the nucleus migrates to the posterior end of the cell, by using three-dimensional electron microscopy (focused ion beam scanning electron microscopy, FIB-SEM) and immunofluorescence assays. Results: The combination of light and electron microscopy approaches provided structural and molecular evidence that the new Flagellum is assembled while the nucleus migrates towards the posterior region of the body. Two major differences with well-known procyclic cells are reported. First, growth of the new Flagellum begins when the associated basal body is found in a posterior position relative to the mature Flagellum. Secondly, the new Flagellum acquires its own flagellar pocket before rotating on the left side of the anterior-posterior axis. FIB-SEM revealed the presence of a structure connecting the new and mature Flagellum and serial sectioning confirmed morphological similarities with the flagella connector of procyclic cells. We discuss the potential function of the flagella connector in trypanosomes from the proventriculus. Conclusions: These findings show that T. brucei finely modulates its cytoskeletal components to generate highly variable morphologies.
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A key regulatory protein for Flagellum length control in stable flagella
bioRxiv, 2019Co-Authors: Madison Atkins, Philippe Bastin, Samuel Dean, Eloïse Bertiaux, Jiri Tyc, Shahaan Shafiq, Manu Ahmed, Artur Leonel De Castro Neto, Jack Sunter, Sue VaughanAbstract:Summary: Cilia and flagella are highly conserved microtubule-based organelles that have important roles in cell motility and sensing [1]. They can be highly dynamic and short lived such as primary cilia or Chlamydomonas [2] or very stable and long lived such as those in spermatozoa [3] photoreceptors [4] or the flagella of many protist cells [3,4]. Although there is a wide variation in length between cell types, there is generally a defined length for a given cell type [1]. Many unicellular flagellated and ciliated organisms have an additional challenge as they must maintain flagella/cilia at a defined length whilst also growing new flagella/cilia in the same cell. It is not currently understood how this is achieved. A grow-and-lock model was proposed for the maintenance of stable flagella where a molecular lock is applied to prevent Flagellum length change after assembly [5]. The molecular mechanisms of how this lock operates are unknown, but could be important in cells where an existing Flagellum must be maintained whilst a new Flagellum assembles. Here we show that Cep164C contributes to the locking mechanism at the base of the Flagellum in Trypanosoma brucei. It is only localised on the transition fibres of basal bodies of fully assembled flagella and missing from assembling flagella. In fact, basal bodies only acquire Cep164C in the third cell cycle after they assemble in trypanosomes. Depletion leads to dysregulation of Flagellum growth with both longer and shorter flagella; consistent with defects in a Flagellum locking mechanism. By controlling delivery of components into the old assembled Flagellum, maintenance of stable flagella can occur but limits further growth. This offers an important explanation for how many eukaryotic unicellular cells maintain their existing flagella whilst growing new ones before these cells divide. This work also reveals additional regulatory roles for Cep164 in eukaryotic organisms.
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Control of Flagellum Length by a Grow-and-Lock Model
bioRxiv, 2018Co-Authors: Eloïse Bertiaux, Thierry Blisnick, Brice Rotureau, Benjamin Morga, Philippe BastinAbstract:Several cell types such as photoreceptors and spermatozoa possess very stable cilia and flagella, a feature also encountered in numerous protists. We tested an original model for the control of Flagellum length in such cells, using Trypanosoma brucei as an experimental system. The grow-and-lock model proposes that the Flagellum elongates at a linear rate and that a locking event takes place in a timely defined manner preventing further elongation or shortening. We show that the total amount of IFT material increases during Flagellum elongation, ensuring a constant concentration per unit of length and the ability to provide a constant delivery of precursors in agreement with a linear growth rate. Reducing the IFT rate by RNAi knockdown of the IFT kinesin motors slows down the growth rate and results in the assembly of shorter flagella. The Flagellum is locked after cell division in an irreversible process and even subsequent increase in the IFT rate does not lead to further elongation. Other models (limitation by the soluble pool of tubulin, equilibrium between assembly and disassembly rates, or morphogenetic control) fail to explain the experimental data. The locking event is associated to the addition of the FLAM8 molecular marker at the distal end of the Flagellum and is initiated prior cell division, leading to an arrest of elongation in the daughter cell. These results provide support for the grow-and-lock model as a new paradigm for the control of organelle length.
