Abstract
Trypanosomatidae are protozoans that include monogenetic parasites, such as the Blastocrithidia and Herpetomonas genera, as well as digenetic parasites, such as the Trypanosoma and Leishmania genera. Their life cycles alternate between insect vectors and mammalian hosts. The parasite’s life cycle involves symmetrical division and different transitional developmental stages. In trypanosomatids, the cytoskeleton is composed of subpellicular microtubules organized in a highly ordered array of stable microtubules located beneath the plasma membrane, the paraflagellar rod, which is a lattice-like structure attached alongside the flagellar axoneme and a cytostome-cytopharynx. The complex life cycle, the extremely precise cytoskeletal organization and the single copy structures present in trypanosomatids provide interesting models for cell biology studies. The introduction of molecular biology, FIB/SEM (focused ion beam scanning electron microscopy) and electron microscopy tomography approaches and classical methods, such as negative staining, chemical fixation and ultrafast cryofixation have led to the determination of the three-dimensional (3D) structural organization of the cells. In this chapter, we highlight the recent findings on Trypanosomatidae cytoskeleton emphasizing their structural organization and the functional role of proteins involved in the biogenesis and duplication of cytoskeletal structures. The principal finding of this review is that all approaches listed above enhance our knowledge of trypanosomatids biology showing that cytoskeleton elements are essential to several important events throughout the protozoan life cycle.
Keywords
- trypanosomatids
- cytoskeleton
- ultrastructure
- microscopy
- three-dimensional reconstruction
1. Introduction
Trypanosomatids are uniflagellated protozoan parasites belonging to the Kinetoplastid order. They are the etiological agents of several diseases [1] and exhibit particular features that differentiate them significantly from their mammalian host [2]. First, they have subpellicular microtubules (SPMT), which are a network of organized stable microtubules that are closely associated with the plasma membrane and to each other forming a corset that confers rigidity to the cell body and help to determine the shape of the cell. Second, trypanosomatids have the microtubule quartet (MtQ) of the flagellar pocket (FP) that encircles the flagellar pocket in a helicoidal pattern [3]. Third, they have microtubule sets of cytostome-cytopharynx that forms a gutter in this funnel invagination [4]. Fourth, trypanosomatids have a flagellum attachment zone (FAZ) where the flagellum emerges from the flagellar pocket and remains attached to the cell body [5]. Finally, they have a paraflagellar rod (PFR), a lattice-like structure that runs parallel to the axoneme from the flagellar pocket to the flagellar tip [6].
Recent studies using techniques, such as electron tomography and focused ion beam-scanning electron microscopy, that allow three-dimensional reconstruction of whole protozoan, allowed for the accurate understanding of their subcellular morphology. High-resolution microscopy studies have provided detailed cellular information regarding protein localization and phenotype after cytoskeleton-protein depletion aiming at the elucidation of protein function during life cycle of trypanosomatids. Using FIB/SEM, one can examine a large number of whole cells at the same time, which enables qualitative and quantitative studies about different morphological aspects of cytoskeleton elements during the life cycle of trypanosomatids. This chapter aims to provide an overview of the topographical relationship among the cytoskeletal elements throughout the protozoan life cycle.
2. Subpellicular microtubules
The shape of cells in trypanosomatids is defined by SPMT. SPMT are a cage of stable microtubules located underneath the plasma membrane and composed of α/β tubulin [7]. High-resolution field emission scanning electron microscopy (FESEM) revealed a helicoidal pattern of SPMT in the promastigote form of nonpathogenic
In contrast to the microtubules of mammalian cells, SPMT are resistant to low temperatures and drugs that usually promote microtubule depolymerization [10]; thus, SPMT contribute to the stabilization of the trypanosomatids shape. Transversal ultrathin sections of
These SPMT are cross-linked to each other by short 6 nm thick filaments and to the plasma membrane [9, 13] (Figure 1D). Molecular studies using
Due to the rigidity of the SPMT cage beneath the cell membrane, endocytic events are limited to sites where these microtubules are absent: the flagellar pocket and the complex cytostome-cytopharynx.
