Trypanosome cell structure and function

1. Trypanosome cell structure and function Medical Parasitology, CBIO 4500/6500 Spring 2010 Silvia N.J. Moreno

2. Trypanosome forms • Trypomastigotes: 15-80 m single flagellum posterior to the nucleus. Found in the mammalian host in the blood. Also as metacyclics in the insect vector. Undulating membrane. T. cruzi and T. brucei • Epimastigotes: flagellum anterior to the nucleus. Present in the insect vector. T. brucei and T. cruzi Anterior Posterior • Amastigotes: short or no flagellum. Intracellular form in mammalian cells. T. cruzi and leishmania. • Promastigotes: short flagellum. No undulating membrane. Extracellular form in the insect vector. Leishmania parasites

3. Schematic of the T. brucei life cycle. • Two major switches studied in detail: • slender to stumpy bloodstream form: density sensing could be important or “differentiation division” • the bloodstream form to procyclic (tse tse midgut form):The in vitro process is stimulated by citrate and cis-aconitate and a reduction in Temp from 37 to 27 C Profound changes in morphology, surface coat and biochemistry (as exemplified by mitochondrial function) occur during the trypanosome life cycle. The coordinated control of cell cycle and differentiation events are essential to life cycle progression. Metacyclic and short stumpy trypanosomes are non-replicating forms arrested in the G0/G1 phase of the cell cycle and are pre-adapted for survival in the ensuing host.

4. Trypanosome structures • Cytoskeleton: • Subpellicular microtubules • The flagellum • The plasma membrane and the surface coat: Surface membrane is densely packed with a protein called VSG (variant surface glycoprotein) • A unique mitochondrial DNA architecture: The kinetoplast • Complex and energy-consuming mitochondrial RNA editing • The compartmentation of glycolysis: glycosome

5. Microtubules • Microtubules (MT) form part of the cytoskeleton that gives structure and shape to a cell, and also serve as conveyor belts moving other organelles throughout the cytoplasm. Microtubules are polymers of α- and β-tubulin dimers. These dimers polymerize end to end in protofilaments which bundle into hollow cylindrical filaments. The head-to-tail association of the αβ heterodimers makes microtubules polar structures. A microtubule is a polar structure, its polarity arising from the head-to-tail arrangement of the α- and β-tubulin dimers in a protofilament. Because all protofilaments in a microtubule have the same orientation, one end of a microtubule is ringed by α-tubulin, while the opposite end is ringed by β-tubulin. • Trypanosomes have 3 different populations of MTs: • The highly labile MTs of the mitotic apparatus. • The stable MTs of the membrane skeleton. • The flagellar axoneme The organization of tubulin subunits in a microtubule. The subunits are aligned end to end into a protofilament (magenta highlight). The side-by-side packing of protofilaments forms the wall of the microtubule. In this model, the protofilaments are slightly staggered so that α-tubulin in one protofilament is in contact with α-tubulin in the neighboring protofilaments. http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406&rendertype=figure&id=A5409

6. Trypanosome cytoskeleton Functions: Maintaining the shape and form of the cell Modulation of cell shape between different life cycle stages Motility and attachment to host cell surfaces Components: Microtubular subpellicular corset The flagellum contain two major structural components: the axoneme and paraflagellar rod

7. Subpellicular Microtubules • A layer of microtubules localized below the plasma membrane and closely associated with it. • Cage-likearray which encloses the entire body. This structure gives rigidity to the cell. • MTs are regularly spaced and their number is related to the diameter of the cell. • Cross-links between each other and to the inner face of the PM • Absence of trans-cellular cytoskeletal structures • Most subpellicular microtubules extend the entire length of the cell. Different views of the association of the sub-pellicular microtubules with each other and with the plasma membrane.

8. Trypanosome microtubules • The 24-nm-diameter microtubules form a helical pattern along the long axis of the cell. • Individual microtubules vary in length. When a microtubule stops, those on either side of it continue and become connected partners. • This construction allows for shape changes along the length of the parasite and between life cycle stages. • The cortical microtubules are very stable and remain attached to the plasma membrane during cell fractionation studies. Flagellum Microtubules

9. T. brucei microtubules • Resistant to de-polymerization by low temperatures and to antimicrotubular drugs with activity against higher eukaryotes MTs: colchicine • Microtubule associated proteins: (MAPs): EM shows that the MTs are interconnected by MPAs and also connected to the PM. • Microtubules have an intrinsic polarity reflecting their constituent (αβ-tubulin) heterodimers. • The MTs of the T. brucei cortex appear to have the same polarity—with their plus (+) ends at the posterior of the cell. • The subpellicular corset does not break down during cell division. New microtubules are added and intercalated into this array during the cell cycle, and a complete microtubule corset is inherited by each daughter cell at cytokinesis.

