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Motility and more: the flagellum of Trypanosoma brucei Gerasimos Langousis1 and Kent L. Hill1,2
Abstract | 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. Kinetoplastid A term used to describe a group of flagellated protozoa within the phylum Euglenozoa. The defining feature of kinetoplastids is that their mitochondrial DNA is arranged into a tightly packed network that is known as the kinetoplast.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, USA. 2 Molecular Biology Institute, University of California, Los Angeles, California 90095, USA. Correspondence to K.L.H. e-mail: [email protected] doi:10.1038/nrmicro3274 1
African trypanosomes, such as Trypanosoma brucei and related species, are kinetoplastid parasites1, which also include the human pathogens Trypanosoma cruzi and Leishmania spp. Together, these protozoan parasites cause substantial human morbidity and mortality worldwide and limit economic development in some of the most impoverished regions of the world2–4. In this Review, we focus on T. brucei, which is the causative agent of African trypanosomiasis (also known as sleeping sickness) in humans. The parasite is transmitted to humans by tsetse flies (BOX 1), and the disease proceeds in two clinical stages: the haemolymphatic stage, during which parasites replicate in the blood and lymph, causing clinical manifestations of recurrent fever and general malaise; and the invasive phase, during which parasites move out of the bloodstream and into extravascular spaces, including cardiac tissue and the central nervous system (CNS). This second stage is marked by meningoencephalitis, headaches and severe neurological changes that disrupt the sleep–wake cycle, and if left untreated, it is typically followed by coma and death5,6. Sleeping sickness poses a threat to an estimated 60 million people. There is currently no vaccine available and treatments are antiquated, toxic and increasingly ineffective7. T. brucei has a single flagellum, which is present throughout the cell cycle and during all stages of development (BOX 1). The flagellum is essential for viability8, is the sole means of motility and has emerged as a key player in multiple facets of development, transmission and pathogenesis. In fact, the distinctive auger-like motility of T. brucei (Supplementary information S1 (movie)) provided the basis for naming the genus. In
1843, in one of the earliest descriptions of T. brucei, Gruby9 observed that the organism, which he identified in frog blood, “ … turns two or three times around its axis, like a drill or a corkscrew, which is why I propose to name this haematozoan ‘Trypanosoma’. ” The name combines the Greek words trypanon (which means auger) and soma (which means body), so literally translated, the trypanosome is an ‘auger body’. Since then, trypanosome motility has captured the attention of many scientists, and an ‘undulating membrane’ (REF. 10) (now known to be the flagellum) is a prominent feature of most descriptions. Trypanosome movements to specific host tissues are defining events in pathogenesis and transmission (BOX 1), thus emphasizing the importance of the motility function of the flagellum. In addition to its canonical role in motility, the T. brucei flagellum is important for morphogenesis and cell division, and it is a crucial host–parasite interface that mediates attachment to host tissues and provides a scaffold for the assembly of signalling proteins and virulence factors. In this Review, we outline the structure and assembly of the T. brucei flagellum and discuss how this organelle functions in motility, morphogenesis and host–parasite interactions. Where relevant, we point out eukaryote-conserved versus trypanosome-specific flagellum features.
Flagellum structure The trypanosome flagellum emerges from the posterior of the cell and defines the anterior–posterior axis (FIG. 1). It is built on a canonical 9 + 2 axoneme, which contains nine doublet microtubules that are
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REVIEWS Box 1 | Trypanosoma brucei life cycle A generalized life cycle of Trypanosoma brucei is shown in the figure, part a. Infection of a mammalian host initiates when a tsetse fly bite delivers growth-arrested metacyclic trypomastigotes to the mammalian bloodstream. Metacyclic trypomastigotes then differentiate into proliferating long slender forms that establish and maintain a bloodstream infection. Parasites eventually penetrate the blood vessel endothelium and invade extravascular tissues, including the central nervous system (CNS). Little is known about specific developmental forms that are present in extravascular compartments. In the bloodstream, a quorum sensing-like mechanism elicits the differentiation of long slender forms into short stumpy forms that are cell-cycle arrested and pre-adapted for survival in the tsetse fly136. When an infected host is bitten by a tsetse fly, parasites are taken up with the blood meal into the midgut, where short stumpy forms differentiate into procyclic trypomastigotes that resume cell division and establish a midgut infection. Midgut procyclic trypomastigotes then embark on an epic migration (see the figure, part b) that takes them through the peritrophic matrix, along the foregut to the proventriculus, and from there onwards through the mouthparts, salivary ducts and ultimately into the salivary gland, where they attach to the salivary gland epithelium. In the proventriculus, procyclic trypomastigotes undergo extensive restructuring, coupled to an asymmetric division, to generate one long epimastigote and one short epimastigote. The short epimastigote attaches to epithelial cells following arrival in the salivary gland. Attached epimastigotes replicate and ultimately complete the life cycle via an asymmetric division82 to generate metacyclic trypomastigotes that are free in the salivary gland lumen and are uniquely adapted to survive in the mammalian host. The dividing forms that replicate via binary fission are indicated with a circular arrow. Parasite movements within the tsetse fly (see the figure, part b) and mammalian host (see the figure, part c) are illustrated. The blue line in part b depicts the route taken from the midgut to the salivary gland and the red line indicates the route from the salivary gland to the mouthparts. Parasites move out of blood vessels, penetrating the blood–brain barrier to enter the CNS (see the figure, part c).