3. Cytoskeletal elements associated with endocytic entry sites
3.1. Microtubules
The FP is an invagination close to the basal body and surrounds the site of flagellum exit. It is found at the anterior and posterior regions of the cell body of
Several studies with
Although
Electron microscopy tomography and dual beam scanning electron microscopy are used to observe epimastigotes of
The same methodology was used to obtain 3D reconstructions of the ultrastructural changes present in the intermediate forms of
3.2. Microfilaments
The presence of actin has been confirmed in some trypanosomatids species. Nevertheless, little is known about microfilament function or localization [7, 42]. In trypanosomatids, it does not appear to polymerize into highly structured cytoskeletal microfilaments [43].
The trypanosomatids genome contains putative actin and actin-binding protein sequences [44]. However, to date, few studies have successful visualized microfilaments in trypanosomatids. Sahasrabuddhe et al. visualized actin filaments in
Molecular methods confirmed the expression of actin in trypanosomes, although the role of actin in
Using cytochalasin to depolymerize actin resulted in morphological changes to the cytoskeletal elements associated with the cytostome-cytopharynx of
4. Paraflagellar structure and flagellum attachment zone
The trypanosomatid flagellum consists of an evolutionarily conserved axoneme. It is also composed of peculiar elements such as the FAZ and the paraflagellar rod (PFR), a structure that is attached to the flagellum. FAZ is a specialized cytoskeletal region that links most of the flagellar membrane to the membrane of the cell body [5] (Figure 5A–C). The FAZ filament, the MtQ and the bilobe structure also help to link the flagellum to the cell surface [34, 50] (Figure 2A).
This complex has a left-handed, helical conformation around the cell body and within the microtubule array [51]. This region is considered a junctional complex and formed by lined apposed macular structures. The FAZ is an essential structure; disruption of FAZ assembly can lead to dramatic changes in morphogenesis.
The flagellum adhesion glycoprotein 1 (FLA1) was identified in
The first molecular component of the
Recently, depletion of ClpGM6, a calpain-like protein localized to the FAZ of
In live parasites, flagellar beating produces a wave that gives the appearance of an “undulating membrane” on the sides of the cell body linked to flagellum as consequence of the FAZ arrangement. A detailed study using high-resolution microscopy to compare the swimming behavior of several trypanosome species that infect livestock showed that the waveforms are distinctive for each trypanosome species. This is due to variations in the microenvironment, such as differences in viscosity [61]. Inside the flagellar membrane of
Replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated
The paraflagellar major proteins are PFR1 and PFR2; null mutant and RNAi ablation of PFR2 demonstrated that this protein is required for efficient flagellar beating in
The PFR in
5. Conclusions
All the cytoskeletal structures and related proteins covered here are essential for the biology of trypanosomatids. Advances in high-resolution microscopies and molecular biology have provided more information regarding protein localization and function in these protozoa. This chapter provided an overview of unique and essentials cytoskeleton elements and proteins in trypanosomatids that may provide alternative targets in the future for chemotherapeutic drugs.
References
- 1.
Morriswood B. Form, fabric and function of a flagellum-associated cytoskeletal structure. Cells. 2015;4(4):726–47. - 2.
Bastin P, Pullen TJ, Moreira-Leite FF, Gull K. Inside and outside of the trypanosome flagellum: a multifunctional organelle. Microbes and Infection/Institute Pasteur. 2000;2(15):1865–74. - 3.
Lacomble S, Vaughan S, Gadelha C, Morphew MK, Shaw MK, McIntosh JR, et al. Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography. Journal of Cell Science. 2009;122(Pt 8):1081–90. - 4.
Alcantara CL, Vidal JC, de Souza W, Cunha ESNL. The three-dimensional structure of the cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes. Journal of Cell Science. 2014;127(Pt 10):2227–37. - 5.
Vickerman K. On the surface coat and flagellar adhesion in trypanosomes. Journal of Cell Science. 1969;5(1):163–93. - 6.
de Souza W, Souto-Padron T. The paraxial structure of the flagellum of trypanosomatidae. The Journal of Parasitology. 1980;66(2):229–36. - 7.
Gull K. The cytoskeleton of trypanosomatid parasites. Annual Review of Microbiology. 1999;53:629–55. - 8.
Sant'Anna C, Campanati L, Gadelha C, Lourenco D, Labati-Terra L, Bittencourt-Silvestre J, et al. Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM). Histochemistry and Cell Biology. 2005;124(1):87–95. - 9.
