Centrioles are small cylindrical organelles composed of an array of stabilized microtubules that forms around a 9-fold symmetric central hub called the cartwheel. In dividing cells, centrioles recruit pericentriolar material to form centrosomes that contribute to spindle assembly and positioning through their ability to nucleate and anchor microtubules. Centrioles duplicate precisely once per cell cycle to ensure that each mitotic cell contains only two centrosomes. Supernumerary centrosomes are a common feature of cancer cells and their presence causes chromosomal instability, leading to the hypothesis that misregulation of centriole duplication is a driving force during tumorigenesis. The proposed research combines an in vitro approach with in vivo work in the model system C. elegans to elucidate the pathways that control centriole duplication and centrosome assembly. Understanding these processes is essential to understand the failure to control centrosome number and morphology in cancer and to define the contexts in which it will be useful to target centriole duplication and/or centrosome assembly for therapeutic purposes. During the initiation of centriole assembly, the conserved centriole component SPD-2/Cep192 targets the ZYG-1/Plk4 kinase to the mother centriole. ZYG-1/Plk4 initiates polymerization of the centriolar scaffold protein SAS-6 to form the cartwheel. In Specific Aim 1, we elucidate the mechanisms that control this initiation reaction by performing a parallel in vivo/in vitro analysis of physical interactions and phosphoregulation involving SPD-2, ZYG-1 and SAS-6. SAS- 6 and SAS-4 are components of a universally conserved module required for centriole assembly. Purified SAS-6 dimers can associate with each other through their N-terminal globular domains to form a 9-fold symmetric circular plate-like structure. The important questions are how SAS-6 polymerization is controlled, how the plate-like SAS-6 assemblies stack to form the cylindrical cartwheel, and how the cartwheel directs formation of the outer centriole wall. In Specific Aim 2, we address these questions by defining the nature and significance of interactions of SAS-6 with: i) SAS-5, a conserved protein that functions with SAS-6 to promote cartwheel assembly; and ii) SAS-4, a key component of the outer centriole wall that controls assembly of the centriolar microtubules. We also investigate the significance of a folded conformation of SAS-6. The centriole outer wall directs the recruitment of pericentriolar material (PCM) to form centrosomes that nucleate and anchor microtubules. The amount of PCM increases five to ten-fold during mitotic entry in a process known as centrosome maturation. PCM amplification is controlled by phosphoregulation mediated by a conserved module that includes the bifunctional centrosome protein SPD-2/Cep192 and the mitotic kinases PLK-1 and Aurora A. Specific Aim 3 dissects this phosphoregulation to elucidate the mechanisms that control centrosome size.