a
Metacyclic trypomastigote Salivary gland
Bloodstream
Long slender form
Attached epimastigote
Tsetse Fly
Mammal CNS
Short epimastigote Long epimastigote
Short stumpy form
Proventriculus
Bloodstream Midgut
b
Procyclic trypomastigote
c
Proventriculus Salivary gland
Salivary duct Mouthparts
Midgut
Blood–brain barrier
symmetrically arranged around a central pair of singlet microtubules11. The axoneme is anchored in the cytoplasm at the posterior of the cell via the basal body, which is a barrel-like structure that contains nine peripheral triplet microtubules and has no central pair. As the basal body extends outwards, triplet microtubules become doublets, forming the axoneme transition zone. Filaments connect transition-zone microtubules to the surrounding membrane, forming a ciliary necklace12,13, and tubular structures on the external face of the membrane form the collarette13,14. The transition zone ends at the basal plate, which marks the site at which the centralpair microtubules, and hence the 9 + 2 axoneme, begin. The transition zone and the first part of the axoneme exit the cytoplasm through a specialized invagination of the plasma membrane, which is known as the flagellar pocket. The flagellar pocket corresponds to a gap in the subpellicular microtubules and is the exclusive site of endocytosis and secretion, which makes it a key portal for host–parasite interactions15. The T. brucei flagellar pocket is quite pronounced compared with what is observed in many other flagellated eukaryotes16, which perhaps reflects the demands of a pathogenic lifestyle and the role of the flagellar pocket in virulence and immune evasion15. The site at which the axoneme exits the flagellar pocket is known as the flagellar pocket collar, which is marked by a fibrous cytoskeletal structure that holds the flagellar membrane and cell membrane in close apposition and is required for flagellar pocket biogenesis17. From the collar onwards, the axoneme is surrounded by its own membrane, which has a lipid and protein composition that is distinct from that of the flagellar pocket membrane and cell membrane18,19. Most flagellated cells maintain flagellum connections to the cell membrane within the flagellar pocket16. However, the T. brucei flagellum is also laterally connected to the cell body along almost its entire length, and only the distal tip extends free of the cell body (FIG. 1). Lateral flagellum attachment is mediated by junctional complexes that use proteins in the flagellum and cell body to hold the flagellum membrane and plasma membrane in tight apposition20–23. These junctional complexes constitute a specialized ‘flagellum attachment zone’ (FAZ) that extends from the flagellar pocket to the anterior end of the cell. The FAZ includes a cytoplasmic FAZ filament24,25, together with a specialized quartet of subpellicular microtubules (known as the microtubule quartet) that are associated with a membranous reticulum26, the FAZ endoplasmic reticulum (FER) that is contiguous with the ER. Lateral attachment of the flagellum to the cell body has consequences for cell motility and cell division, as described below. Moreover, this unique architecture divides the cell surface into several discrete membrane subdomains27,28 (FIG. 1) and thereby imposes specialized demands on protein targeting to the host– parasite interface. Trafficking mechanisms that enable protein targeting to each of these specific subdomains remain enigmatic. The axoneme. The fundamental unit of flagellum motility is the 9 + 2 axoneme, which consists of nine outer
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REVIEWS 92 + 2
92 + 0 Flagellar membrane
Axoneme
Flagellar pocket membrane Flagellar membrane Ciliary necklace
PFR
Collarette
A lethal disease that is prevalent in sub-Saharan Africa. Two specific subspecies of Trypanosoma brucei, known as Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, cause disease in humans. A third subspecies, Trypanosoma brucei brucei, and the related trypanosomes Trypanosoma congolense and Trypanosoma vivax, infect only non-primates, causing wasting disease, which limits economic development in endemic areas.
Cell membrane
Ciliary necklace
Kinetoplast
A specialized region of the flagellar or ciliary membrane that surrounds the transition zone; it is defined by chalice-shaped filaments that extend outwards from the axoneme and form indentations in the ciliary membrane.
Subpellicular microtubules
92 + 2
93 + 0
African trypanosomiasis
Cell membrane
Microtubule quartet FER
Flagellar membrane
FAZ filament
Cell membrane Basal plate Flagellar membrane Axoneme
Flagellum
PFR FAZ
Tripartite attachment complex
Flagellar pocket
Basal body
Flagellar pocket collar
FAZ-associated flagellar membrane
FAZ filament
FAZ-associated cell body membrane
Flagellar pocket membrane
Transition zone Transition fibres
1 μm Flagellum
Subpellicular microtubules A cage-like array of microtubules that subtend the plasma membrane (pellicle) and run parallel to the long axis of the cell.
Microtubule quartet Four specialized subpellicular microtubules that extend from the basal body to the anterior of the cell and subtend the region of plasma membrane where the flagellum attaches to the cell body. These four microtubules constitute part of the flagellum attachment zone, are associated with a subdomain of the smooth endoplasmic reticulum and are antiparallel to the other subpellicular microtubules.
