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Studies on pericentrin in regulation of centriole separation : 중심립 유리과정에서 pericentrin 역할 연구
DC Field | Value | Language |
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dc.contributor.advisor | 이건수 | - |
dc.contributor.author | 김재연 | - |
dc.date.accessioned | 2017-10-27T17:12:49Z | - |
dc.date.available | 2017-10-27T17:12:49Z | - |
dc.date.issued | 2017-08 | - |
dc.identifier.other | 000000145695 | - |
dc.identifier.uri | https://hdl.handle.net/10371/137152 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 자연과학대학 생명과학부, 2017. 8. 이건수. | - |
dc.description.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. | - |
dc.description.tableofcontents | BACKGROUND 1
1. Structure of centrosome 1 1.1 Discovery of centrosome 1 1.2 Centrioles 1 1.3 Pericentriolar material (PCM) 2 2. Functions of centrosome 6 2.1 Microtubule network formation in interphase 6 2.2 Spindle formation during mitosis 6 2.3 Primary cilia formation in quiescent cells 7 3. Centrosome cycle 12 3.1 Initiation of centriole duplication 12 3.2 Centriole elongation 13 3.3 Centrosome maturation 13 3.4 Centrosome separation 14 3.5 Bipolar spindle formation 14 3.6 Centriole disengagement 14 4. Licensing mechanism for centriole duplication: Centriole engagement and disengagement 19 PURPOSE 23 CHAPTER 1. PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit 24 ABSTRACT 25 INTRODUCTION 26 MATERIAL AND METHODS 29 Plasmids, siRNA, and cell culture 29 Antibodies 30 Immunoprecipitation and immunoblot analyses 31 Immunostaining analysis 33 Statistical analysis 34 RESULTS 35 BI2536 blocks both PCM disassembly and centriole separation 35 PLK1 phosphorylation is necessary for PCNT cleavage 36 PLK1 phosphorylates PCNT in vivo 38 PCNT phosphorylation is necessary for centriole separation 40 PCNT and CEP215 are essential for centriole association 41 Dual functions of PLK1 phosphorylation of PCNT 43 Discussion 60 CHAPTER 2. Phenotypic analyses of PCNT-deleted cells 64 ABSTRACT 65 INTRODUCTION 66 MATERIAL AND METHODS 68 Plasmids, siRNA, and cell culture 68 Generation of knockout cell lines with CRISPR/CAS9 system 69 Antibodies 69 Immunoblot analysis 70 Immunostaining analysis 71 Statistical analysis 72 RESULTS 73 Generation of PCNT | - |
dc.description.tableofcontents | TP53 double knockout cell lines 73
Interphase centrosomes in the PCNT-deleted cells 73 Spindle poles in the PCNT-deleted mitotic cells 74 Active role of PCNT for centriole association 77 DISCUSSION 94 PERSPECTIVE 98 REFERENCES 101 ABSTRACT IN KOREAN (국문 초록) 108 | - |
dc.format | application/pdf | - |
dc.format.extent | 11768559 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Pericentrin | - |
dc.subject | 중심체 | - |
dc.subject | 중심립 유리과정 | - |
dc.subject.ddc | 570 | - |
dc.title | Studies on pericentrin in regulation of centriole separation | - |
dc.title.alternative | 중심립 유리과정에서 pericentrin 역할 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Jaeyoun Kim | - |
dc.description.degree | Doctor | - |
dc.contributor.affiliation | 자연과학대학 생명과학부 | - |
dc.date.awarded | 2017-08 | - |
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