Centrosomes are microtubule-organizing centers (MTOCs) consisting of two centrioles surrounded by pericentriolar material. Accurate centrosome duplication precisely once per cell cycle is required for formation of a bipolar mitotic spindle, the apparatus responsible for proper segregation of chromosomes during mitosis. Centrosome defects cause genetic instability and are evident in certain cancers. Centriole duplication is a critical event in centrosome duplication, and the events of centriole assembly are mechanistically similar to those of another MTOC, the basal body, which nucleates cilia or flagella. Centrioles and basal bodies are similar in structure, share many components, and the assembly of the two organelles require many of the same genes. We will investigate basal body assembly in the ciliated protozoan Tetrahymena thermophila. Among the conserved proteins found at centrioles and basal bodies is centrin, a small calcium-binding EF hand protein. We have studied four centrin genes in Tetrahymena. Of these, Cen1 localizes to basal bodies, and a CEN1 null allele reveals defects in basal body assembly and maintenance. We have identified additional components of basal bodies by Multidimensional Protein Identification Technololgy (MudPIT). This analysis has described well over 50 candidate basal body components which we propose to study. Initially, we will localize tagged versions of the proteins to confirm that they are basal body components. New components will be localized by immuno-EM to identify those that localize to structures important in basal body assembly. These components will be subjected to genetic analysis, as done with Cen1, to determine whether they indeed have a role in basal body assembly or have another function at the basal body. Finally, the development of an in vitro assembly assay will contribute to a detailed understanding of centriole duplication. We believe that Tetrahymena is ideally suited for such an approach. We have established an in vitro centrin binding assay that has led to the identification of alpha and beta-tubulin as potential centrin binding partners at basal bodies, and we propose to study this interaction. We also will characterize an in vitro system that forms an early and important intermediate in basal body assembly. Overall, our approaches using Tetrahymena will identify new basal body and centriole components and define mechanisms for their roles in basal body and centriole assembly.
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