Trypomastigotes Parasite morphotypes in which the basal body is posterior to the nucleus.
Epimastigote A parasite morphotype in which the basal body is anterior to the nucleus.
Cell body
Posterior
Anterior
Flagellum tip
Figure 1 | Overview of the Trypanosoma brucei flagellum. A scanning electron micrograph of a Trypanosoma brucei Nature Reviews | Microbiology procyclic form is shown (lower panel). The main direction of cell propulsion (arrow) is with the flagellum tip leading. The upper panel shows a diagram of flagellum emergence from the flagellar pocket at the posterior end of the cell (boxed region in lower panel). The flagellum is built around a core of microtubules that are arranged in characteristic patterns, as shown above the corresponding cross sections. The flagellar axoneme emanates from the basal body via the transition zone and is laterally connected to the cell membrane via the flagellum attachment zone (FAZ). Extra-axonemal structures inside the flagellar membrane include the paraflagellar rod (PFR) and the ciliary necklace. A tripartite attachment complex links the basal body to the kinetoplast, and transition fibres connect the basal body to the flagellar pocket. The distinctive architecture of the trypanosome flagellum is dictated by specialized membrane and cytoskeletal features, such as the flagellar pocket collar and the FAZ filament. Adjacent to the FAZ filament are the subpellicular microtubules, the microtubule quartet and the FAZ endoplasmic reticulum (FER). For clarity, some axonemal structures, such as dyneins and radial spokes are not depicted.
doublet microtubules that surround a central pair of singlet microtubules29 (FIG. 2). Each of the outer doublet microtubules consists of an A‑tubule and a B‑tubule, which provide a scaffold for the assembly of inner-arm and outer-arm dynein motors. Adjacent doublets are connected via the nexin–dynein regulatory complex (NDRC)30,31,32. Radial spokes extend inwards from each
outer doublet and converge on the central-pair apparatus, which includes the central-pair microtubules surrounded by a sheath-like structure33. Components of the T. brucei radial spokes and central-pair apparatus remain mostly uncharacterized, but the few examples that have been studied show that these structures are critical for normal motility34–36. In a landmark study, axoneme biogenesis
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REVIEWS 96 nm Outer-arm dynein Outer doublet microtubules
NDRC
B
A
Inner-arm dynein
Radial spoke
Outer doublet microtubules
A
B 1
2
9
3
8
Central pair apparatus
Outer-arm dynein NDRC Inner-arm dynein
Radial spoke 4
7 6
5 Axoneme–PFR connectors Proximal domain Intermediate domain
PFR Distal domain
Figure 2 | The axoneme and PFR structure. The upper panel shows a longitudinal Nature Reviews | Microbiology view of the axonemal repeating unit on one outer doublet microtubule, which is oriented with the basal body end on the left-hand side. The lower panel is a diagram showing transverse section detail of the axoneme and paraflagellar rod (PFR), as viewed from posterior to anterior (lower panel) . Prominent axonemal structures are labelled and include outer doublet and central-pair microtubules, outer-arm dyneins and inner-arm dyneins, radial spokes and the nexin–dynein regulatory complex (NDRC), which connects adjacent doublets. Outer doublet microtubules are numbered 1–9, as indicated. Proximal, intermediate and distal domains of the PFR are shown.
Propulsive parasite motility A sustained, forwards movement of a parasite. Propulsive motility is distinguished from general writhing of the parasite, which is generated by unregulated beating of the flagellum.
in T. brucei was shown to be essential and to depend on intraflagellar transport (IFT)8 (BOX 2). When viewed longitudinally, substructures on outer doublet microtubules are arranged in a repeating unit with a periodicity of approximately 96 nm37. In T. brucei, the axonemal repeat unit includes three radial spokes, four outer-arm dyneins, inner-arm dyneins and the NDRC30 (FIG. 2). The stoichiometry of inner-arm dyneins and the NDRC in T. brucei has not been determined. The basic architecture of the axoneme repeating unit is conserved among eukaryotes that have motile flagella. However, there are several differences in T. brucei compared with Chlamydomonas reinhardtii, which is considered to be a reference organism for axoneme structure37,38.
