The Trypanosoma brucei flagellum: moving parasites in new directions

Abstract

African trypanosomes are devastating human and animal pathogens. Trypanosoma brucei rhodesiense and T. b. gambiense subspecies cause the fatal human disease known as African sleeping sickness. It is estimated that several hundred thousand new infections occur annually and the disease is fatal if untreated. T. brucei is transmitted by the tsetse fly and alternates between bloodstream-form and insect-form life cycle stages that are adapted to survive in the mammalian host and the insect vector, respectively. The importance of the flagellum for parasite motility and attachment to the tsetse fly salivary gland epithelium has been appreciated for many years. Recent studies have revealed both conserved and novel features of T. brucei flagellum structure and composition, as well as surprising new functions that are outlined here. These discoveries are important from the standpoint of understanding trypanosome biology and identifying novel drug targets, as well as for advancing our understanding of fundamental aspects of eukaryotic flagellum structure and function.

Figure 1

The trypanosome flagellum. (a) Scanning electron micrograph (EM) of a procyclic trypanosome. The arrow indicates the single flagellum. (b) Transmission EM showing a cross-section of the flagellum and its attachment to the cell body as viewed looking from posterior toward anterior. (c) Schematic representation of the micrograph in panel b. Major flagellar substructures are indicated and outer doublet microtubules are numbered according to convention. Flagellar substructures that are broadly conserved among eukaryotes are in blue, and structures that are unique to trypanosomes and closely related organisms are in green. Abbreviations: DRC, dynein regulatory complex; FAZ, flagellum attachment zone; IFT, intraflagellar transport; MT, microtubule; PFR, paraflagellar rod. Adapted from Reference , with permission.

Figure 2

Ultrastructure of the trypanosome flagellum. (a) Schematic cut-out view of the flagellar pocket area. Note that because the mitochondrion is not shown, only a partial TAC, consisting of the exclusion zone filaments extending from the basal body toward the mitochondrion, is represented in this schematic. (b–h) Electron micrographs of longitudinal (b) and transverse (c–h) sections of procyclic cells, from posterior (c) to anterior (h). (b) Longitudinal section showing the flagellar apparatus (blue arrow) and FP (orange arrow). The positions of the transverse sections in panels c–h are indicated with dashed lines, with panel h farther anterior than panel g. (c) The basal body apparatus. (d) The 9 + 0 transition zone within the FP; note the filaments of the ciliary necklace radiating from the doublet microtubules. (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). Note that FAZ connections to the axoneme initiate proximal to the initiation of the PFR (not shown). Panel a is adapted from Reference , with permission. Abbreviations: 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.

Figure 3

The flagellum attachment zone (FAZ). (a) Transverse electron micrograph (EM) section of the Trypanosoma brucei rhodesiense 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). (b) Longitudinal section of T. vivax, showing a series of macula adherens (MA) with their anchoring strands passing to longitudinally oriented filaments. (c) Whole mount of an intact, detergent-extracted T. brucei cytoskeleton, with the MA indicated with arrows. (d) RNAi knockdown of Fla1 in bloodstream-form T. brucei cells (Fla1-) leads to flagellar detachment (arrow). (e) RNAi knockdown of FAZ1 in procyclic T. brucei cells (FAZ1-) leads to abnormalities in FAZ ultrastructure, including unusually wide FAZ filaments (bracket). Arrows point to the four specialized PMTs of the FAZ. Panels a and b are adapted from Reference , panel c from Reference , panel d from Reference , and panel e from Reference , with permission.

Figure 4

Trypanosome flagellum biogenesis. (a,b) Scanning electron micrographs of procyclic-form cells show that the distal tip of the new flagellum (arrow) remains associated with the old flagellum during cell division. The distal tip of the new flagellum is indicated (arrow), the flagellar pockets are marked (asterisks), and the cell posterior is indicated (P). (c,d) Low (c) and high (d) magnification transmission electron micrographs of a detergent-extracted procyclic cytoskeleton showing the flagellar connector (arrow) that is present at the tip of the new flagellum (NF) and contacts the old flagellum (OF). (e) Scanning electron micrograph of a procyclic-form IFT80 RNAi knockdown mutant. A flagellar membrane sleeve (FS) of the new flagellum (NF) extends toward the lateral aspect of the old flagellum (OF). (f) Scanning electron micrograph of a bloodstream-form (BSF) cell during cell division, demonstrating that the tip of the new flagellum (NF) does not appear to connect to the old flagellum (OF) during elongation. Panels a–d are adapted from Reference , panel e from Reference , and panel f from Reference , with permission.

