Molecular Biology and Genetics MS Thesis Defense by Efraim Culfa

KOÇ UNIVERSITY

GRADUATE SCHOOL OF SCIENCES & ENGINEERING

MOLECULAR BIOLOGY AND GENETICS

MS THESIS DEFENSE BY EFRAİM CULFA

Title: Mechanistic Dissection of the Centriole Duplication and Ciliogenesis Proximity Interaction Maps

Speaker: Efraim Culfa

Time: 10:00, 09.07.2018

Place: ENG 208

Koç University

Rumeli Feneri Yolu

Sariyer, Istanbul

Thesis Committee Members:

ASSIST. PROF. Elif Nur Fırat Karalar (Advisor, Koç University)

ASSOC. PROF. Nurhan Özlü (Koç University)

ASSIST. PROF. Umut Şahin (Boğaziçi University)

Abstract: The centrosome is the main microtubule-organizing center in animal cells which function in key processes including cell division and transmission of signaling pathways including Hedgehog signaling. Centrosome and cilium dysfunction is 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. To this end, 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. 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 pattern during the cell cycle. 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 and suggested possible functions for CEP103 in centriole duplication and centrosome cohesion. Moreover, using BioID mapping and co-immunoprecipitation experiments, we showed that the retinal degeneration gene product CCDC66 forms a complex with the ciliogenesis proteins PCM1 and CEP290, providing insight into the underlying molecular defects associated with retinal degeneration. 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