Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Mark Winey

Second Advisor

Gia Voeltz

Third Advisor

Michael Klymkowsky

Fourth Advisor

Jeffrey Moore

Fifth Advisor

Andreas Hoenger

Abstract

Basal bodies are microtubule-based structures which template, anchor, and orient cilia at the cell surface. Although basal bodies contribute to vital cell functions, the molecular contributors of their assembly and maintenance are poorly understood. Previous studies in Tetrahymena thermophila revealed important roles for centrins in basal body assembly, separation of new basal bodies, and stability. Here, I characterized the basal body function of two centrin-binding proteins, Sfr1 and Poc5, in Tetrahymena. Sfr1 is the only centrin-binding protein in Tetrahymena that localizes to all cortical row and oral apparatus basal bodies. Poc5, on the other hand, transiently localizes to basal bodies with enrichment in assembling basal bodies and removal prior to the onset of cilia formation. On an ultrastructural level, Sfr1 resides at the microtubule scaffold from the proximal cartwheel to the distal transition zone. Tetrahymena Poc5 ultrastructural localization is not fully characterized, but localization was observed in the proximal and distal ends of the basal body microtubule scaffold. Complete genomic knockouts of SFR1 (sfr1Δ) and POC5 (poc5Δ) caused a significant increase in cortical row basal body density, contributing to an overall overproduction of basal bodies. Reintroduction of SFR1 in sfr1Δ cells led to a marked reduction in basal body density and total cortical row basal bodies. Therefore, Sfr1 directly modulates cortical row basal body production. This study revealed an inhibitory role for Sfr1 and Poc5 (and potentially centrins) in Tetrahymena basal body production. Given the modulatory roles of Sfr1 and Poc5 in basal body production, a double knockout was generated that resulted in an exacerbated basal body overproduction phenotype in poc5Δ; sfr1Δ cells. This Tetrahymena work highlights a redundant role for Sfr1 and Poc5 in modulating the production of basal bodies along cortical rows. Through a collaborative effort, Xenopus Poc5 appears to have an important role in ciliogenesis since depletion of Poc5 resulted in ciliopathy-like defects in wholemount embryos and a reduction in the number of ciliated cells in the ciliated epithelium of Xenopus. Collectively, this work shed light on the function of Sfr1 and Poc5 in Tetrahymena basal bodies and Xenopus cilia formation, of which nothing was previously known.

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