For example, the T. brucei axoneme has three radial spokes instead of two, each outer-arm dynein has two heavy chains instead of three and the T. brucei axoneme has specialized NDRC subunits and central-pair microtubules that retain a fixed orientation relative to outer doublets30,34,36,39. At the molecular level, flagellum motility is powered by ATP-dependent structural changes in axonemal dynein motors that are permanently attached to the A‑tubule of each outer doublet microtubule. These structural changes cause dyneins to reversibly attach to the B-tubule of the neighbouring doublet and drive the sliding of adjacent doublets. Separate connections, such as the NDRC, limit sliding, and this generates doublet bending40. Harnessing flagellum beating to drive propulsive parasite motility requires the propagation of localized bending of axonemal doublets along the length of the axoneme — this process requires spatial and temporal regulation of thousands of dynein motors. The precise mechanisms for regulating axonemal dynein activity are not clear, but they involve various axonemal protein complexes41,42. Among these, the NDRC is perhaps the best characterized32. The NDRC is a large (>1 MDa) complex that is present in almost all motile axonemes and contains conserved as well as organism-specific subunits43–46. In T. brucei, the NDRC includes trypanin36 and component of motile flagella 70 (CMF70)44, as well as candidate subunits, trypanin-related protein, CMF46, CMF40 and CMF22 (REFS 36,43,47). Pioneering studies in C. reinhardtii show that the NDRC functions together with the radial spokes and central-pair apparatus as a reversible inhibitor of dynein32,48. Functional analysis of NDRC subunits supports a similar role in T. brucei36,44. The architecture and exact subunit composition of the NDRC and other axonemal regulatory systems remain unknown and this should be the focus of future studies. The paraflagellar rod. As the flagellum extends beyond the flagellar pocket, an extra-axonemal structure called the paraflagellar rod (PFR) is formed49. The PFR is a massive, lattice-like filament that runs alongside the axoneme and is connected to axonemal doublets 4–7. It is restricted to kinetoplastids and a few other organisms49 and is essential for normal motility in T. brucei50. In cross-section, the PFR has three structural domains that are proximal, intermediate and distal to the axoneme– PFR interface (FIG. 2). Three recent electron tomography studies reported that a repeating unit within the distal domain has 51–57 nm longitudinal periodicity21,30,51, and PFR connections to the axoneme were observed to occur with a longitudinal periodicity of 56–57 nm. The periodicity of the connectors corresponds well to the PFR repeat-unit size and is a multiple of the 8 nm unit of tubulin dimers that make up the axonemal microtubules, which suggests that the connectors might be elaborated from intrinsic structural repeats. The size and complexity of the PFR–axoneme superstructure makes structural studies technically demanding, and more work is needed to define a detailed structure as well as to elucidate the assembly mechanisms of the PFR (BOX 2).
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REVIEWS Box 2 | Flagellum assembly in Trypanosoma brucei Trypanosoma brucei flagellum assembly initiates at the basal body and depends on intraflagellar transport (IFT)8,137,138, which is a bidirectional transport system that operates along outer axonemal doublet microtubules to deliver proteins into and out of the flagellum. Complexes of IFT proteins assemble into particles that mediate the delivery of nascent flagellum subunits from their cytoplasmic site of synthesis into the flagellum. Kinesin motors transport IFT particles and their cargo to the flagellar tip (via anterograde transport), where cargo is released for assembly into the growing flagellum. IFT particles are returned to the base of the flagellum (via retrograde transport) by dynein motors. IFT was originally identified in Chlamydomonas reinhardtii and is now known to be an almost universal feature of eukaryotic flagella137. T. brucei encodes all of the IFT proteins that are required for flagellum assembly139. Live imaging of IFT in T. brucei (Supplementary information S2 (movie)) suggests that there are two pools of IFT particles at the base of the flagellum and that only one of these actively participates in IFT140. These studies also revealed two distinct populations of anterograde IFT particles, each of which moves at a different speed. In addition to IFT, eukaryotes depend on a specialized protein complex, which is known as the BBSome, for trafficking of particular membrane proteins into and out of the flagellum. The BBSome is a multimeric complex of highly conserved proteins that is found only in ciliated organisms141. In humans, mutations in BBSome genes result in Bardet–Biedl syndrome (BBS), which is a pleiotropic disease that is caused by defective cilia141. T. brucei has homologues of all BBSome subunits and there is indirect evidence that links them to flagellum assembly142, although their direct involvement has not been tested. In addition to having characteristics in common with other eukaryotes, T. brucei flagellum assembly has parasite-specific features. First, the trypanosome flagellum is retained throughout the cell cycle, which differs from most other organisms that have been studied, and assemblies of the new and old flagellum seem to be controlled separately. Second, the flagellum tip harbours a disorganized axoneme during flagellum biogenesis143. Third, growth of each new flagellum follows a path that is defined by the old flagellum86, and the presence of the paraflagellar rod (PFR) seems to limit which outer doublet microtubules may be used for IFT139. Thus, T. brucei flagellum assembly must accommodate spatial constraints that are not seen in other organisms. The T. brucei flagellum also undergoes major changes in position and length throughout the parasite life cycle (BOX 1; FIG. 3), which indicates a need for plasticity in the assembly systems that control flagellum size and position. Furthermore, axoneme assembly must be coordinated with assembly of the PFR and flagellum attachment zone (FAZ) (FIG. 1). The regulatory systems that are responsible, as well as their relationships to canonical IFT and BBSome systems, remain to be discovered. PFR assembly depends on axoneme biogenesis, but axoneme biogenesis does not depend on PFR assembly, as IFT mutants fail to assemble both structures, whereas PFR mutants assemble normal axonemes8,144. The mechanisms of PFR assembly are not known, but, in addition to the major subunits PFR1 and PFR2 (REFS 50,145), assembly depends on calmodulin95 and involves a specific kinesin146.
