Golgi Duplication in Trypanosoma brucei Requires Centrin2

Abstract

Centrins are highly conserved components of the centrosome, which in the parasitic protozoan T. brucei comprises the basal body and nucleates the flagellum used for locomotion. Here, we found TbCentrin2 in an additional bi-lobed structure near to the Golgi apparatus. One lobe was associated with the old Golgi, and the other became associated with the newly forming Golgi as the cell grew. Depletion of TbCentrin1 inhibited duplication of the basal body, whereas depletion of TbCentrin2 also inhibited duplication of the Golgi. Thus, a Centrin2-containing structure distinct from the basal body appears to mark the site for new Golgi assembly.

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

“…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.…”

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

“…SPBB1 is the first basal body protein identified as a TbPLK substrate. TbCentrin2, a bilobe protein involved in bilobe biogenesis, Golgi duplication, and flagellum adhesion, is also a TbPLK substrate (8,16,17). TbPLK is known to differ significantly from its yeast and animal homologs in terms of subcellular localization and cellular functions (7)(8)(9)15), and previous studies have demonstrated that TbPLK is required for basal body segregation, bilobe duplication, flagellum attachment, and cytokinesis initiation (7,8,14,15,36).…”

“…A number of proteins have been localized to the basal body in T. brucei, but only a few of them are localized to both the probasal body and the mature basal body, which includes TbCentrin2 (17,36), TbCentrin4 (38), , and an unknown protein that is recognized by a monoclonal antibody BBA4 (39). Centrins and SAS-6 are evolutionarily conserved components of the centriole/basal body, and are present in all of the eukaryotic organisms investigated so far (40,41).…”

“…Depletion of SPBB1 Restricts TbPLK in the Basal Body and the Bilobe Structure-To test whether SPBB1 RNAi might affect the localization of TbPLK, non-induced control and SPBB1 RNAi cells induced for 4 days were co-immunostained with anti-TbPLK antibody and the 20H5 antibody, which stains the basal body and the bilobe structure in T. brucei (17). In noninduced control cells, TbPLK was localized to the basal body and the bilobe structure in G 1 cells, and was enriched at the anterior tip of the new FAZ filament during G 2 to early mitotic phases (metaphase and early anaphase) (Fig.…”

“…Phosphorylation of TbCentrin2 by TbPLK occurs only on the bilobe structure and appears to be required to maintain flagellum attachment (16). However, RNAi of TbCentrin2 does not cause flagellum detachment (17). To better understand the function of TbPLK in regulating basal body segregation, flagellum attachment, and cytokinesis, we sought to identify additional substrates of TbPLK and characterize their function.…”