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Intraflagellar transport is required for the maintenance of the trypanosome Flagellum composition but not its length.
Journal of Cell Science, 2016Co-Authors: Cécile Fort, Linda Kohl, Serge Bonnefoy, Philippe BastinAbstract:Intraflagellar transport (IFT) is required for construction of most cilia and flagella. Here, we used electron microscopy, immunofluorescence and live video microscopy to show that IFT is absent or arrested in the mature Flagellum of Trypanosoma brucei upon RNA interference (RNAi)-mediated knockdown of IFT88 and IFT140, respectively. Flagella assembled prior to RNAi did not shorten, showing that IFT is not essential for the maintenance of flagella length. Although the ultrastructure of the axoneme was not visibly affected, flagellar beating was strongly reduced and the distribution of several flagellar components was drastically modified. The R subunit of the protein kinase A was no longer concentrated in the Flagellum but was largely found in the cell body whereas the kinesin 9B motor was accumulating at the distal tip of the Flagellum. In contrast, the distal tip protein FLAM8 was dispersed along the Flagellum. This reveals that IFT also functions in maintaining the distribution of some flagellar proteins after construction of the organelle is completed.
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A novel kinesin involved in Flagellum attachment and positioning in Trypanosoma brucei
Cilia, 2015Co-Authors: S Luiggi, Thierry Blisnick, Philippe Bastin, Sylvie Perrot, A Raïa, L Petit, L Pao, P Grellier, Linda KohlAbstract:Results In procyclic cells KIN5 is localised in the Flagellum (probably the axoneme), with a strong fluorescent signal at the distal tip. Depletion of KIN5 results in cells with a mispositioned and partially detached Flagellum. The Flagellum of the cells is still beating, but the cells are unable to swim. Nevertheless they divide normally indicating that they have adapted to the partially detached Flagellum, probably by modifications of the intracellular organisation. Preliminary data indicate that the filament of the Flagellum Attachment Zone is smaller in length in cells depleted of KIN5.
Kent L. Hill - One of the best experts on this subject based on the ideXlab platform.
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APEX2 proximity proteomics resolves Flagellum subdomains and identifies Flagellum tip-specific proteins in Trypanosoma brucei
bioRxiv, 2020Co-Authors: Daniel E. Vélez-ramírez, Michelle M. Shimogawa, Sunayan S. Ray, Andrew Lopez, Shima Rayatpisheh, Gerasimos Langousis, Marcus Gallagher-jones, Samuel Dean, James A. Wohlschlegel, Kent L. HillAbstract:Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single Flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in Flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall Flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual Flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei Flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the Flagellum. We found that DRC1-APEX2 directs Flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 9proximity proteome9 showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the Flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the Flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for Flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of Flagellum subdomains that cannot themselves be readily purified.
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Motility and more: the Flagellum of Trypanosoma brucei
Nature Reviews Microbiology, 2014Co-Authors: Gerasimos Langousis, Kent L. HillAbstract:The protozoan parasite Trypanosoma brucei has a single Flagellum that is present in all of its different developmental stages. In this Review, Langousis and Hill discuss the structural and functional features of the Flagellum and highlight its central role in the virulence and transmission of this important human pathogen. The African trypanosome Trypanosoma brucei is a unicellular pathogen that causes lethal sleeping sickness in humans, which is a devastating and neglected tropical disease that is endemic to vast regions of Africa. T. brucei also infects wild and domestic livestock, which limits sustainable development, and it is thus considered to be both a cause and consequence of poverty. T. brucei has a single Flagellum that is present throughout the parasite and its life cycle. The Flagellum has conserved and unique features. It emerges from a membrane invagination at the posterior end of the cell and remains attached to the cell body for most of its length. The Flagellum contains cytoskeletal structures, which are ensheathed by a specialized flagellar membrane that interfaces with the external environment and that has a protein and lipid composition that is distinct from the rest of the cell surface. The T. brucei Flagellum has multiple functions and is essential for parasite motility, viability, transmission and pathogenesis. Flagellum-mediated motility is powered by the axoneme, which is a biological machine that converts dynein motor structural changes into Flagellum beating and parasite propulsion. T. brucei motility is crucial for movement through host tissues and provides a surprising immune-evasion mechanism. In addition to motility, the T. brucei Flagellum is an important morphogenetic hub that controls cell shape and size, directs organelle segregation and governs cell division. These functions are modulated during developmental transitions of the parasite and are achieved by the direct or indirect physical connections of the Flagellum to other cellular elements. The Flagellum is a crucial host–pathogen interface that has important roles in parasite transmission and virulence. Flagellar proteins mediate attachment to host tissues, carry out uptake of host growth factors and promote parasite survival by inhibiting host immunity. T. brucei is an excellent model system to study the biology of the highly conserved eukaryotic Flagellum and offers valuable insights into how flagella assemble, move and sense the environment. Continued studies of the T.brucei Flagellum hold the promise of having a great impact on human health, as human flagella are paramount in human development and physiology. In addition, the flagella of many human pathogens are salient but unexplained structures that await further study. Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single Flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome Flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host–parasite interactions.
