Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei

Abstract

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1-S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

Fig. 1.

Repositioning of the basal body. Negatively stained whole-mount cytoskeletons illustrate a repositioning of the new basal body. (A) A G1 cell with a single flagellum nucleated from the basal body (BB). The probasal body (PBB) is located anterior to the basal body. (B) Pre-mitotic cell with two flagella. The new flagellum is positioned posterior to the old flagellum. (C)A higher magnification view of a G1 cell to illustrate the positioning of the PBB relative to the BB, the axoneme (Ax) and collar (arrow). The microtubule quartet (labelled *) initiates between the BB and PBB. (D) The first marker of initiation of cytoskeletal morphogenesis is a new microtubule quartet (labelled **). (E) The probasal body has now matured to the point that it can form a new flagellum. There is an extension of the transition zone (white arrowhead), and a very short axoneme distal to the transition zone, yet the connection of the new flagellum to the old via the flagella connector (white arrow) has already been established The new flagellum basal body (NBB) is positioned anterior to the old flagellum basal body (OBB). (F) The new flagellum basal body (NBB) is now positioned posterior to the old flagellum basal body (OBB), even though the new flagellum is still short (white arrow). Scale bars: 2 μm (A,B), 500 nm (C–F).

Fig. 2.

Flagellar pocket duplication. Representative, thin-section, transmission electron micrographs illustrating the early stages of flagellar pocket duplication. (A) A single flagellum (F) extends from a basal body (BB) within the flagellar pocket (FP). A probasal body (PBB) is located in an anterior position relative to the flagellum. (B) The probasal body has matured and has initiated a new flagellum (NF), which has invaded the existing flagellar pocket. The new flagellum is still located anterior to the old flagellum (OF). (C) The new flagellum (NF) is now posterior to the old flagellum (OF). A membrane profile, termed the ridge (arrow), protrudes into the existing flagellar pocket between the two flagella. (D) The ridge has now developed further, and there is more separation of the basal bodies. (E) Transverse section of a flagellar pocket containing two flagella and two closely associated microtubule quartets (black bars). (F) Transverse section of the exit point from the flagellar pocket illustrating two flagella each surrounded by an electron-dense collar structure. Scale bars: 200 nm.

Fig. 3.

Rotation of the new basal body around the old flagellum. Three representative tomograms (A–C) reveal different stages of basal body and flagellar pocket morphogenesis and demonstrate the rotation. A1,B1,C1 show slices through the flagellar pockets from the original tomograms. Tomogram models (A2–C3) contain Cartesian axes previously described (Lacomble et al., 2009). (A2,A3) 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. (B2–B3) 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 A2 and A3: quadrant 2. (C2,C3) 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. Please see text for explanation of the rotation. See supplementary material Table S1 for colour key. Scale bars: 200 nm.

Fig. 4.

New flagellar pocket morphogenesis. Two views of a model of a tomogram illustrating the formation of a flagellar pocket. (A) A nascent pocket has formed around the new flagellum (NF), along with a new microtubule quartet (NMtQ). (B) Position of the old and new microtubule quartets and old and new collars. See supplementary material Table S1 for colour key. Scale bars: 200 nm.

Fig. 5.

Flagellar pocket morphogenesis in the bloodstream form. The basic mechanics described for the procyclic form are also found in the bloodstream form. (A) G1 cell with a single flagellum nucleated from the single basal body (BB). The probasal body (PBB) is located anterior to the basal body. (B) Pre-mitotic cell with two flagella. The new flagellum and basal body (NBB) are posterior to the old flagellum basal body (OBB). (C1,C2) Alternative views of a model of a tomogram with a flagellar pocket containing a single flagellum, a probasal body positioned in quadrant 2 and the origin of the microtubule quartet between the two basal bodies. (D1,D2) Alternative views of a model of a tomogram where the new flagellum basal body has rotated around the old flagellum basal body and a new nascent flagellar pocket has formed. Scale bars: 200 nm (A,B), 250 nm (C1–D2).