The flagellum of Trypanosoma brucei: new tricks from an old dog

Abstract

African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. T. brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Therefore, understanding unique aspects of the T. brucei flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei flagellum are directly relevant to understanding fundamental aspects of flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei flagellum contains a canonical 9+2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the flagellum. Most notably, the flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei flagellum.

Fig. 1

The trypanosome flagellum. (A) Scanning electron micrograph of a procyclic trypanosome. An arrow indicates the single flagellum that is attached along the cell body. (B) Transmission electron micrograph, showing a cross-section of the flagellum and its attachment to the cell body as viewed from posterior toward anterior. (C) Schematic representation of the micrograph in B. Major flagellar sub-structures are indicated and outer doublet microtubules are numbered according to conventional nomenclature. The four specialized sub-pellicular microtubules that are part of the FAZ are indicated in blue. Abbreviations: DRC, dynein regulatory complex; IFT, intraflagellar transport; FAZ, flagellum attachment zone; PFR, paraflagellar rod.

Fig. 2

RNA interference (RNAi) knockdown of the conserved component of motile flagella 9 (CMF9) protein results in loss of inter-doublet connections. Transverse (A, B) and longitudinal (C) sections of procyclic cells in which CMF9 is knocked down with RNAi. White arrows point to misplaced outer doublets Scale bars are 100 nm (A), 50 nm (B) and 200 nm (C). Adapted from Baron et al., 2007b, with permission.

Fig. 3

Simultaneous RNA interference (RNAi) knockdown of the parkin co-regulated genes PACRGA and PACRGB results in loss of outer doublets. Serial transverse sections (proceeding from posterior to anterior (A, B, C, respectively) of a procyclic cell in which both PACRGA and B have been knocked down with RNAi. Black arrows indicate the outer doublet that is lost toward the distal tip of the axoneme. The white arrow indicates the electron dense lumen of the A-tubule. Adapted from Dawe et al., 2005; reproduced with permission of the Company of Biologists.

Fig. 4

Central pair orientation is fixed in wild type cells and is dependent upon the central pair protein “paralyzed flagella 16” (PF16). (A, B) Transverse sections of PF16 knockdowns grown in the absence (−) or presence (+) of tetracycline to induce RNA interference knockdown of PF16. In control cells (-Tet) central pair microtubules lie in a plane that is roughly parallel to the paraflagellar rod, whereas in PF16 knockdown mutants (+ Tet) central pair orientation is variable. (C, D) Schematic with lines depicting the plane of central pair microtubules measured in control (C) and PF16 knockdown (D) cells, n = 26. Outer doublet number 1 is labeled for reference. Scale bar is 100 nm. Adapted from Ralston et al., 2006, with permission.

Fig. 5

RNA interference (RNAi) knockdown of dynein light chain 1 (LC1) or axonemal dynein intermediate chain 1 (DNAI1) results in loss of outer dynein arms. Transverse sections of flagella (A, B) or flagellar cytoskeleton (C) from LC1 (A, B) or DNAI1 (C) knockdown lines grown in the absence (−) or presence (+) of tetracycline. Arrows point to positions of outer dynein arms, which are absent in LC1 and DNAI1 knockdowns. Scale bar is 100 nm. Adapted from Baron et al., 2007a (A); and Branche et al., 2006 (C), reproduced with permission of the Company of Biologists.

Fig. 6

Importance of flagellum motility revealed by procyclic motility mutants. Schematic representations of motility phenotypes in procyclic Trypanosoma brucei flagellum mutants. Wild-type (WT) cell movement is helical with the flagellum tip leading. RNA interference knockdown of flagellar protein expression leads to a variety of motility phenotypes. “Dizzy” mutants, such as trypanin knockdown mutants (Hutchings et al., 2002), retain a vigorously beating flagellum, but are unable to move directionally, and instead tumble in place. “Reverse” motility is observed when subunits of the outer dynein motors, such as DNAI1 (Branche et al., 2006) or LC1 (Baron et al., 2007a) are targeted. These mutants exhibit a reverse, base-to-tip beat and move backward with the flagellum tip trailing. “Paralyzed” mutants, such as central pair and radial spoke mutants (Branche et al., 2006; Ralston et al., 2006) are incapable of movement at the cellular level. Some paralyzed mutants have flagella that are able to beat, while other mutants have more severe defects and are only capable of erratic twitching motions. “Lethal” defects arise in mutants with severe flagellar paralysis (Baron et al., 2007b).

Fig. 7

Cartoon depicting major stages of the Trypanosoma brucei cell division cycle in procyclic lifecycle stages. The cell cycle begins with replication of the basal body and initiation of new flagellum (blue) biogenesis. The new flagellum extends along a path defined by the old flagellum (green), while the basal bodies and associated kinetoplasts (small gray circles) migrate away from each other. In a closed mitotic cycle, the nucleus (large gray circle) is replicated and the new nucleus assumes a position between the new and old kinetoplast/basal body apparatus. Cytokinesis initiates at the tip of the new flagellum (black arrowhead) and cleavage furrow ingression proceeds to the cell posterior, separating the two daughter cells between the new and old flagellum. Ultimately, daughter cells are oriented in opposite directions, with their flagella exerting rotational and pulling forces that facilitate final cell separation.

Fig. 8

Flagellum attachment zone (FAZ) length defines cell cleavage site. (A, B) Immunofluorescence (A) and merged (B) image of a dividing procyclic cell following RNA interference knockdown of intraflagellar transport protein 88 (IFT88), showing the paraflagellar rod (green), FAZ (red) and nucleus (blue). (C) Cartoon depicting the result of improper cell cleavage in an IFT88 knockdown (left), resulting in one normal-sized daughter cell (right, top) and one short daughter cell (right, bottom). The site of cell cleavage corresponds to the end of the new FAZ filament (white arrowheads in A and B. Adapted from Kohl et al., 2003, with permission.

Fig. 9

There is a dichotomy in the requirement of the flagellum in procyclic versus bloodstream form cells. (A) DIC images of uninduced (-Tet) and tetracycline-induced (+Tet) procyclic pfr2 knockdown mutants. Induced cells fail in the completion of cytokinesis, and accumulate as clusters of distinct cell bodies that remain physically attached at their posterior ends (arrowhead). (B) Phase-contrast images of uninduced and tetracycline-induced trypanin knockdown mutants in the procyclic (PCF) and bloodstream (BSF) lifecycle stages. Procyclic trypanin mutants are viable with little or no division defect, while bloodstream mutants fail in the initiation of cytokinesis and accumulate as amorphous masses with multiple flagella. (C) Immunofluorescence images of uninduced and tetracycline-induced bloodstream form trypanin mutants. Phase contrast images are shown in the left panels and staining for the FAZ (red) and DNA (blue) is shown in the right panels. Uninduced cells undergoing division have a maximum of two FAZ structures, which have a parallel configuration until cytokinesis is initiated. Induced cells have more than two parallel FAZ structures, which retain a parallel configuration, indicating that failure to initiate cytokinesis in these cells is not due to failed organelle replication or positioning. Adapted from Ralston et al., 2006 and Ralston and Hill, 2006, with permission.