We will investigate the early steps of basal body assembly by utilizing the cytology and molecular genetic advantages of the ciliate, Tetrahymena thermophila. Basal bodies have the defining structural characteristic of a conserved radial array of nine microtubule structures and serve to nucleate cilia and flagella. Dysfunction in basal body or ciliary components results in pleiotropic human diseases, collectively known as ciliopathies. Ciliopathies affect several organ systems and developmental processes, reflecting the importance of basal bodies and cilia to a wide range of cell types. Furthermore, most cell types interconvert basal bodies and centrioles, the latter of which function in organizing centrosomes. Many tumor cells have aberrant centriole structure and number, which contributes to genetic instability during cell division. Despite the importance of basal bodies and centrioles, our understanding of their molecular composition, architecture, and assembly remains sparse. Basal body assembly has been monitored by electron microscopy, revealing that the site of nascent basal body assembly is associated with an amorphous accumulation of proteins from which the cartwheel, the first recognizable structural intermediate of the basal body, is formed. In the first aim, we will study the role of centrins and their binding partners, Sfi1-repeat proteins, at the site of nascent basal body assembly. We will determine which centrin/Sfi1-repeat complex(es) is at this site and investigate the importance of the interaction between centrin and its cognate Sfi1-repeat protein during basal body assembly. The second aim focuses on the cartwheel structure, which consists of a circular hub and nine spokes that nucleate the first of the triplet microtubules. We have shown that the conserved proteins Sas6a and Sas6b localize to the hub of the cartwheel and required for its assembly. We have identified candidate cartwheel proteins in complex with Sas6a. We will verify whether these proteins are cartwheel components, establishing whether they function in cartwheel assembly, and determine if they bind Sas6a directly. Furthermore, we will establish whether vertebrate orthologs of the new cartwheel proteins are found in vertebrate basal bodies or centrioles and function in these structures. Finally, we have demonstrated that many basal body components have both a stably incorporated population and a dynamically exchanging population. In the third aim, we propose to explore the in vivo and in vitro requirements for the dynamic exchange of cartwheel components, starting with Sas6a, to determine the mechanisms of exchange in basal body function. We will also ask whether mechanisms of dynamic exchange are shared with those for stable incorporation during basal body assembly, answering the question of whether a component has a single or multiple mechanisms of incorporation into a basal body. The proposed research program will elucidate key components and mechanisms for the early stages of basal body and centriole assembly, and for exchange of proteins that may contribute to maintaining the integrity of these structures.