Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella

Abstract

Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites-Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines.

Keywords: Axoneme; Basal body; Electron tomography; Flagellum; Leishmania; SBFSEM; SEM; Trypanosoma.

FIGURE 1. Morphological forms in the life cycle of Trypanosoma brucei and Leishmania mexicana

(A) Cell morphologies observed in the T. brucei life cycle. (1) Procyclic trypomastigote in the tsetse fly midgut. (2) Elongated trypomastigote, which migrates to the proventriculus where an asymmetric division (3) produces one short epimastigote (4) and one long epimastigote (thought to decay; not shown). (5) Salivary gland epimastigote. (6) Mammalian-infective metacyclic trypomastigote. (7) Long slender bloodstream form. (8) Short stumpy bloodstream form, which is preadapted to differentiate to procyclic forms following ingestion by a tsetse fly. Curved arrows denote stages undergoing proliferative cell cycles; the asterisk indicates a single asymmetric division. (B) Morphological forms in the life cycle of L. mexicana. (1) Procyclic promastigotes in the abdominal (posterior) midgut. (2) Slender nectomonad promastigotes, which migrate toward the thoracic (anterior) midgut. (3) Leptomonad promastigotes. (4) Broad haptomonad promastigote forms are found attached via their flagella to the chitin lining of the stomodeal valve; their precise relationship with other developmental forms is currently unclear. Leptomonad promastigotes differentiate to mammalian-infective metacyclic promastigotes (5). (6) Amastigote forms, which replicate in the phagolysosome of mammalian phagocytes. Curved arrows denote stages undergoing proliferative cell cycles.

FIGURE 2. Ultrastructural characteristic of the Trypanosoma brucei flagellum

(A) Transverse section through a procyclic form flagellum and cell body, illustrating the 9 + 2 axoneme, PFR, and position of the FAZ filament and MtQ. (B) and (C) are the same transverse section through a cell with two flagella. The new flagellum is located to the left of the old flagellum—see text. (B) The old flagellum has been tilted on the TEM to bring the old flagellum into view. Arrowheads in the inset highlight the ponticuli within the B-tubule of each outer doublet microtubule. (C) The new flagellum has been tilted on the TEM and does not contain ponticuli. Scale bar: 100 nm.

FIGURE 3. Differences in external morphology during the cell division cycle of Trypanosoma brucei procyclic and bloodstream forms

Composite scanning electron micrograph images of stages of procyclic (A–H) and bloodstream (I–O) form cell division. (A and I) G1 cell with a single attached flagellum. The exit point of the flagellum from the flagellar pocket is arrowed and the anterior and posterior indicated. (B and J) A new flagellum has grown to extend from the flagellar pocket (arrow). The distal tip of the new flagellum is laterally connected to the old flagellum in the procyclic form (B) and is laterally embedded in the groove structure in the flank of the cell in the bloodstream form (J), indicated by dashed circles. (C, K–L) The flagellar pocket associated with the new flagellum (arrowed) is positioned posterior to the flagellar pocket associated with the old flagellum (arrowhead). The new flagellum is located to the left of the old when viewed looking from posterior to anterior. (D and M) A division fold is evident between the two flagella (arrowed) which is located along the long axis and begins to define the daughter cell shape. There are two distinct posterior end profiles. The new-flagellum daughter inherits the existing posterior end and a new posterior end is formed for the old-flagellum daughter (circled). The new flagellum is still attached to the old by the flagella connector in the procyclic form (D), but has grown free of the cell body in the bloodstream form (M), indicated by dashed circles. (E–F, N) A division cleft has opened up between the daughters (arrow) and the new flagellum tip remains attached to the old flagellum by the flagella connector in the procyclic form (E–F). (G–H, O) Preabscission stage. In the procyclic form (G–H) the two daughter cells are attached by the posterior end of the old-flagellum daughter (circled) to the side of the new-flagellum daughter by a cytoplasmic bridge connection. In the bloodstream form (O) a posterior-to-posterior (circled) configuration is typical (Wheeler, Scheumann, et al., 2013).

FIGURE 4. Differences in morphogenesis of the bloodstream and procyclic forms of Trypanosoma brucei through the cell division cycle

Major morphogenetic events are shown alongside cartoon representations of the associated cell morphologies. Vertical spacing is purely illustrative and does not represent relative time spent during each of these stages (Wheeler, Scheumann, et al., 2013).

FIGURE 5. Scanning electron micrographs of trypanosome forms found in the tsetse fly proventriculus and salivary glands

(A) Long trypomastigote. The asterisk indicates the exit point of the flagellum from the flagellar pocket and the arrowhead points to the distal tip of the long flagellum. (B) Posterior end of an asymmetrically dividing cell with a clearly visible cleavage fold. Arrows point to the old (OF) and new flagellum (NF). (C) Short epimastigote found in the proventriculus. (D) Epimastigotes in the salivary gland are attached to the gland epithelium via their flagellum. Long protrusions extending from the posterior end of the epimastigote cells are marked with arrowheads. Arrows indicate the region of flagellar attachment to the tsetse salivary gland cells. (E) Metacyclic trypomastigote. Scale bar represents 1 μm.

