Life with eight flagella: flagellar assembly and division in Giardia

Abstract

Flagellar movement in Giardia, a common intestinal parasitic protist, is crucial to its survival in the host. Each axoneme is unique in possessing a long, cytoplasmic portion as well as a membrane-bound portion. Intraflagellar transport (IFT) is required for the assembly of membrane-bound regions, yet the cytoplasmic regions may be assembled by IFT-independent mechanisms. Steady-state axoneme length is maintained by IFT and by intrinsic and active microtubule dynamics. Following mitosis and before their segregation, giardial flagella undergo a multigenerational division cycle in which the parental eight flagella migrate and reposition to different cellular locations; eight new flagella are assembled de novo. Each daughter cell thus inherits four mature and four newly synthesized flagella.

Figure 1. Giardial axonemes are characterized by long cytoplasmic regions

A ventral view of a Giardia trophozoite, visualized using SEM, is shown in (A) (scale bar = 5 μm). The characteristic teardrop shape and ventral disc (vd) as well as the four flagellar pairs (afl = anterior, pfl = posteriorlateral, vfl = ventral, cfl = caudal) are visible; image courtesy of Joel Mancuso, UC Berkeley. The distal flagellar tip (B) is shown in TEM and demonstrates the continuation of the A and B tubules close to the tip. In panel C, electron density around the flagellar pore (fp) region of the ventral axonemes is shown. A caudal axoneme basal body (E) and associated axonemal cytoplasmic region (D) shows the presence of the outer doublet MTS as well as the central pair MTs. Scale bars in B–E = 200 nm. The long cytoplasmic regions (F, G) and the membrane-bound portions (H) of all eight axonemes are visible using DIC and tubulin-immunostaining (F = DIC; G = anti-tubulin immunostaining, red; H = anti-alpha14-annexin which labels the membrane-bound axonemes [82] in green, red = anti-tubulin, blue = DAPI; mb = median body). Scale bars = 5 μm.

Figure 2. Flagellar contributions to complex movements in attached and unattached trophozoites

Each pair of giardial flagella (anterior = afl, posteriolateral = pfl, ventral = vfl, and caudal = cfl, fn = funis) contributes specifically to various modes of flagellar motility through differential movements and the action of axoneme-associated structures. In attached cells (A), flagellar motility is primarily evident in the ventral (white arrow) or anterior flagella (black arrow) (see Supplemental Movie 1). “Dorsal/lateral tail flexion”, or the lateral and/or dorsal flexing of the posterior end of the cell, has been attributed to the bending of the funis (i.e. caudal complex) that encircles the caudal flagella and may modulate their beating (see Supplemental Movie 2 and Supplemental Movie 3)[21,83]. Dorsal tail flexion has been associated with detachment essentially by breaking the “seal” of the ventral disc on a surface [84]. In unattached cells (B), left-right directional movement has been attributed to the anterior flagella, and forward or downward movement to the anterior and ventral flagella (see Supplemental Movie 3). Rotational or tumbling movement has been ascribed to anterior and/or posteriolateral flagellar beating, although ventral flagellar beating is during tumbling is also apparent and may contribute to these movements (see Supplemental Movie 4).

Figure 3. Unique axoneme-associated structures in Giardia

Panels A and D show transmission electron micrographs of the marginal plate (mp) and striated fibers (sf), repetitive structures of unknown function associated with the cytoplasmic regions of the anterior axonemes (afl) that may modulate attachment via the ventral disc (vd). In B, caudal axoneme basal bodies (cbb) are shown to nucleate the two spiral arrays of the ventral disc (B). Electron dense structures, i.e., “fins” (black arrows) associate with the membrane-bound portions of the ventral flagella (C); image courtesy of Cindi Schwartz, CU Boulder. In panel E, the MTs and fibers of the funis (fn) are shown radiating from the caudal axonemes toward the cell periphery (vfl = ventral flagella). The funis is proposed to modulate caudal axonemal beating resulting in dorsal/lateral tail flexion (see Figure 2).

Figure 4. Putative IFT-dependent and IFT-independent mechanisms of flagellar assembly and maintenance

In terms of assembly of the membrane-bound axonemal regions, the anterograde kinesin-II complex likely assembles and loads on cytoplasmic axonemes rather than the basal bodies (BB) or transition zones (TZ), accumulates at the flagellar pore region (FP), and transports IFT raft particles (A and B complexes) as well as tubulin subunits to the flagellar tip. Retrograde transport is mediated by IFT dynein. As in other organisms, the giardial BBSome may be involved in linking the A and B raft complexes, and IFT-complex B associated proteins may facilitate assembly or docking at the flagellar pores. Cytoplasmic regions of axonemes may be assembled by IFT-independent mechanisms. The MT destabilizing kinesin-13 is present at the distal tips and promotes depolymerization of axonemes. Switching of anterograde to retrograde transport also occurs at the distal tip. EB1 is present at the distal tips and flagellar pores (and may mark microtubule plus ends and/or recruit kinesin-13 to tips). An aurora-like kinase (AK) may regulate axonemal disassembly through its regulation of kinesin-13 at the distal flagellar tips. Giardial homologs of IFT, BBSome or IFT-complex B associated proteins are indicated.