Trypanosomatids A group of parasitic protozoa that infect mammals (Trypanosoma spp. and Leishmania spp.), plants (Phytomonas spp.) and insects (Crithidia spp.).
Reynolds number A dimensionless number that describes the relative contribution of inertial and viscous forces to cell movement. Microorganisms operate at low Reynolds numbers, for example,
V E C TO R - B O R N E D I S E A S E S
Motility and more: the flagellum of Trypanosoma brucei Gerasimos Langousis1 and Kent L. Hill1,2
Abstract | 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. Kinetoplastid A term used to describe a group of flagellated protozoa within the phylum Euglenozoa. The defining feature of kinetoplastids is that their mitochondrial DNA is arranged into a tightly packed network that is known as the kinetoplast.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, USA. 2 Molecular Biology Institute, University of California, Los Angeles, California 90095, USA. Correspondence to K.L.H. e-mail: [email protected] doi:10.1038/nrmicro3274 1
African trypanosomes, such as Trypanosoma brucei and related species, are kinetoplastid parasites1, which also include the human pathogens Trypanosoma cruzi and Leishmania spp. Together, these protozoan parasites cause substantial human morbidity and mortality worldwide and limit economic development in some of the most impoverished regions of the world2–4. In this Review, we focus on T. brucei, which is the causative agent of African trypanosomiasis (also known as sleeping sickness) in humans. The parasite is transmitted to humans by tsetse flies (BOX 1), and the disease proceeds in two clinical stages: the haemolymphatic stage, during which parasites replicate in the blood and lymph, causing clinical manifestations of recurrent fever and general malaise; and the invasive phase, during which parasites move out of the bloodstream and into extravascular spaces, including cardiac tissue and the central nervous system (CNS). This second stage is marked by meningoencephalitis, headaches and severe neurological changes that disrupt the sleep–wake cycle, and if left untreated, it is typically followed by coma and death5,6. Sleeping sickness poses a threat to an estimated 60 million people. There is currently no vaccine available and treatments are antiquated, toxic and increasingly ineffective7. T. brucei has a single flagellum, which is present throughout the cell cycle and during all stages of development (BOX 1). The flagellum is essential for viability8, is the sole means of motility and has emerged as a key player in multiple facets of development, transmission and pathogenesis. In fact, the distinctive auger-like motility of T. brucei (Supplementary information S1 (movie)) provided the basis for naming the genus. In
1843, in one of the earliest descriptions of T. brucei, Gruby9 observed that the organism, which he identified in frog blood, “ … turns two or three times around its axis, like a drill or a corkscrew, which is why I propose to name this haematozoan ‘Trypanosoma’. ” The name combines the Greek words trypanon (which means auger) and soma (which means body), so literally translated, the trypanosome is an ‘auger body’. Since then, trypanosome motility has captured the attention of many scientists, and an ‘undulating membrane’ (REF. 10) (now known to be the flagellum) is a prominent feature of most descriptions. Trypanosome movements to specific host tissues are defining events in pathogenesis and transmission (BOX 1), thus emphasizing the importance of the motility function of the flagellum. In addition to its canonical role in motility, the T. brucei flagellum is important for morphogenesis and cell division, and it is a crucial host–parasite interface that mediates attachment to host tissues and provides a scaffold for the assembly of signalling proteins and virulence factors. In this Review, we outline the structure and assembly of the T. brucei flagellum and discuss how this organelle functions in motility, morphogenesis and host–parasite interactions. Where relevant, we point out eukaryote-conserved versus trypanosome-specific flagellum features.
Flagellum structure The trypanosome flagellum emerges from the posterior of the cell and defines the anterior–posterior axis (FIG. 1). It is built on a canonical 9 + 2 axoneme, which contains nine doublet microtubules that are
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REVIEWS Box 1 | Trypanosoma brucei life cycle A generalized life cycle of Trypanosoma brucei is shown in the figure, part a. Infection of a mammalian host initiates when a tsetse fly bite delivers growth-arrested metacyclic trypomastigotes to the mammalian bloodstream. Metacyclic trypomastigotes then differentiate into proliferating long slender forms that establish and maintain a bloodstream infection. Parasites eventually penetrate the blood vessel endothelium and invade extravascular tissues, including the central nervous system (CNS). Little is known about specific developmental forms that are present in extravascular compartments. In the bloodstream, a quorum sensing-like mechanism elicits the differentiation of long slender forms into short stumpy forms that are cell-cycle arrested and pre-adapted for survival in the tsetse fly136. When an infected host is bitten by a tsetse fly, parasites are taken up with the blood meal into the midgut, where short stumpy forms differentiate into procyclic trypomastigotes that resume cell division and establish a midgut infection. Midgut procyclic trypomastigotes then embark on an epic migration (see the figure, part b) that takes them through the peritrophic matrix, along the foregut to the proventriculus, and from there onwards through the mouthparts, salivary ducts and ultimately into the salivary gland, where they attach to the salivary gland epithelium. In the proventriculus, procyclic trypomastigotes undergo extensive restructuring, coupled to an asymmetric division, to generate one long epimastigote and one short epimastigote. The short epimastigote attaches to epithelial cells following arrival in the salivary gland. Attached epimastigotes replicate and ultimately complete the life cycle via an asymmetric division82 to generate metacyclic trypomastigotes that are free in the salivary gland lumen and are uniquely adapted to survive in the mammalian host. The dividing forms that replicate via binary fission are indicated with a circular arrow. Parasite movements within the tsetse fly (see the figure, part b) and mammalian host (see the figure, part c) are illustrated. The blue line in part b depicts the route taken from the midgut to the salivary gland and the red line indicates the route from the salivary gland to the mouthparts. Parasites move out of blood vessels, penetrating the blood–brain barrier to enter the CNS (see the figure, part c).