Figure 5

Trypanosoma brucei cell motility. (a) Motility traces of wild-type procyclic cells. The positions of individual cells are plotted at 1-s intervals (indicated by dots). Starting positions are marked with open circles; ending positions are marked with arrowheads, and the time in seconds that each cell was within the field of view is indicated. (b) Schematic representations of motility phenotypes in procyclic motility mutants. Wild-type (WT) cell movement is helical with the flagellum tip leading. Dizzy mutants, such as trypanin knockdown mutants (58), retain a vigorously beating flagellum but are unable to move directionally. Reverse mutants, such as DNAI1 (15) or LC1 (9) knockdown 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 (15, 114), are incapable of movement at the cellular level. Lethal defects arise in mutants with severe flagellar paralysis (10). (c) Time series (0.66-s intervals) of a WT procyclic cell moving in liquid medium. The movements of the posterior tip of the cell body are traced with a red dashed line. Note that periods of running (0–4.66 and 8–9.33 s) alternate with periods of tumbling (5.33–7.33 s). Panel a is adapted from Reference , and panel b from Reference , with permission.

Figure 6

The flagellum mediates attachment to the tsetse fly salivary gland. (a) Transverse electron micrograph section of a Trypanosoma 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. (b) Electron micrograph section showing three T. brucei flagella (F₁, F₂, and F₃), each with extensive flagellar outgrowths. For emphasis, the outgrowths of F₁ are outlined in red. (c) Detail of the attachment between the flagellum and the host cell (HC). Note that APs are found on the flagellar membrane only where the membrane is indented by the MV, leading to a much smaller gap between host and parasite membranes in this region (small arrowheads) than in the spaces that lack APs (large arrowheads). (d) Diagram of the developmental changes that occur while the parasite is attached to the salivary gland epithelium. Cells from left to right are epimastigote, premetacyclic, nascent metacyclic, and metacyclic forms. VSG, variant surface glycoprotein. Adapted from Reference , with permission.

Figure 7

There is a dichotomy in the requirement of the flagellum in procyclic versus bloodstream-form cells. Panels a and b show phenotypes commonly observed in procyclic (PCF) and bloodstream-form (BSF) cells when flagellum proteins are knocked down. (a) Differential interference contrast (DIC) images of uninduced (−Tet) and tetracycline-induced (+Tet) procyclic pfr2 knockdown mutants (PCF Mot −). 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 −Tet and +Tet bloodstream-form trypanin knockdown mutants (BSF Mot −). Induced cells fail in the initiation of cytokinesis and accumulate as amorphous masses with multiple flagella. (c) Schematic representation of the cell cycle and the differing cell cycle blocks in procyclic versus bloodstream flagellum mutants. The cell cycle begins with replication of the basal body and initiation of new flagellum biogenesis. The new flagellum extends along a path defined by the old flagellum, 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 flagella. Ultimately, daughter cells are oriented in opposite directions, with their flagella exerting rotational and pulling forces that facilitate final cell separation. Panel a is adapted from Reference , panel b from Reference , and panel c from Reference , with permission.

Figure 8

Trypanosoma brucei as a model system for flagellum structure and function studies. Artistic representation of the trypanosome, emphasizing the evolutionarily conserved character of the 9 + 2 flagellar axoneme, with various ciliated organisms representing the radial spokes. (Clockwise from top: Ciona intestinalis, Danio rerio, Drosophila melanogaster, Gallus gallus, Chlamydomonas reinhardtii, Homo sapiens, Caenorhabditis elegans, Mus musculus, and Asterias forbesi.) The paraflagellar rod, unique to trypanosomes, is not shown.