Souto-Padron T, de Souza W, Heuser JE. Quick-freeze, deep-etch rotary replication of Trypanosoma cruzi and Herpetomonas megaseliae. Journal of Cell Science. 1984;69:167–78. - 10.
MacRae TH, Gull K. Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei. The Biochemical Journal. 1990;265(1):87–93. - 11.
Soares TC, de Souza W. Fixation of trypanosomatids for electron microscopy with the glutaraldehyde-tannic acid method. Zeitschrift fur Parasitenkunde. 1977;53(2):149–54. - 12.
Robinson DR, Sherwin T, Ploubidou A, Byard EH, Gull K. Microtubule polarity and dynamics in the control of organelle positioning, segregation and cytokinesis in the trypanosome cell cycle. The Journal of Cell Biology. 1995;128(6):1163–72. - 13.
de Souza W, Sant'Anna C, Cunha-e-Silva NL. Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa. Progress in Histochemistry and Cytochemistry. 2009;44(2):67–124. - 14.
Vedrenne C, Giroud C, Robinson DR, Besteiro S, Bosc C, Bringaud F, et al. Two related subpellicular cytoskeleton-associated proteins in Trypanosoma brucei stabilize microtubules. Molecular Biology of the Cell. 2002;13(3):1058–70. - 15.
De Souza W. From the cell biology to the development of new chemotherapeutic approaches against trypanosomatids: dreams and reality. Kinetoplastid Biology and Disease. 2002;1(1):3. - 16.
Pimenta PF, De Souza W. Fine structure and cytochemistry of the endoplasmic reticulum and its association with the plasma membrane of Leishmania mexicana amazonensis. Journal of Submicroscopic Cytology. 1985;17(3):413–9. - 17.
Meyer H, De Souza W. Electron microscopic study of Trypanosoma cruzi periplast in tissue cultures. I. Number and arrangement of the peripheral microtubules in the various forms of the parasite's life cycle. The Journal of Protozoology. 1976;23(3):385–90. - 18.
Sheriff O, Lim LF, He CY. Tracking the biogenesis and inheritance of subpellicular microtubule in Trypanosoma brucei with inducible YFP-alpha-tubulin. BioMed Research International. 2014;2014:893272. - 19.
Robinson DR, Gull K. Basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle. Nature. 1991;352(6337):731–3. - 20.
Wheeler RJ, Scheumann N, Wickstead B, Gull K, Vaughan S. Cytokinesis in Trypanosoma brucei differs between bloodstream and tsetse trypomastigote forms: implications for microtubule-based morphogenesis and mutant analysis. Molecular Microbiology. 2013;90(6):1339–55. - 21.
Borst P, Fairlamb AH. Surface receptors and transporters of Trypanosoma brucei. Annual Review of Microbiology. 1998;52:745–78. - 22.
Landfear SM, Ignatushchenko M. The flagellum and flagellar pocket of trypanosomatids. Molecular and Biochemical Parasitology. 2001;115(1):1–17. - 23.
Field MC, Carrington M. Intracellular membrane transport systems in Trypanosoma brucei . Traffic. 2004;5(12):905–13. - 24.
Tardieux I, Webster P, Ravesloot J, Boron W, Lunn JA, Heuser JE, et al. Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells. Cell. 1992;71(7):1117–30. - 25.
Webster P, Russell DG. The flagellar pocket of trypanosomatids. Parasitology Today. 1993;9(6):201–6. - 26.
De Souza W, Bunn MM, Angluster J. Demonstration of concanavalin A receptors on Leptomonas pessoai cell membrane. The Journal of Protozoology. 1976;23(2):329–33. - 27.
Coppens I, Opperdoes FR, Courtoy PJ, Baudhuin P. Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei. The Journal of Protozoology. 1987;34(4):465–73. - 28.
Soares MJ, de Souza W. Endocytosis of gold-labeled proteins and LDL by Trypanosoma cruzi . Parasitology Research. 1991;77(6):461–8. - 29.
Bonhivers M, Nowacki S, Landrein N, Robinson DR. Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated. PLoS Biology. 2008;6(5):e105. - 30.
Gadelha C, Rothery S, Morphew M, McIntosh JR, Severs NJ, Gull K. Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(41):17425–30. - 31.