FIGURE 6. Cell cycle of Leishmania mexicana promastigotes

(A–D) Scanning electron micrographs showing progression of L. mexicana promastigote forms through the cell cycle. The arrows in (C) point to the two flagella. The scale bar represents 5 μm. (E) Cartoons of the major morphological forms during L. mexicana cell cycle progression and their approximate timing relative to the G1, S, and post-S phases of the nucleus [N] and kinetoplast [K]. Approximate timings are indicated for [F₁] the start of axoneme extension from the basal body, [B₁] new probasal body formation, [B₂] probasal body rotation, [F₂] emergence of the new flagellum from the flagellar pocket, [M] DNA segregation during mitotic anaphase, and [D] kinetoplast division. Figure adapted from Wheeler et al. (2011).

FIGURE 7. Structure of the Leishmania flagellum in amastigotes and promastigotes

Scanning electron micrographs show the difference in flagellum length between Leishmania mexicana promastigote (A) and axenic amastigote (B), where only the tip of the short amastigote flagellum is exposed to the environment (arrowhead). Differences between promastigote and amastigote axoneme architecture are clearly visible in transmission electron micrographs of flagellum cross sections (C and E, respectively). Prominent structural features of the promastigote flagellum (C) include the canonical “9 + 2” microtubule axoneme, forming a ring of nine outer microtubule (MT) doublets around a pair of central MT (CP), with associated outer and inner dynein arms, and the PFR (shown in cartoon form in D). In contrast the amastigote axoneme (E) consists of a bundle of nine doublet MT and there is no PFR. Scale bars represent 1 μm (A, B) and 0.1 μm (C–E) Figure adapted from (Gluenz, Ginger, et al., 2010).

FIGURE 8. Cellular electron tomography of the Trypanosoma brucei flagellar pocket

(A) A scanning electron micrograph illustrates the position of the flagellar pocket region. The flagellum exit point on the cell surface is labeled with an asterisk. (B) The three-dimensional model illustrates the relationship of the cytoskeletal and membrane structures associated with the pocket. Abbreviations: BB, basal body; PBB, probasal body; FP, flagellar pocket; PFR, paraflagellar rod; MtQ, microtubule quartet; FAZ, flagellum attachment zone; ER, endoplasmic reticulum. (C) This cartoon defines the axes that we used to position tomograms. The origin point is defined by the center of the basal body at its most proximal end. The z-axis runs up the length of the axoneme; the x-axis is defined by the plane of the central pair microtubules at the point at which they are nucleated; finally, the y-axis points toward the probasal body. (D) A model with many of the components excluded has been orientated such that the view is along the z-axis. This allows the definition of four quadrants in the cell useful for positioning organelles and structures and comparison of tomograms. Scale bars: 200 nm (Lacomble et al., 2009). Three representative tomograms (E–G) reveal different stages of basal body and flagellar pocket morphogenesis and demonstrate the rotation. (E1, F1, and G1) show slices through the flagellar pockets from the original tomograms. Tomogram models (E2,3) contain the Cartesian axes described above (Lacomble et al., 2009). (E2,3) Two views of the model of a tomogram illustrating a cell in which the probasal body is located on the bulge side of the flagellar pocket in quadrant 2. The origin of the microtubule quartet lies between the two basal bodies. (F2,3) In this cell, the probasal body has matured and has subtended a new flagellum (NF) that has invaded the existing flagellar pocket and connected to the old flagellum (OF). The new flagellum is still positioned essentially as in (E2 and 3): quadrant 2. (G2,3) A later stage in the cell cycle just before flagellar pocket division. The new flagellum is now in a more posterior location and lies in quadrant 4 (Lacomble et al., 2010). (See color plate)

FIGURE 9. SBFSEM of bloodstream form Trypanosoma brucei

(A) A single slice (100 nm thick) from an SBFSEM dataset, illustrating organelles and cytoskeletal structures of the cell. (B) Surface volume rendering of the SBFSEM dataset containing a whole cell with a transverse slice from the dataset to illustrate the rendering process. (C) A whole-cell rendering of a G1 cell. Index: mitochondrion (M)—green; nucleus (N)—blue; old flagellum (F)—purple; new flagellum (arrow in B)—red; glycosomes (G)—orange; acidocalcisomes (A)—white; vesicles—yellow. Scale bar: 500 nm. (See color plate)

FIGURE 10. Combining cellular electron tomography and SBFSEM to analyze the ultrastructure of the groove

(A) Surface rendering of segmented data illustrating the location of the distal tip of the new flagellum in an indentation of the cell body membrane called the groove. Subpellicular microtubules (green) surround the groove and there is close association of the old MtQ with the groove. (Ai, Aii) Selected z-slices of the tomogram ~6 nm thick in (A). Index: old flagellum (OF)—purple; new flagellum (NF)—red; subpellicular microtubules—green; MtQ—light green. Asterisks illustrate short microtubules that finish in the tomogram; (B) surface rendering of a whole cell to illustrate the distal tip of the new flagellum located inside the cell body; (C) same cell as (B) with a slice from the SBFSEM data to illustrate the groove. Scale bar: 200 nm. (See color plate)