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.
- three-dimensional reconstruction
Trypanosomatids are uniflagellated protozoan parasites belonging to the Kinetoplastid order. They are the etiological agents of several diseases  and exhibit particular features that differentiate them significantly from their mammalian host . 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 . Third, they have microtubule sets of cytostome-cytopharynx that forms a gutter in this funnel invagination . Fourth, trypanosomatids have a flagellum attachment zone (FAZ) where the flagellum emerges from the flagellar pocket and remains attached to the cell body . 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 .
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 . 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 ; 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
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
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
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 .
The trypanosomatids genome contains putative actin and actin-binding protein sequences . 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  (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 . 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 . 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
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.
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