Sherwin T, Gull K. The cell division cycle of Trypanosoma brucei : timing of event markers and cytoskeletal modulations. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences. 1989;323(1218):573–88. - 32.
Field MC, Carrington M. The trypanosome flagellar pocket. Nature Reviews Microbiology. 2009;7(11):775–86. - 33.
Morriswood B, He CY, Sealey-Cardona M, Yelinek J, Pypaert M, Warren G. The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein. Molecular and Biochemical Parasitology. 2009;167(2):95–103. - 34.
Esson HJ, Morriswood B, Yavuz S, Vidilaseris K, Dong G, Warren G. Morphology of the trypanosome bilobe, a novel cytoskeletal structure. Eukaryotic Cell. 2012;11(6):761–72. - 35.
Morriswood B, Schmidt K. A MORN repeat protein facilitates protein entry into the flagellar pocket of Trypanosoma brucei . Eukaryotic Cell. 2015;14(11):1081–93. - 36.
Porto-Carreiro I, Attias M, Miranda K, De Souza W, Cunha-e-Silva N. Trypanosoma cruzi epimastigote endocytic pathway: cargo enters the cytostome and passes through an early endosomal network before storage in reservosomes. European Journal of Cell Biology. 2000;79(11):858–69. - 37.
Soares MJ. Endocytic portals in Trypanosoma cruzi epimastigote forms. Parasitology Research. 2006;99(4):321–2. - 38.
Milder R, Deane MP. The cytostome of Trypanosoma cruzi andT. conorhini . The Journal of Protozoology. 1969;16(4):730–7. - 39.
Attias M, Vommaro RC, de Souza W. Computer aided three-dimensional reconstruction of the free-living protozoan Bodo sp. (Kinetoplastida: Bodonidae). Cell Structure and Function. 1996;21(5):297–306. - 40.
Alcantara CL, Vidal JC, de Souza W, Cunha ESNL. The cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes disassembles during cell division. Journal of Cell Science. 2016. DOI: 10.1242/jcs.187419 - 41.
Vidal, J.C., et al., Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J Struct Biol, 2016. 196(3):319–328. - 42.
De Souza W. Basic cell biology of Trypanosoma cruzi . Current Pharmaceutical Design. 2002;8(4):269–85. - 43.
Cevallos AM, Segura-Kato YX, Merchant-Larios H, Manning-Cela R, Alberto Hernandez-Osorio L, Marquez-Duenas C, et al. Trypanosoma cruzi : multiple actin isovariants are observed along different developmental stages. Experimental Parasitology. 2011;127(1):249–59. - 44.
El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, et al. The genome sequence of Trypanosoma cruzi , etiologic agent of Chagas disease. Science. 2005;309(5733):409–15. - 45.
Sahasrabuddhe AA, Bajpai VK, Gupta CM. A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules. Molecular and Biochemical Parasitology. 2004;134(1):105–14. - 46.
Garcia-Salcedo JA, Perez-Morga D, Gijon P, Dilbeck V, Pays E, Nolan DP. A differential role for actin during the life cycle of Trypanosoma brucei. The EMBO Journal. 2004;23(4):780–9. - 47.
De Melo LD, Sant'Anna C, Reis SA, Lourenco D, De Souza W, Lopes UG, et al. Evolutionary conservation of actin-binding proteins in Trypanosoma cruzi and unusual subcellular localization of the actin homologue. Parasitology. 2008;135(8):955–65. - 48.
Correa JR, Atella GC, Batista MM, Soares MJ. Transferrin uptake in Trypanosoma cruzi is impaired by interference on cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis. Experimental Parasitology. 2008;119(1):58–66. - 49.
Souza LC, Pinho RE, Lima CV, Fragoso SP, Soares MJ. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea). Memorias do Instituto Oswaldo Cruz. 2013;108(5):631–6. - 50.
Gheiratmand L, Brasseur A, Zhou Q, He CY. Biochemical characterization of the bi-lobe reveals a continuous structural network linking the bi-lobe to other single-copied organelles in Trypanosoma brucei . The Journal of Biological Chemistry. 2013;288(5):3489–99. - 51.
Portman N, Gull K. Proteomics and the Trypanosoma brucei cytoskeleton: advances and opportunities. Parasitology. 2012;139(9):1168–77. - 52.