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motility and more the Flagellum of trypanosoma brucei
Nature Reviews Microbiology, 2014Co-Authors: Gerasimos Langousis, Kent L. HillAbstract:The protozoan parasiteTrypanosoma bruceihas a single Flagellum that is present in all of its different developmental stages. In this Review, Langousis and Hill discuss the structural and functional features of the Flagellum and highlight its central role in the virulence and transmission of this important human pathogen. Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single Flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome Flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host–parasite interactions.
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independent analysis of the Flagellum surface and matrix proteomes provides insight into Flagellum signaling in mammalian infectious trypanosoma brucei
Molecular & Cellular Proteomics, 2011Co-Authors: Michael Oberholzer, Michelle M. Shimogawa, Gerasimos Langousis, James A. Wohlschlegel, Hoangkim T Nguyen, Edwin A Saada, Zophonias O Jonsson, Steven M Nguyen, Kent L. HillAbstract: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.
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The Flagellum of Trypanosoma brucei: new tricks from an old dog.
International journal for parasitology, 2008Co-Authors: Katherine S Ralston, Kent L. HillAbstract:African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. T. brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Therefore, understanding unique aspects of the T. brucei Flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei Flagellum are directly relevant to understanding fundamental aspects of Flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei Flagellum contains a canonical 9+2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome Flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the Flagellum. Most notably, the Flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei Flagellum.
Keith Gull - One of the best experts on this subject based on the ideXlab platform.
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Protein diversity in discrete structures at the distal tip of the trypanosome Flagellum.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Vladimir Varga, Flavia Moreira-leite, Neil Portman, Keith GullAbstract:The distal end of the eukaryotic Flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific Flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome Flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during Flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling Flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature Flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.
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The Flagellum Attachment Zone: 'The Cellular Ruler' of Trypanosome Morphology.
Trends in Parasitology, 2016Co-Authors: Jack Sunter, Keith GullAbstract:A defining feature of Trypanosoma brucei cell shape is the lateral attachment of the Flagellum to the cell body, mediated by the Flagellum attachment zone (FAZ). The FAZ is a complex cytoskeletal structure that connects the Flagellum skeleton through two membranes to the cytoskeleton. The FAZ acts as a ‘cellular ruler’ of morphology by regulating cell length and organelle position and is therefore critical for both cell division and life cycle differentiations. Here we provide an overview of the advances in our understanding of the composition, assembly, and function of the FAZ.
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a dynamic coordination of Flagellum and cytoplasmic cytoskeleton assembly specifies cell morphogenesis in trypanosomes
Journal of Cell Science, 2015Co-Authors: Jack Sunter, Vladimir Varga, Samuel Dean, Keith GullAbstract:Plasma membrane‐to‐plasma membrane connections are common features of eukaryotic cells, with cytoskeletal frameworks below the respective membranes underpinning these connections. A defining feature of Trypanosoma brucei is the lateral attachment of its single Flagellum to the cell body, which is mediated by a cytoskeletal structure called the Flagellum attachment zone (FAZ). The FAZ is a key morphogenetic structure. Disruption of FAZ assembly can lead to Flagellum detachment and dramatic changes in cell shape. To understand this complex structure, the identity of more of its constituent proteins is required. Here, we have used both proteomics and bioinformatics to identify eight new FAZ proteins. Using inducible expression of FAZ proteins tagged with eYFP we demonstrate that the site of FAZ assembly is close to the flagellar pocket at the proximal end of the FAZ. This contrasts with the Flagellum, which is assembled at its distal end; hence, these two interconnected cytoskeletal structures have distinct spatially separated assembly sites. This challenging result has many implications for understanding the process of cell morphogenesis and interpreting mutant phenotypes.
