중심체 복제 및 기능 조절에서의 Cdc6의 역할에 대한 연구

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 2. 황덕수.The centrosome functions as major microtubule organizing center. Microtubules play critical roles in chromosome segregation, cell shape and motility, vesicular transport, and signal transduction. The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. I demonstrated a novel function of Cdc6 in controlling microtubule formation at the centrosomes. A conserved centrosome localization signal allowed Cdc6 to localize to S- and G2-phase centrosomes in a cell cycle-dependent manner. Cdc6 depletion increased the amounts of the integral proteins Cep215/CDK5RAP2 and Cep192 of the pericentriolar material (PCM), promoting the gamma-tubulin ring complex (gamma-TuRC) recruitment and microtubule formation, suggesting that Cdc6 modulates the PCM to control gamma-TuRC recruitment. Furthermore, Cdc6 depletion increased cell adhesion and spreading, and Cdc6 overexpression had the opposite effect. These results demonstrate that Cdc6 contributes to control of cell cycle-regulated microtubule formation. Centrosome is duplicated through cell cycle and its integrity is critical for chromosome segregation that is tightly linked to chromosome stability. I addressed that Cdc6 negatively regulated centrosome amplification and Grp78 was required for centrosome amplification. Whereas Cdc6 depletion induced centrosome amplification, Cdc6 overexpression suppressed hydroxyurea (HU)-induced centrosome amplification. The centrosome amplification arising by Cdc6 depletion was caused by centrosome over-duplication rather than centriole fragmentation. Amino acid residues 197 to 214 of Cdc6 (Cdc6-SCA: suppression of centrosome amplification) was responsible for suppression of centrosome amplification. The Cdc6-SCA contained a FHA domain protein binding motif and mediated interaction with Grp78, which is a molecular chaperon of HSP70 family. Grp78 localized to interphase centrosomes and was necessary for centrosome amplification by Cdc6 depletion or HU treatment. My results suggest that interaction between Cdc6 and Grp78 is involved in controlling centrosome duplication. Through my graduate works, I have demonstrated novel functions of Cdc6 on the regulation of microtubule formation and centrosome duplication. My achievements will contribute to understanding centrosome function and cell cycle control.ABSTRACT 1 TABLE OF CONTENTS 3 LIST OF FIGURES 6 LIST OF ABBREVIATIONS 7 CHAPTER 1. Cdc6 Localizes to S and G2 Phase Centrosomes to Control Microtubule Formation 9 Abstract 10 Introduction 11 Materials and Methods 13 DNA construction and transfection 13 Short interfering RNA (siRNA) transfection 13 Cell culture and cell line construction 13 Immunofluorescence microscopy 14 Microtubule regrowth assay 14 Fluorescence-activated cell sorting (FACS) analysis 15 Statistical analysis 15 Results 16 Cdc6 localizes to the centrosomes of S- and G2-phase cells 16 Cdc6 centrosome localization signal 19 Cdc6 depletion increases regrowth of centrosomal microtubules 22 Inhibition of microtubule formation by Cdc6 requires centrosomal localization of Ccd6 25 Cdc6 participates in the control of gamma-TuRC recruitment to the centrosomes 28 Cdc6 controls cell spreading 28 Discussion 34 CHAPTER 2. Cdc6 Contributes to Maintaining Centrosome Integrity 37 Abstract 38 Introduction 39 Materials and Methods 42 DNA construction and transfection 42 Short interfering RNA (siRNA) transfection 42 Cell culture and cell line construction 42 In vivo 32P-orthophosphate incorporation assay 43 Tandem affinity purification 43 Results 45 Cdc6 prevents centrosome amplification 45 Overexpression of Cdc6 suppresses hydroxyurea-induced centrosome amplification 50 FHA protein binding motif of Cdc6-SCA is involved in suppression of centrosome amplification 55 Cdc6 interacts with Grp78 through Cdc6-SCA motif 60 Grp78 participates in centrosome amplification 60 Discussion 70 REFERENCES 73 ABSTRACT IN KOREAN 82 ACKNOWLENGMENTSDocto

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