LaCount DJ, Barrett B, Donelson JE. Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis. The Journal of Biological Chemistry. 2002;277(20):17580–8. - 53.
Cooper R, de Jesus AR, Cross GA. Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion. The Journal of Cell Biology. 1993;122(1):149–56. - 54.
Vaughan S, Kohl L, Ngai I, Wheeler RJ, Gull K. A repetitive protein essential for the flagellum attachment zone filament structure and function in Trypanosoma brucei . Protist. 2008;159(1):127–36. - 55.
Sun SY, Wang C, Yuan YA, He CY. An intracellular membrane junction consisting of flagellum adhesion glycoproteins links flagellum biogenesis to cell morphogenesis in Trypanosoma brucei . Journal of Cell Science. 2013;126(Pt 2):520–31. - 56.
Zhou Q, Liu B, Sun Y, He CY. A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei . Journal of Cell Science. 2011;124(Pt 22):3848–58. - 57.
Hayes P, Varga V, Olego-Fernandez S, Sunter J, Ginger ML, Gull K. Modulation of a cytoskeletal calpain-like protein induces major transitions in trypanosome morphology. The Journal of Cell Biology. 2014;206(3):377–84. - 58.
Zhou Q, Gu J, Lun ZR, Ayala FJ, Li Z. Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(12):3287–92. - 59.
Zhou Q, Hu H, Li Z. An EF-hand-containing protein in Trypanosoma brucei regulates cytokinesis initiation by maintaining the stability of the cytokinesis initiation factor CIF1.The Journal of Biological Chemistry, 2016; 291(28):14395–409. - 60.
Sunter JD, Gull K. The flagellum attachment zone: 'the cellular ruler' of Trypanosome morphology. Trends in Parasitology. 2016;32(4):309–24. - 61.
Bargul JL, Jung J, McOdimba FA, Omogo CO, Adung'a VO, Kruger T, et al. Species-specific adaptations of Trypanosome morphology and motility to the mammalian host. PLoS Pathogens. 2016;12(2):e1005448. - 62.
Schlaeppi K, Deflorin J, Seebeck T. The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical, tandemly linked genes. The Journal of Cell Biology. 1989;109(4 Pt 1):1695–709. - 63.
Hyams JS. The Euglena paraflagellar rod: structure, relationship to other flagellar components and preliminary biochemical characterization. Journal of Cell Science. 1982;55:199–210. - 64.
Cachon M, Cosson MP. Ciliary and flagellar apparatuses and their associated structures. Biology of the Cell/Under the Auspices of the European Cell Biology Organization. 1988;63(2):115. - 65.
Farina M, Attias M, Souto-Padrón T, de Souza W. Further studies on the organization of the paraxial rod of Trypanosomatids. J. Protozool, 1986;33:552–557. - 66.
Rocha GM, Teixeira DE, Miranda K, Weissmuller G, Bisch PM, de Souza W. Structural changes of the paraflagellar rod during flagellar beating in Trypanosoma cruzi . PloS One. 2010;5(6):e11407. - 67.
Gadelha AP, Cunha-e-Silva NL, de Souza W. Assembly of the Leishmania amazonensis flagellum during cell differentiation. Journal of Structural Biology. 2013;184(2):280–92. - 68.
Santrich C, Moore L, Sherwin T, Bastin P, Brokaw C, Gull K, et al. A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts. Molecular and Biochemical Parasitology. 1997;90(1):95–109. - 69.
Portman N, Gull K. The paraflagellar rod of kinetoplastid parasites: from structure to components and function. International Journal for Parasitology. 2010;40(2):135–48. - 70.
Ginger ML, Collingridge PW, Brown RW, Sproat R, Shaw MK, Gull K. Calmodulin is required for paraflagellar rod assembly and flagellum-cell body attachment in trypanosomes. Protist. 2013;164(4):528–40. - 71.
Luhrs KA, Fouts DL, Manning JE. Immunization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection. Vaccine. 2003;21(21–22):3058–69. - 72.
Kurup SP, Tarleton RL. The Trypanosoma cruzi flagellum is discarded via asymmetric cell division following invasion and provides early targets for protective CD8(+) T cells. Cell Host & Microbe. 2014;16(4):439–49.