The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

Abstract

The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.T rypanosoma brucei is an early branching microbial eukaryote and the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. A trypanosome cell possesses a motile flagellum that is nucleated by the basal body, the cell's microtubule organizing center (MTOC), exits the cell body through the flagellar pocket, and is attached to the cell body along most of its length via a specialized cytoskeletal structure termed the flagellum attachment zone (FAZ). The flagellum is composed of a canonical 9 ϩ 2 microtubule axoneme and an extra-axoneme structure termed the paraflagellar rod (PFR), and it is required for cell morphogenesis, cell motility, and cytokinesis (1, 2). Early in the cell cycle, a trypanosome cell possesses a basal body, which nucleates the flagellum axoneme, and an associated probasal body. The probasal body matures to a basal body when the cell proceeds to S phase of the cell cycle; subsequently, two probasal bodies are formed, each of which associates with each of the two mature basal bodies (3, 4). A new flagellum then is assembled from the newly matured basal body, which then undergoes a rotation toward the posterior of the old basal body. Following the elongation of the new flagellum, the new basal body/probasal body pair moves toward the posterior portion of the cell, which constitutes one of the first cytoskeletal events in the cell cycle of T. brucei (3, 4). Movement of the basal body is required for cell morphogenesis (4) and for segregation of the kinetoplast, the cell's mitochondrial genome that is attached to the basal body through a struc...

SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament

“…In addition to mediating flagellum-cell body attachment, the FAZ filament also defines the cytokinesis cleavage plane (11,12), with the distal tip of the new FAZ filament marking the site of cytokinesis initiation (18 -20). Moreover, during the cell cycle in the procyclic form, the elongation of the FAZ filament positions the newly assembled basal body toward the cell posterior (21), which is crucial for organelle segregation and cell division (10,(22)(23)(24)(25).Trypanosomatids, a group of kinetoplastid parasites consisting of T. brucei, Trypanosoma cruzi, and Leishmania spp., appear in a variety of different morphological forms during their life cycle and are distinguished by the relative position of the kinetoplast (mitochondrial DNA) and the nucleus and by the position, length, and cell body attachment of the flagellum (26). In the tsetse fly vector, T. brucei differentiates from the trypomastigote form to the epimastigote form, which undergoes asymmetrical cell division to produce a long epimastigote cell and a short epimastigote cell (26).…”

“…In addition to mediating flagellum-cell body attachment, the FAZ filament also defines the cytokinesis cleavage plane (11,12), with the distal tip of the new FAZ filament marking the site of cytokinesis initiation (18 -20). Moreover, during the cell cycle in the procyclic form, the elongation of the FAZ filament positions the newly assembled basal body toward the cell posterior (21), which is crucial for organelle segregation and cell division (10,(22)(23)(24)(25).…”

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

“…The results showed that 3HA-SPBB1 was localized to the basal body as verified by co-localization with TbSAS-6, the basal body cartwheel protein in both the mature basal body and the probasal body (25) (Fig. 2A).…”

“…Fixed cells were then rehydrated with PBS and incubated in blocking buffer (1% BSA and 0.1% Triton X-100 in PBS) for 1 h at room temperature. Cells were then incubated with the primary antibody diluted in PBS containing 1% BSA at room temperature for 1 h. The following primary antibodies were used: FITC-conjugated anti-HA mAb (1:400 dilution, Sigma-Aldrich), anti-protein A pAb (1: 400 dilution, SigmaAldrich), anti-TbPLK pAb (1:1,000 dilution), L3B2 (1:20 dilution) (22), YL 1/2 mAb (1:1,000 dilution) (23), 20H5 (anti-Centrin mAb, 1:400 dilution) (24), PS54 (anti-phospho-Ser-54 of TbCentrin2, 1: 30,000 dilution) (16), and anti-TbSAS-6 pAb (1:1,000 dilution) (25). After three washes with wash buffer (0.1% Triton X-100 in PBS), cells were incubated with FITCconjugated or Cy3-conjugated secondary antibody at room temperature for another hour.…”

“…To test this hypothesis, we co-immunostained the cells with the YL 1/2 antibody, which labels the transition fibers near the mature basal body and serves as a marker for the mature basal body (34,35), and anti-TbSAS-6, which detects the basal body cartwheel protein in both the mature basal body and the probasal body (25). We then quantified the numbers of mature basal bodies (mBB) and total basal bodies (BB) in 2N1K, 2N2K, and multi-nucleated (Ͼ2N) cells from SPBB1 RNAi and in 2N2K cells from the non-induced control.…”