10. THE FLAGELLUM The internal structure is composed of a central axonemeand an outer sheath that is the continuation of the plasma membrane. The axoneme consists of nine peripheral (doublets) and one central pair of microtubules (singlets). The force for propulsion is provided by dynein (motor proteins) anchored in rows along one side of each doublet, which can walk along the microtubule of the adjacent doublet. In order to produce coordinated bending of the flagellum, these dynein motors – organized into multi-headed complexes called the inner and outer dynein arms – must produce their power strokes in synchrony. http://www.northland.cc.mn.us/biology/Biology1111/animations/flagellum.swf

11. T. brucei flagellum • FLAGELLUM FUNCTIONS: • Motility • Adhesion to various cell types and surfaces • Sensory Perception • Cell Morphogenesis • Cell Division • Motility and cytokinesis The single flagellum emerges from an invagination called the flagellar pocket and remains attached to the cell body along its length. Important structural elements: The microtubular axoneme with the canonical eukaryotic 9+2 and The paraflagellar rod (PFR): a lattice like structure highly organized of unknown function. (a) Scanning electron micrograph (EM) of a procyclic trypanosome. The arrow indicates the single flagellum. (b) Cross-section of the flagellum and its attachment to the cell body as viewed looking from posterior toward anterior. (c) Major flagellar substructures are indicated and outer doublet microtubules are numbered according to convention. Abbreviations: DRC, dynein regulatory complex; FAZ, flagellum attachment zone; IFT, intraflagellar transport; MT, microtubule; PFR, paraflagellar rod.

12. T. brucei flagellum structure The basal body is a barrel-shaped structure comprising nine triplet microtubules analogous to the mammalian centriole (9 triplets of MTs). BBs provide MT organizing centers for axonemal and subpellicular MTs while controlling the position and segregation of the mitochondrial genome. Triplet microtubules of the mature basal body extend to become doublets, forming a 9 + 0 transition zone between the basal body and the basal plate that demarcates the beginning of the 9 + 2 axoneme. (b) Longitudinal section showing the flagellar apparatus (blue arrow) and FP (orange arrow). (c) The basal body apparatus. (d) The 9 + 0 transition zone within the FP; (e) The 9 + 2 flagellar axoneme within the FP. (f) The flagellum exiting the FP through the FPC. (g) The flagellum outside of the FP but not yet having a PFR. (h) The flagellum outside of the FP, containing both an axoneme and a PFR (asterisk). AX, 9 + 2 axoneme. BB, basal body; FAZ, flagellum attachment zone; FAZ PMT, the four specialized subpellicular microtubules of the flagellum attachment zone; FP, flagellar pocket; FPC, flagellar pocket collar; PFR, paraflagellar rod; TAC, tripartite attachment complex; TZ, transition zone.

13. T. brucei flagellum Axoneme: an evolutionarily-conserved microtubule-based structure that provides the scaffold for all eukaryotic flagella. As in most motile eukaryotic flagella, the trypanosome axoneme consists of nine outer doublet microtubules (ODs) surrounding a central pair apparatus (CP) of singlet microtubules. Radial spokes extend inward from each outer doublet toward the CP. ATP-dependent dynein motor proteins attached to outer doublets generate the sliding forces that underlie flagellar movement. Outer arm and inner arm dyneins extend from each outer doublet A-tubule and provide the driving force for motility.

14. T. brucei flagellum The Paraflagellar Rod • The PFR is a large paracrystalline filament extending alongside the axoneme from the flagellar pocket to the flagellum tip. • The PFR is unique to kinetoplastids, euglenoids, and dinoflagellates. • In cross-section the PFR has a lattice-like appearance with three distinct regions, proximal, intermediate, and distal, defined by their structural morphologies and positions relative to the axoneme.

