Striking one in 1000 live births, autosomal dominant PKD (ADPKD) is among the most common monogenetic disorders. Half of the patients will progress to end stage renal disease by age 60. Recent results suggest that the cilium functions as a sensor for environmental anti-proliferation signals and that cilial defects can cause cyst formation. Given the importance of cilia in PKD, dissecting cilia formation and maintenance is critically important for understanding PKD pathogenesis. In an insertional mutagenesis screen for cystic kidney mutants in zebrafish, a novel gene Sco (Scorpion) was identified along with Pkd2 and three IFT (intraflagellar transport) genes, which are thought to be required for the formation and maintenance of cilia. Sco encodes a protein with a small GTPase domain at the N-terminus and a coiled-coil domain and a proline-rich region in the C-terminal half. Preliminary analysis showed that Sco is required for renal cilia formation and that Sco protein is located on cilia. This project is designed to test the hypothesis that Sco is a signaling molecule that regulates cilia assembly during kidney development. First, the phenotypes of sco mutant and the subcellular localization of Sco will be examine in detail to address the question whether Sco is directly involved in regulating cilia formation. Then the functions of the small GTPase domain and the C-terminal region of Sco will be studied in detail via biochemical assays and biological tests with over-expression, deletion analysis, dominant active and negative constructs. Furthermore, binding partners for different regions of Sco will be identified with yeast two-hybrid screen and tandem affinity purification. These experiments will reveal how Sco functions as a signal transducer in the genetic network that regulates cilia formation during zebrafish kidney development. Finally, the functional conservation of Sco will be analyzed by examining its expression and subcellular localization in mouse kidney and by analyzing its function with RNAi and dominant constructs in mouse cells.