a
Metacyclic trypomastigote Salivary gland
Bloodstream
Long slender form
Attached epimastigote
Tsetse Fly
Mammal CNS
Short epimastigote Long epimastigote
Short stumpy form
Proventriculus
Bloodstream Midgut
b
Procyclic trypomastigote
c
Proventriculus Salivary gland
Salivary duct Mouthparts
Midgut
Blood–brain barrier
symmetrically arranged around a central pair of singlet microtubules11. The axoneme is anchored in the cytoplasm at the posterior of the cell via the basal body, which is a barrel-like structure that contains nine peripheral triplet microtubules and has no central pair. As the basal body extends outwards, triplet microtubules become doublets, forming the axoneme transition zone. Filaments connect transition-zone microtubules to the surrounding membrane, forming a ciliary necklace12,13, and tubular structures on the external face of the membrane form the collarette13,14. The transition zone ends at the basal plate, which marks the site at which the centralpair microtubules, and hence the 9 + 2 axoneme, begin. The transition zone and the first part of the axoneme exit the cytoplasm through a specialized invagination of the plasma membrane, which is known as the flagellar pocket. The flagellar pocket corresponds to a gap in the subpellicular microtubules and is the exclusive site of endocytosis and secretion, which makes it a key portal for host–parasite interactions15. The T. brucei flagellar pocket is quite pronounced compared with what is observed in many other flagellated eukaryotes16, which perhaps reflects the demands of a pathogenic lifestyle and the role of the flagellar pocket in virulence and immune evasion15. The site at which the axoneme exits the flagellar pocket is known as the flagellar pocket collar, which is marked by a fibrous cytoskeletal structure that holds the flagellar membrane and cell membrane in close apposition and is required for flagellar pocket biogenesis17. From the collar onwards, the axoneme is surrounded by its own membrane, which has a lipid and protein composition that is distinct from that of the flagellar pocket membrane and cell membrane18,19. Most flagellated cells maintain flagellum connections to the cell membrane within the flagellar pocket16. However, the T. brucei flagellum is also laterally connected to the cell body along almost its entire length, and only the distal tip extends free of the cell body (FIG. 1). Lateral flagellum attachment is mediated by junctional complexes that use proteins in the flagellum and cell body to hold the flagellum membrane and plasma membrane in tight apposition20–23. These junctional complexes constitute a specialized ‘flagellum attachment zone’ (FAZ) that extends from the flagellar pocket to the anterior end of the cell. The FAZ includes a cytoplasmic FAZ filament24,25, together with a specialized quartet of subpellicular microtubules (known as the microtubule quartet) that are associated with a membranous reticulum26, the FAZ endoplasmic reticulum (FER) that is contiguous with the ER. Lateral attachment of the flagellum to the cell body has consequences for cell motility and cell division, as described below. Moreover, this unique architecture divides the cell surface into several discrete membrane subdomains27,28 (FIG. 1) and thereby imposes specialized demands on protein targeting to the host– parasite interface. Trafficking mechanisms that enable protein targeting to each of these specific subdomains remain enigmatic. The axoneme. The fundamental unit of flagellum motility is the 9 + 2 axoneme, which consists of nine outer
Nature Reviews | Microbiology 506 | JULY 2014 | VOLUME 12
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REVIEWS 92 + 2
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A lethal disease that is prevalent in sub-Saharan Africa. Two specific subspecies of Trypanosoma brucei, known as Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, cause disease in humans. A third subspecies, Trypanosoma brucei brucei, and the related trypanosomes Trypanosoma congolense and Trypanosoma vivax, infect only non-primates, causing wasting disease, which limits economic development in endemic areas.
Cell membrane
Ciliary necklace
Kinetoplast
A specialized region of the flagellar or ciliary membrane that surrounds the transition zone; it is defined by chalice-shaped filaments that extend outwards from the axoneme and form indentations in the ciliary membrane.
Subpellicular microtubules
92 + 2
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African trypanosomiasis
Cell membrane
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Cell membrane Basal plate Flagellar membrane Axoneme
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Tripartite attachment complex
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Basal body
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FAZ-associated flagellar membrane
FAZ filament
FAZ-associated cell body membrane
Flagellar pocket membrane
Transition zone Transition fibres
1 μm Flagellum
Subpellicular microtubules A cage-like array of microtubules that subtend the plasma membrane (pellicle) and run parallel to the long axis of the cell.