15. (2) (2) (3) (1) (1) (4) The Flagellar Pocket The FP (1) forms from an invagination of the cell membrane where the proximal end (4) of the flagellum emerges from the cytoplasm. The FP is not completely membrane enclosed, electron-dense adhesion zones (2) hold the flagellum in tight apposition to the cell membrane and demarcate the boundary of the FP membrane, causing a constriction termed the flagellar pocket neck or collar. The lumen of the flagellar pocket (1) is an extracellular compartment that is secluded from hostile host environments. The invagination that forms the flagellar pocket also forms the lumen of the flagellum through which the flagellar axoneme passes (3). Landfear and Ignatushchenko, Mol. Biochem. Parasitol. 115, 1-17, 2001

16. The flagellar pocket • The flagellar pocket is free of sub-pellicular microtubules. • The FP is the sole site of endocytosis and exocytosis. • The FP must accommodate export of abundant variant surface glycoprotein (VSG) or procyclin surface proteins and simultaneously mediate uptake of extracellular macromolecules. As the sole site of surface protein turnover and nutrient uptake, the FP represents a key portal for host-parasite interaction. Cut-out view of the FP area. The FP is formed by an invagination of the plasma membrane where the flagellum emerges from the basal body complex.

17. T. brucei flagellum The flagellar attachment zone: FAZ Transverse electron micrograph (EM) section of the T. brucei bloodstream form, showing the flagellum (Flag) and cell body (Body). The electron-dense filament (Fil), macula adherens (small arrowheads) and membranous compartment (MC) of the FAZ are indicated. Large arrowheads point out the gap between the flagellar and cell body membranes. Also indicated are the subpellicular microtubules (PMT), the paraflagellar rod (PFR), and the granular endoplasmic reticulum (GR). • The FAZ is an adhesion region where the flagellum emerges from the FP and remains attached to the cell body. • On the cytoplasmic side, the FAZ is defined by an electron-dense filament of unknown composition and by four specialized microtubules of the subpellicular corset that are associated with a membranous compartment. • A network of regularly spaced filaments links the FAZ filament in the cell body to both the axoneme and the PFR in the flagellum, abutting the flagellar membrane and plasma membrane into macula adherens.

18. FLAGELLUM FUNCTION Motility:African trypanosomes are highly motile, moving at speeds of up to 20 um s−1. They exhibit alternating periods of translational cell movement and tumbling, which causes reorientation. The dominant waveform is a tractile beat that initiates at the tip of the flagellum and propagates toward the base. Pathogenic features of sleeping sickness are directly linked to migration of the parasite to specific host tissues and parasite traversal of the blood brain barrier is a critical and defining step of disease progression.

19. The flagellum mediates attachment to the tsetse fly salivary gland. Diagram of the developmental changes that occur while the parasite is attached to the salivary gland epithelium. Transverse electron micrograph section of a T. brucei epimastigote showing the flagellum (F) with flagellar outgrowths (FO) intercalating with the microvilli (MV) of the tsetse fly salivary gland epithelium. Note the attachment plaques (APs) that form junctions with MV. The mitochondrion (M) is indicated. FLAGELLUM FUNCTION Host cell attachment • The final step in maturation to infectious bloodstream forms, including acquisition of the VSG coat, occurs following parasite attachment to the tsetse fly salivary gland. This attachment is mediated by the flagellum, which forms outgrowths of membrane and cytoskeletal material that penetrate into the spaces between microvilli of the salivary gland epithelium. • Flagellar and host cell membranes are held in close apposition by hemidesmosome-like attachment plaques. • Attached epimastigotes cease division and lose the extensive flagellar outgrowths but retain the attachment plaques. • VSG-coated metacyclic parasites are released into the lumen of the salivary gland for transmission to a new mammalian host.

20. FLAGELLUM FUNCTION Endocytosis and immune evasion:it has been demonstrated that flagellar beating is required for endocytosis of VSG complexed with immunoglobulin. This strategy avoids destruction by the host immune system. Flagellar motility would contribute to immune evasion and persistent infection. Sensory Perception:Flagella are well known for their sensory roles in other organisms. Few evidences indicate that this could be the case in Trypanosomes Cell Morphogenesis:The flagellum is connected to several subcellular organelles and structures, e.g., FAZ, flagellar pocket, kinetoplast, and mitochondrion, and dictates their arrangement. Cell Division:The T. brucei cleavage furrow initiates between the tips of the new and old flagella/FAZ and progresses posteriorly, suggesting an important role for the flagellum/FAZ in directing cleavage furrow formation Motility and cytokinesis: pulling and rotational forces supplied by flagellar beating contribute to normal cytokinesis.