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a cell body groove housing the new Flagellum tip suggests an adaptation of cellular morphogenesis for parasitism in the bloodstream form of trypanosoma brucei
Journal of Cell Science, 2013Co-Authors: Louise Hughes, Keith Gull, Katie Towers, Tobias Starborg, Sue VaughanAbstract: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.
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a repetitive protein essential for the Flagellum attachment zone filament structure and function in trypanosoma brucei
Protist, 2008Co-Authors: Sue Vaughan, Linda Kohl, Ian Ngai, Richard J Wheeler, Keith GullAbstract: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.
Ingyu Hwang - One of the best experts on this subject based on the ideXlab platform.
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regulation of polar Flagellum genes is mediated by quorum sensing and flhdc in burkholderia glumae
Molecular Microbiology, 2007Co-Authors: Yong Sung Kang, Ok Hee Choi, Yeonhwa Jeong, Jaeeun Jeong, Hiroaki Suga, Jae Sun Moon, Ingyu HwangAbstract:Summary The bacterium Burkholderia glumae causes rice grain rot by producing toxoflavin, whose expression is regulated by quorum sensing (QS). We report a major deviation from the current paradigm for the regulation of bacterial polar Flagellum genes. The N-octanoyl homoserine lactone (C8-HSL)-deficient mutant of B. glumae is aflagellate and has lost the ability to swim and swarm at 37°C. Mutagenesis of the bacterium with the mini-Tn5rescue identified an IclR-type transcriptional regulator, called QsmR, which is important for Flagellum formation. TofR, which is a cognate C8-HSL receptor, activated qsmR expression by binding directly to the qsmR promoter region. From the Flagellum gene cluster, we identified flhDC homologues that are directly activated by QsmR. FlhDC in turn activates the expression of genes involved in Flagellum biosynthesis, motor functions and chemotaxis in B. glumae. Non-motile qsmR, fliA and flhDC mutants produced toxoflavin, but lost pathogenicity for rice. The unexpected discovery of FlhDC in a polarly flagellate bacterium suggests that exceptions to the typical regulatory mechanisms of Flagellum genes exist in Gram-negative bacteria. The finding that functional flagella play critical roles in the pathogenicity of B. glumae suggests that either QS or Flagellum formation constitutes a good target for the control of rice grain rot.
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Regulation of polar Flagellum genes is mediated by quorum sensing and FlhDC in Burkholderia glumae.
Molecular Microbiology, 2007Co-Authors: Jinwoo Kim, Yong Sung Kang, Ok Hee Choi, Yeonhwa Jeong, Jaeeun Jeong, Hiroaki Suga, Jae Sun Moon, Jae Yun Lim, Minkyun Kim, Ingyu HwangAbstract:The bacterium Burkholderia glumae causes rice grain rot by producing toxoflavin, whose expression is regulated by quorum sensing (QS). We report a major deviation from the current paradigm for the regulation of bacterial polar Flagellum genes. The N-octanoyl homoserine lactone (C8-HSL)-deficient mutant of B. glumae is aflagellate and has lost the ability to swim and swarm at 37 degrees C. Mutagenesis of the bacterium with the mini-Tn5rescue identified an IclR-type transcriptional regulator, called QsmR, which is important for Flagellum formation. TofR, which is a cognate C8-HSL receptor, activated qsmR expression by binding directly to the qsmR promoter region. From the Flagellum gene cluster, we identified flhDC homologues that are directly activated by QsmR. FlhDC in turn activates the expression of genes involved in Flagellum biosynthesis, motor functions and chemotaxis in B. glumae. Non-motile qsmR, fliA and flhDC mutants produced toxoflavin, but lost pathogenicity for rice. The unexpected discovery of FlhDC in a polarly flagellate bacterium suggests that exceptions to the typical regulatory mechanisms of Flagellum genes exist in Gram-negative bacteria. The finding that functional flagella play critical roles in the pathogenicity of B. glumae suggests that either QS or Flagellum formation constitutes a good target for the control of rice grain rot.