Microtubule quartet Four specialized subpellicular microtubules that extend from the basal body to the anterior of the cell and subtend the region of plasma membrane where the flagellum attaches to the cell body. These four microtubules constitute part of the flagellum attachment zone, are associated with a subdomain of the smooth endoplasmic reticulum and are antiparallel to the other subpellicular microtubules.
Trypomastigotes Parasite morphotypes in which the basal body is posterior to the nucleus.
Epimastigote A parasite morphotype in which the basal body is anterior to the nucleus.
Cell body
Posterior
Anterior
Flagellum tip
Figure 1 | Overview of the Trypanosoma brucei flagellum. A scanning electron micrograph of a Trypanosoma brucei Nature Reviews | Microbiology procyclic form is shown (lower panel). The main direction of cell propulsion (arrow) is with the flagellum tip leading. The upper panel shows a diagram of flagellum emergence from the flagellar pocket at the posterior end of the cell (boxed region in lower panel). The flagellum is built around a core of microtubules that are arranged in characteristic patterns, as shown above the corresponding cross sections. The flagellar axoneme emanates from the basal body via the transition zone and is laterally connected to the cell membrane via the flagellum attachment zone (FAZ). Extra-axonemal structures inside the flagellar membrane include the paraflagellar rod (PFR) and the ciliary necklace. A tripartite attachment complex links the basal body to the kinetoplast, and transition fibres connect the basal body to the flagellar pocket. The distinctive architecture of the trypanosome flagellum is dictated by specialized membrane and cytoskeletal features, such as the flagellar pocket collar and the FAZ filament. Adjacent to the FAZ filament are the subpellicular microtubules, the microtubule quartet and the FAZ endoplasmic reticulum (FER). For clarity, some axonemal structures, such as dyneins and radial spokes are not depicted.
doublet microtubules that surround a central pair of singlet microtubules29 (FIG. 2). Each of the outer doublet microtubules consists of an A‑tubule and a B‑tubule, which provide a scaffold for the assembly of inner-arm and outer-arm dynein motors. Adjacent doublets are connected via the nexin–dynein regulatory complex (NDRC)30,31,32. Radial spokes extend inwards from each
outer doublet and converge on the central-pair apparatus, which includes the central-pair microtubules surrounded by a sheath-like structure33. Components of the T. brucei radial spokes and central-pair apparatus remain mostly uncharacterized, but the few examples that have been studied show that these structures are critical for normal motility34–36. In a landmark study, axoneme biogenesis
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Figure 2 | The axoneme and PFR structure. The upper panel shows a longitudinal Nature Reviews | Microbiology view of the axonemal repeating unit on one outer doublet microtubule, which is oriented with the basal body end on the left-hand side. The lower panel is a diagram showing transverse section detail of the axoneme and paraflagellar rod (PFR), as viewed from posterior to anterior (lower panel) . Prominent axonemal structures are labelled and include outer doublet and central-pair microtubules, outer-arm dyneins and inner-arm dyneins, radial spokes and the nexin–dynein regulatory complex (NDRC), which connects adjacent doublets. Outer doublet microtubules are numbered 1–9, as indicated. Proximal, intermediate and distal domains of the PFR are shown.
Propulsive parasite motility A sustained, forwards movement of a parasite. Propulsive motility is distinguished from general writhing of the parasite, which is generated by unregulated beating of the flagellum.
in T. brucei was shown to be essential and to depend on intraflagellar transport (IFT)8 (BOX 2). When viewed longitudinally, substructures on outer doublet microtubules are arranged in a repeating unit with a periodicity of approximately 96 nm37. In T. brucei, the axonemal repeat unit includes three radial spokes, four outer-arm dyneins, inner-arm dyneins and the NDRC30 (FIG. 2). The stoichiometry of inner-arm dyneins and the NDRC in T. brucei has not been determined. The basic architecture of the axoneme repeating unit is conserved among eukaryotes that have motile flagella. However, there are several differences in T. brucei compared with Chlamydomonas reinhardtii, which is considered to be a reference organism for axoneme structure37,38.