21. The Kinetoplast DNA with a peculiar network structure: 20-25% of the total DNA of some of the parasites Disk shape structure near the flagellar basal body Lu et al. Mol. Cell. Biol.(1998)18, 2309-2323

22. KDNA components The network contains 5-20 x103 catenated minicircles (0.8-2.5 kb) and 20-50 catenated maxicircles (23-36 kb). Maxicircles: 20-40 kb. Functional homologue of mitochondrial DNA: Region encoding ribosomal RNAs, subunits of respiratory complexes and a variable region non-transcribed. Maxicircles transcripts undergo RNA editing. Minicircles: 0.5-2.5 kb. Heterogenous in sequence. Encode the guide RNAs. All minicircles share the Universal minicircle sequence (UMS) a 12 nt motif, part of the replication origin and conserved in all trypanosomatids EMs of Crithidia fasciculata kDNA. Segment of intact network. Small loops are minicircles, and longer strands (yellow arrow) are parts of maxicircles. The entire network is elliptical, planar and 10 μm by 15 μm.

23. KDNA Network Organization Isolated Network 10×15 μm in size for the isolated network. Diagram of isolated planar kDNA network, valence~3. Minicircles in the network are relaxed and singly interlocked to each other. Network in vivo Network condensed into a disk. Vertical bar represents the axis of the disk

24. Images of kinetoplasts of Crithidia fasciculata (a) and Trypanosoma avium (b) A: Longitudinal section through the classical disk-shaped kDNA of C. fasciculata. The disk thickness is about half the minicircle circumference (2.5 kB) B:Longitudinal section through the kDNA of T. avium. The disk appears cylindrical due to the large minicircle size (10 kB) The kDNA disk is positioned in a fixed region of the mitochondrial matrix, near the basal body of he flagellum. The axis of the disk aligns with the axis of the flagellum to which is physically linked. Eukaryotic Cell 1: 495

25. Replication of the kDNA network • kDNA replication involves doublingof the number of minicircles and maxicircles and distributing the progeny into two daughter networks which are identical to the parent network. • Both minicircles and maxicircles replicate only once per generation during S phase, synchrony with nuclear DNA synthesis. • kDNA surrounded by proteins that catalyze various steps in replication • Minicircles do not replicate while attached to the disk • Minicircles contain a nick or gap after replication that persists until all minicircles have undergone replication

26. Minicircles replication • Minicircles replicate by theta (mechanism • Minicircles: decatenate—replicate---catenate---fill gaps--- scission • Replication initiates at a 12-nucleotide sequence called “universal minicircle sequence” (UMS)-- GGGTTGGTGTA • Replication is unidirectional, with the leading strand synthesized continuously and lagging strand synthesized in short Okazaki fragments Book-keeping Final stages As the minicircle number increases in the same space, it becomes crowded. When replication is complete, the crowdedness decreases and the network surface area doubles. After the nicks and gaps are repaired, the network splits in two.

27. kDNA-replication model Kinetoflagellar zone Liu et al, Trends in Parasitol. (2005) 21:363 The kDNA disk, organized with minicircles stretched parallel to its axis, is surrounded by replication proteins. Covalently closed minicircles are released from the network into the KFZ, in which they initiate replication as θ structures. The progeny free minicircles then migrate to the antipodal sites at which the next stages of replication occur. The minicircles (still containing at least one nick or gap) are then linked to the network periphery by topoisomerase II (Topo II). DNA polymerase β-PAK and DNA ligase kα are probably involved in the repair of the remaining minicircle gaps when replication is complete. The figure shows the filament system linking the kDNA to the flagellar basal body. These filaments, together with the replication machinery, emphasize the structural complexity of the KFZ.

