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

Abstract

Background:The substrates of Polo-like kinase in Trypanosoma brucei are mostly not identified. Results: A basal body protein was identified as a substrate of Polo-like kinase and is required for basal body segregation and flagellum adhesion. Conclusion: Polo-like kinase regulates a basal body protein.Significance: Dissecting the Polo-like kinase pathway is crucial for understanding its cellular functions.

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

“…To identify the factor(s) that interacts with both TbPLK and TbAUK1, we carried out yeast two-hybrid library screening using the kinase-dead TbPLK (TbPLK-K70R) and TbAUK1 as baits. A hypothetical protein (Tb927.11.15800), which was previously identified as a partner of TbPLK-K70R by us (14), was also identified as a partner of TbAUK1 (Fig. S1 A and B).…”

“…The procyclic 427 cell line and 29-13 cell line (34) were cultured according to our published procedures (14). To knock down CIF1, a fragment of CIF1 gene was cloned into the pZJM vector (35) and transfected into the 29-13 cell line.…”

“…To knock down CIF1, a fragment of CIF1 gene was cloned into the pZJM vector (35) and transfected into the 29-13 cell line. Selection of transfectants, cloning, and RNAi induction were carried out as described previously (14). TbPLK RNAi cell line and TbAUK1 RNAi cell line have been reported previously (12,17).…”

“…Cells were fixed with cold methanol and incubated with the primary antibody for 1 h at room temperature. The following primary antibodies were used: FITC-conjugated anti-HA antibody, anti-Protein A antibody, anti-CC2D antibody (6), 20H5 (36), and anti-TbPLK antibody (14). Cells were then incubated with FITC-conjugated anti-mouse IgG or Cy3-conjugated anti-rabbit IgG for 1 h at room temperature, mounted with DAPI-containing VectaShield mounting medium (Vector Labs), and imaged using an inverted fluorescence microscope (Olympus IX71) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60 × 1.42-NA lens.…”

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).…”