For example, the T. brucei axoneme has three radial spokes instead of two, each outer-arm dynein has two heavy chains instead of three and the T. brucei axoneme has specialized NDRC subunits and central-pair microtubules that retain a fixed orientation relative to outer doublets30,34,36,39. At the molecular level, flagellum motility is powered by ATP-dependent structural changes in axonemal dynein motors that are permanently attached to the A‑tubule of each outer doublet microtubule. These structural changes cause dyneins to reversibly attach to the B-tubule of the neighbouring doublet and drive the sliding of adjacent doublets. Separate connections, such as the NDRC, limit sliding, and this generates doublet bending40. Harnessing flagellum beating to drive propulsive parasite motility requires the propagation of localized bending of axonemal doublets along the length of the axoneme — this process requires spatial and temporal regulation of thousands of dynein motors. The precise mechanisms for regulating axonemal dynein activity are not clear, but they involve various axonemal protein complexes41,42. Among these, the NDRC is perhaps the best characterized32. The NDRC is a large (>1 MDa) complex that is present in almost all motile axonemes and contains conserved as well as organism-specific subunits43–46. In T. brucei, the NDRC includes trypanin36 and component of motile flagella 70 (CMF70)44, as well as candidate subunits, trypanin-related protein, CMF46, CMF40 and CMF22 (REFS 36,43,47). Pioneering studies in C. reinhardtii show that the NDRC functions together with the radial spokes and central-pair apparatus as a reversible inhibitor of dynein32,48. Functional analysis of NDRC subunits supports a similar role in T. brucei36,44. The architecture and exact subunit composition of the NDRC and other axonemal regulatory systems remain unknown and this should be the focus of future studies. The paraflagellar rod. As the flagellum extends beyond the flagellar pocket, an extra-axonemal structure called the paraflagellar rod (PFR) is formed49. The PFR is a massive, lattice-like filament that runs alongside the axoneme and is connected to axonemal doublets 4–7. It is restricted to kinetoplastids and a few other organisms49 and is essential for normal motility in T. brucei50. In cross-section, the PFR has three structural domains that are proximal, intermediate and distal to the axoneme– PFR interface (FIG. 2). Three recent electron tomography studies reported that a repeating unit within the distal domain has 51–57 nm longitudinal periodicity21,30,51, and PFR connections to the axoneme were observed to occur with a longitudinal periodicity of 56–57 nm. The periodicity of the connectors corresponds well to the PFR repeat-unit size and is a multiple of the 8 nm unit of tubulin dimers that make up the axonemal microtubules, which suggests that the connectors might be elaborated from intrinsic structural repeats. The size and complexity of the PFR–axoneme superstructure makes structural studies technically demanding, and more work is needed to define a detailed structure as well as to elucidate the assembly mechanisms of the PFR (BOX 2).
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REVIEWS Box 2 | Flagellum assembly in Trypanosoma brucei Trypanosoma brucei flagellum assembly initiates at the basal body and depends on intraflagellar transport (IFT)8,137,138, which is a bidirectional transport system that operates along outer axonemal doublet microtubules to deliver proteins into and out of the flagellum. Complexes of IFT proteins assemble into particles that mediate the delivery of nascent flagellum subunits from their cytoplasmic site of synthesis into the flagellum. Kinesin motors transport IFT particles and their cargo to the flagellar tip (via anterograde transport), where cargo is released for assembly into the growing flagellum. IFT particles are returned to the base of the flagellum (via retrograde transport) by dynein motors. IFT was originally identified in Chlamydomonas reinhardtii and is now known to be an almost universal feature of eukaryotic flagella137. T. brucei encodes all of the IFT proteins that are required for flagellum assembly139. Live imaging of IFT in T. brucei (Supplementary information S2 (movie)) suggests that there are two pools of IFT particles at the base of the flagellum and that only one of these actively participates in IFT140. These studies also revealed two distinct populations of anterograde IFT particles, each of which moves at a different speed. In addition to IFT, eukaryotes depend on a specialized protein complex, which is known as the BBSome, for trafficking of particular membrane proteins into and out of the flagellum. The BBSome is a multimeric complex of highly conserved proteins that is found only in ciliated organisms141. In humans, mutations in BBSome genes result in Bardet–Biedl syndrome (BBS), which is a pleiotropic disease that is caused by defective cilia141. T. brucei has homologues of all BBSome subunits and there is indirect evidence that links them to flagellum assembly142, although their direct involvement has not been tested. In addition to having characteristics in common with other eukaryotes, T. brucei flagellum assembly has parasite-specific features. First, the trypanosome flagellum is retained throughout the cell cycle, which differs from most other organisms that have been studied, and assemblies of the new and old flagellum seem to be controlled separately. Second, the flagellum tip harbours a disorganized axoneme during flagellum biogenesis143. Third, growth of each new flagellum follows a path that is defined by the old flagellum86, and the presence of the paraflagellar rod (PFR) seems to limit which outer doublet microtubules may be used for IFT139. Thus, T. brucei flagellum assembly must accommodate spatial constraints that are not seen in other organisms. The T. brucei flagellum also undergoes major changes in position and length throughout the parasite life cycle (BOX 1; FIG. 3), which indicates a need for plasticity in the assembly systems that control flagellum size and position. Furthermore, axoneme assembly must be coordinated with assembly of the PFR and flagellum attachment zone (FAZ) (FIG. 1). The regulatory systems that are responsible, as well as their relationships to canonical IFT and BBSome systems, remain to be discovered. PFR assembly depends on axoneme biogenesis, but axoneme biogenesis does not depend on PFR assembly, as IFT mutants fail to assemble both structures, whereas PFR mutants assemble normal axonemes8,144. The mechanisms of PFR assembly are not known, but, in addition to the major subunits PFR1 and PFR2 (REFS 50,145), assembly depends on calmodulin95 and involves a specific kinesin146.
Trypanosomatids A group of parasitic protozoa that infect mammals (Trypanosoma spp. and Leishmania spp.), plants (Phytomonas spp.) and insects (Crithidia spp.).
Reynolds number A dimensionless number that describes the relative contribution of inertial and viscous forces to cell movement. Microorganisms operate at low Reynolds numbers, for example,