Gerasimos Langousis - One of the best experts on this subject based on the ideXlab platform.
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APEX2 proximity proteomics resolves Flagellum subdomains and identifies Flagellum tip-specific proteins in Trypanosoma brucei
bioRxiv, 2020Co-Authors: Daniel E. Vélez-ramírez, Michelle M. Shimogawa, Sunayan S. Ray, Andrew Lopez, Shima Rayatpisheh, Gerasimos Langousis, Marcus Gallagher-jones, Samuel Dean, James A. Wohlschlegel, Kent L. HillAbstract:Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single Flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in Flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall Flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual Flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei Flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the Flagellum. We found that DRC1-APEX2 directs Flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 9proximity proteome9 showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the Flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the Flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for Flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of Flagellum subdomains that cannot themselves be readily purified.
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Motility and more: the Flagellum of Trypanosoma brucei
Nature Reviews Microbiology, 2014Co-Authors: Gerasimos Langousis, Kent L. HillAbstract:The protozoan parasite Trypanosoma brucei has a single Flagellum that is present in all of its different developmental stages. In this Review, Langousis and Hill discuss the structural and functional features of the Flagellum and highlight its central role in the virulence and transmission of this important human pathogen. The African trypanosome Trypanosoma brucei is a unicellular pathogen that causes lethal sleeping sickness in humans, which is a devastating and neglected tropical disease that is endemic to vast regions of Africa. T. brucei also infects wild and domestic livestock, which limits sustainable development, and it is thus considered to be both a cause and consequence of poverty. T. brucei has a single Flagellum that is present throughout the parasite and its life cycle. The Flagellum has conserved and unique features. It emerges from a membrane invagination at the posterior end of the cell and remains attached to the cell body for most of its length. The Flagellum contains cytoskeletal structures, which are ensheathed by a specialized flagellar membrane that interfaces with the external environment and that has a protein and lipid composition that is distinct from the rest of the cell surface. The T. brucei Flagellum has multiple functions and is essential for parasite motility, viability, transmission and pathogenesis. Flagellum-mediated motility is powered by the axoneme, which is a biological machine that converts dynein motor structural changes into Flagellum beating and parasite propulsion. T. brucei motility is crucial for movement through host tissues and provides a surprising immune-evasion mechanism. In addition to motility, the T. brucei Flagellum is an important morphogenetic hub that controls cell shape and size, directs organelle segregation and governs cell division. These functions are modulated during developmental transitions of the parasite and are achieved by the direct or indirect physical connections of the Flagellum to other cellular elements. The Flagellum is a crucial host–pathogen interface that has important roles in parasite transmission and virulence. Flagellar proteins mediate attachment to host tissues, carry out uptake of host growth factors and promote parasite survival by inhibiting host immunity. T. brucei is an excellent model system to study the biology of the highly conserved eukaryotic Flagellum and offers valuable insights into how flagella assemble, move and sense the environment. Continued studies of the T.brucei Flagellum hold the promise of having a great impact on human health, as human flagella are paramount in human development and physiology. In addition, the flagella of many human pathogens are salient but unexplained structures that await further study. Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single Flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome Flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host–parasite interactions.
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motility and more the Flagellum of trypanosoma brucei
Nature Reviews Microbiology, 2014Co-Authors: Gerasimos Langousis, Kent L. HillAbstract:The protozoan parasiteTrypanosoma bruceihas a single Flagellum that is present in all of its different developmental stages. In this Review, Langousis and Hill discuss the structural and functional features of the Flagellum and highlight its central role in the virulence and transmission of this important human pathogen. Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single Flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome Flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host–parasite interactions.
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independent analysis of the Flagellum surface and matrix proteomes provides insight into Flagellum signaling in mammalian infectious trypanosoma brucei
Molecular & Cellular Proteomics, 2011Co-Authors: Michael Oberholzer, Michelle M. Shimogawa, Gerasimos Langousis, James A. Wohlschlegel, Hoangkim T Nguyen, Edwin A Saada, Zophonias O Jonsson, Steven M Nguyen, Kent L. HillAbstract: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.