#09-06 STRUCTURAL CHARACTERIZATION OF CENTRIOLE ASSEMBLY

Scripps Research Institute, The, La Jolla CA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Significance: Centrioles are small cylindrical organelles (~100 nm in diameter and ~150 nm in length) whose main distinguishing characteristic is a 9-fold symmetric array of stabilized microtubules. Centrioles have two key functions in eukaryotic cells: (1) They recruit pericentriolar material (PCM) to form centrosomes and (2) they template the formation of cilia. In dividing cells, centrioles, like nuclear DNA, duplicate precisely once per cell cycle in a process that initiates during S-phase. During duplication a single new centriole forms adjacent and at right angles to each parent centriole. Despite their importance, the process of centriole assembly remains largely mysterious. Critical questions include: How is the 9-fold symmetry of the centriole dictated? What limits centriole assembly to a single centriole in the vicinity of the parent? How is centriole length so precisely controlled? Recently, the emergence of Caenorhabditis elegans as a model system provided a major breakthrough in understanding this process by defining a small set of components that constitute the core centriole duplication machinery. Genome wide analysis in C. elegans identified four proteins specifically required for centriole assembly: ZYG-1, SAS-4, SAS-5, and SAS-6. ZYG-1 is a polo family kinase that is thought to play a signaling role to coordinate duplication with the cell cycle and ensure formation adjacent to the parent. SAS-4 and SAS-6 are widely conserved and are thought to be core structural components of the centriole. The work proposed here centers on SAS-6, which is thought to be a component of the first identifiable structural intermediate in centriole assembly, a structure called the central tube/cartwheel. This structural intermediate consists of a nine-fold symmetric array of spokes that emanate from a central tubule. The goal of the proposed work is to use electron microscopy to characterize the structure and assembly of SAS-6 in vitro.