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 Technology (Mud PIT). 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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM074746-01A1
Application #
7094748
Study Section
Cell Structure and Function (CSF)
Program Officer
Deatherage, James F
Project Start
2006-04-05
Project End
2010-03-31
Budget Start
2006-04-05
Budget End
2007-03-31
Support Year
1
Fiscal Year
2006
Total Cost
$285,094
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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Zhao, Ying; Shi, Jianli; Winey, Mark et al. (2016) Identifying domains of EFHC1 involved in ciliary localization, ciliogenesis, and the regulation of Wnt signaling. Dev Biol 411:257-265
Shi, Jianli; Zhao, Ying; Vonderfecht, Tyson et al. (2015) Centrin-2 (Cetn2) mediated regulation of FGF/FGFR gene expression in Xenopus. Sci Rep 5:10283
Square, Tyler; Romášek, Marek; Jandzik, David et al. (2015) CRISPR/Cas9-mediated mutagenesis in the sea lamprey Petromyzon marinus: a powerful tool for understanding ancestral gene functions in vertebrates. Development 142:4180-7
Cole, Eric S; Giddings Jr, Thomas H; Ozzello, Courtney et al. (2015) Membrane dynamics at the nuclear exchange junction during early mating (one to four hours) in the ciliate Tetrahymena thermophila. Eukaryot Cell 14:116-27
Winey, Mark; O'Toole, Eileen (2014) Centriole structure. Philos Trans R Soc Lond B Biol Sci 369:
Winey, Mark; Meehl, Janet B; O'Toole, Eileen T et al. (2014) Conventional transmission electron microscopy. Mol Biol Cell 25:319-23
Shi, Jianli; Zhao, Ying; Galati, Domenico et al. (2014) Chibby functions in Xenopus ciliary assembly, embryonic development, and the regulation of gene expression. Dev Biol 395:287-98
Stemm-Wolf, Alexander J; Meehl, Janet B; Winey, Mark (2013) Sfr13, a member of a large family of asymmetrically localized Sfi1-repeat proteins, is important for basal body separation and stability in Tetrahymena thermophila. J Cell Sci 126:1659-71
Ross, Ian; Clarissa, Christina; Giddings Jr, Thomas H et al. (2013) ?-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies. J Cell Sci 126:3441-51

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