Sentriyol duplikasyonu ve sil biyogenezi proksimite etkileşim haritalarının aydınlatılması

The centrosome is the main microtubule-organizing center in animal cells, participating in a variety of cellular processes from cell polarization to cell division. The core of the centrosome consists of two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Importantly, centrioles function as basal bodies for nucleating the formation of flagella and cilia, including the primary cilium, a sensory organelle for receiving and transmitting developmentally important signaling pathway. Centrosome and cilium dysfunction are associated with a variety of human diseases including cancer and ciliopathies. To develop new diagnostic and therapeutic approaches for these diseases, it is essential to elucidate the mechanisms that regulate the biogenesis and function of the centrosome/cilium complex, which requires the identification of the parts list for the centrosome and mapping the interaction among these parts. In this thesis, we focused on functional characterization of two novel centrosome proteins, CEP103 and CCDC66, which we identified as proximity partners of known centriole duplication and ciliogenesis proteins. CEP103 is upregulated in early differentiation of multiciliated epithelial cells and has high proximity to the de novo centriole duplication protein CCDC67. Supporting these earlier finding in our laboratory, we showed that CEP103 localizes to the centrosome and in part to satellites in a subset of cells. High resolution imaging experiment identified CEP103 as a proximal protein of the PCM, with partial co-localization with centriole fiber proteins. CEP103 has a dynamic localization patter during cell cycle, where it localizes to the spindle poles and microtubules during mitosis and central spindle during telophase. In vivo and in vitro experiments defined CEP103 as a microtubule associated protein. Analyses of the CEP103 truncation mutants revealed that the C terminal half of the protein was required for its localization to the centrosome and microtubules. Proximity dependent biotinylation of CEP103 revealed its extensive interactions with centrosome and centriolar satellite proteins. In o-localization and co-immunoprecipiation experiments, we showed that CEP103 interacts with CCDC67 and CEP63, two centriole duplication proteins that mediate formation of procentrioles during initiation phase. Work from our laboratory identified CCDC66 as a component of the centrosome, cilium and microtubules with functions during cilium formation and trafficking. To gain insight into the molecular mechanism of these functions, we performed immunoprecipiation experiments with the candidate interacting partners of CCDC66 identified by BioID pulldowns and showed that CCDC66 forms a complex with the ciliogenesis proteins PCM1 and CEP290. In structure-function experiments using truncation mutants, the retinal degeneration mutant form of CCDC66 did not interact with PCM1, suggesting that its interaction with PCM1 and proper function at the centrosome is important for retinal development. Together, our work identified new regulatory proteins of centriole duplication and cilium formation and contributed to our understanding of the mechanisms of centrosome/cilium biogenesis and function.