Studies on pericentrin in regulation of centriole separation

Abstract

A centrosome is composed of a centriole surrounded by protein matrix, called pericentriolar materials (PCM). The microtubule organization is the prime function of the centrosome. Cilia formation is another important function of the centrosome in quiescent cells. Duplication and segregation of centrioles occur in tight link to cell cycle. A daughter centriole is assembled next to the mother centriole during S phase, and remained in an engaged state until the cell exits M phase. New daughter centrioles may be generated only after the mother and daughter centrioles in the previous cycle are separated. Therefore, centriole separation is considered a licensing step for centriole duplication. However, it is largely unknown how centriole engagement is maintained and disrupted during the cell cycle. Pericentrin (PCNT) is a PCM protein which is important for maturation process of centrosome to become spindle poles during mitotic entry. PCNT is also involved in induction of centriole separation during mitotic exit. PCNT is specifically cleaved, which is considered an essential step for centriole separation during mitotic exit. The purpose of my research is to elucidate mechanistic aspects of PCNT functions in centriole engagement and separation during M phase. In chapter 1, I report that PCNT has to be phosphorylated by PLK1 in order to be a suitable substrate of separase. The phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of BI2536. Based on these results, I propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit. In chapter 2, I generated PCNT knockout cell lines and analyzed the phenotypes in relation to PCM assembly and centriole association. Deletion of PCNT hardly affected interphase centrosomes but conferred defects in centrosome maturation in cells entering M phase. The centrioles in PCNT–deleted cells were prematurely separated in early phase of mitosis and frequently amplified in M phase-arrested cells. Abnormal multi-nuclear cells repeatedly appeared in PCNT-deleted cells at interphase. My results confirmed that PCNT is critical for centriole association during M phase.