Abstract
The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.
Highlights
The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum
Immunofluorescence microscopy using cells expressing endogenously triple HA-tagged BBP164 protein showed that BBP164 localizes to both mature basal body (mBB) and pro-basal body (pBB), as demonstrated by the co-localization with the basal body cartwheel proteins TbSAS-6 and TbBLD10 and the kinetoplastid-specific basal body protein BBP65 (Fig. 1B)
Among the key regulators that are missing in T. brucei are PLK4/SAK, CP110, SPD2/CEP192, and Asterless/CEP152, which appear to have been added in a taxon-specific manner throughout evolution [47]
Summary
A previous biochemical screen had identified a number of hypothetical proteins as candidate basal body components in T. brucei [34]. Western blotting showed that when BBP65 RNAi was induced for 48 h, BBP164 protein level was reduced to Ͻ5% of the control level and was restored by treatment with MG-132 (Fig. 5C) These results demonstrated that BBP65 is required for the maintenance of BBP164 stability in the existing basal body. Western blotting showed that BBP65 protein was reduced to undetectable level after 48 h of TbBLD10 RNAi, but it was stabilized by MG-132 treatment (Fig. 7C) These results suggest that TbBLD10 is required to maintain BBP65 stability in the existing basal body. Immunofluorescence microscopic analysis of control and TbSAS-6 RNAi cells with anti-TbBLD10 antibody showed that depletion of TbSAS-6 did not affect TbBLD10 localization to the existing basal body (Fig. 8A), and Western blotting showed that TbSAS-6 knockdown did not affect the protein level of TbBLD10 (Fig. 8B). We investigated the functional relationship between TbSAS-6 and TbBLD10, which construct the cartwheel and the
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