Genetic analysis of basal body function

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DESCRIPTION (provided by applicant): In animal cells microtubules are organized by the centrosome, an organelle with a pair of centrioles enclosed in a matrix ofpericentriolar material. Once per cell cycle, the centrosome and centrioles within it duplicate. This event is critical to cell division and genome stability. Duplication involves the separation of the existing centriole pair and the synthesis of a new centriole adjacent to the old centriole. The new pairs of centrioles migrate apart to the poles of the assembling spindle. Centrioles can mature into basal bodies that template flagella and cilia. The unicellular alga, Chlamydomonas provides an outstanding model for investigations into centrioles/basal bodies because of its straightforward cell biological traits and powerful genetics. In the last few years, studies from my laboratory have helped to demonstrate the complexity of basal bodies and to identify critically important roles for two new tubulin family members in centriole/basal body duplication and function. These studies have left unanswered several important questions and we will address three of these questions: 1). What are the molecules needed for early events in centriole duplication? 2). What are the molecules involved in maturation of centrioles through the cell cycle? 3). How do centrioles separate and migrate at mitosis? We will employ mutations, antibodies, and RNA interference in Chlamydomonas to answer these questions. We will test the roles of epsilon- and eta-tubulin in centriole duplication as well as the roles of several suppressors and enhancers of epsilon-tubulin mutations. We hypothesize that at least four genes, delta- tubulin, DT11, DT12, and UN13, are needed to mature centrioles into basal bodies at the M/G1 transition. We will develop antibody markers for maturation and begin to characterize the events and players in maturation. Basal bodies are converted back into centrioles that separate, migrate, and organize events in preprophase of mitosis. Using RNA interference, we will test the roles of several proteins that are important in other organisms and with this knowledge screen for conditional mutants using a GFP-gamma-tubulin reporter to follow centriole separation and migration. We will mine using "comparative genomics" to identify additional genes that are needed for duplication, maturation, and migration of the centrioles/basal bodies.