28. Electron microscopy of Trypanosoma brucei showing cross-sections through KD Early stage of replication Late stage of replication (a) The KD is probably in an early stage of replication. (b) The KD, having expanded laterally, is probably in a late stage of replication. The region below the kDNA disk is the KFZ, where minicircle replication begins. The electron-dense regions flanking the kDNA disk in (a) probably represent the antipodal sites (AS) at which later stages of minicircle replication occur. The dense region in the KFZ in (a) and (b) represents the unilateral filaments that contribute to the linkage between the kDNA and the flagellar basal body; the replication machinery could also contribute to this electron density. The ellipse (red) in the KFZ in (a) represents a free minicircle, drawn approximately to scale. Scale bar=250 nm. Trends in Parasitology 21:363

29. Maintenance of the minicircle repertoire • kDNA networks contain a variety of different minicircle sequences, with each sequence class having a different copy number. Each type of Minicircle encodes a different guide RNAs, complete loss of a minicircle class during replication could prevent proper editing and be lethal to the parasite. • Marking the replicated minicircles with one or more nicks or gaps prevents a second round of replication. • A mechanism ensuring that sister minicircles migrate to different antipodal sites would increase the probability that they ultimately segregate into different daughter networks. This has not been demonstrated experimental but several evidences indicate that this is the case.

30. Maxicircle replication: Theta structure primary • Maxicircles replicate while still attached to the kDNA network • Replication proceeds in theta structure • Initiates in variable region (a noncoding segment characterized by repetitive sequences) • Proceeds unidirectionally clockwise • Share some enzymes with minicircles, such as primase, topo II. mature

31. The peroxisome • Peroxisomes are surrounded by a single membrane and they do not contain DNA or ribosomes. • They acquire their protein through selective import from the cytosol. • They are found in all eukaryotic cells. • They contain oxidative enzymes such as catalase and urate oxidase at such high concentrations that it is possible to see by EM a crystalloid core. • Peroxisomes contain one or more enzymes that use molecular oxygen. • In addition, other oxidative reactions occur in the peroxisome like breakdown of fatty acids. • Peroxisomes are unusually diverse organelles and in different cell types of a single organisms they may contain different sets of enzymes. A particular class of peroxisome, the glycosome, is shown. Bar, 1 mum. ( ER, endoplasmic reticulum; K, kinetoplast; M, mitochondrion; P, peroxisome.) Christian de Duve (1917-)

32. Glycolysis and glycosomes The major part of the glycolytic pathway is compartmentalized in this organelle These are peroxisomes: (1)are bounded by a single phospholipid bilayer membrane, have an electron-dense proteinaceous matrix and do not contain any detectable DNA; (2) although glycolytic enzymes are the most prominent proteins in glycosomes (they may comprise up to 90% of the content in bloodstream forms of T. brucei), some other enzymes or enzyme systems may be present and are shared with peroxisomes: e.g. enzymes of peroxide metabolism, fatty acid oxidation and ether-lipid biosynthesis; (3) glycosomes and other members of the peroxisome family have a homologous process of biogenesis. (4) Content may vary between different organisms http://www.icp.ucl.ac.be/trop/images/glycosomes/glycosomes-Pages/Image0.html

33. The energy metabolism of bloodstream T. brucei • The long-slender bloodstream form T. brucei has a very simple type of energy metabolism: it is entirely dependent on degradation of glucose into pyruvate by glycolysis. • Glucose is degraded to 3-phosphoglycerate inside the glycosomes and this intermediate is then further degraded in the cytosol to pyruvate, the excreted end-product. The redox balance in the glycosome is maintained via a glycerol 3-phosphate shuttle and the alternative oxidase present in the mitochondrion.

34. The energy metabolism of procyclic T. brucei Transformation of bloodstream form T. brucei into the procyclic insect stage is accompanied by striking changes in energy metabolism. The glycosomal metabolism is extended and part of the produced phosphoenolpyruvate is imported from the cytosol and subsequently converted into succinate. In this procyclic insect stage, the end-product of glycolysis, pyruvate, is not excreted but is further metabolized inside the mitochondrion in which it is mainly degraded to acetate. Acetate production occurs by a pathway that generates extra ATP. In addition to carbohydrate degradation, amino acids, mainly proline and threonine, are important substrates for the production of ATP in procyclic insect-stage T. brucei. Procyclics have a more elaborate energy- and carbohydrate metabolic network that also involves the mitochondrial Krebs cycle enzymes and a respiratory chain with coupled transmembrane proton-translocating activity. Two enzymes found that are not present in the bloodstream glycosome: phosphoenol pyruvate carboxykinase (14) and pyruvate phosphate dikinase (18). Phosphoglycerate kinase (7) is re-located to the cytosol so the ATP/ADP balance is maintained. The NAD/NADH is balanced by the malate dehydrogenase (15) and fumarate reductase (17)

35. Glycosomes Parsons et al., Mol. Biochem. Parasitol. 115,19-28, 2001