Roles of mitochondria morphodynamic changes in the DNA damage signaling and cellular stresses
미토콘드리아 형태 변화에 의한 DNA 손상신호 조절에 관한연구
- 주제(키워드) DNA damage signaling , Mitochondria fission , Nuclear , JNK , Jun N-terminal Kinase , SIRT1 , Sirtuin 1 , ATM , ataxia-telangiectasia mutated
- 발행기관 아주대학교
- 지도교수 Hyeseong Cho
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027412
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Mitochondria are dynamic organelles essential for the life and death of the cells. The dynamic nature of mitochondria allows them to organize a network of organelles that dynamically fuse and divide. Mitochondrial morphology determined by the equilibrium betIen fusion and fission processes, is controlled by a family of “mitochondrial shaping” proteins, including hFis1, Drp1, Mfn1/2 and Opa1. In response to cellular stress, signaling cascades require tight coordination between the nucleus and mitochondria to adapt the ever-changing cellular milieu. Despite our increasing knowledge on the core components of the mitochondria-shaping machinery, knowledge regarding to the mitochondrial dynamics and nuclear communication signaling is still scare. Therefore, the aim of this research is to study the role of mitochondria morphodynamic changes in response to DNA damage and cellular stress. It was reported that ablation of mitochondria elongation by hFis1 inhibition (a mitochondria fission factor) triggered nuclear accumulation of γ-H2AX. This was accompanied with a significant reduction in cell cycle regulators that induced cellular senescence. In the first part of this study, I show how mitochondria dynamics communicate with nuclear genome in DNA damage response. Here, I found that mitochondria fragmentation induced by MFN1 depletion significantly decreased γ-H2AX accumulation under DNA damage whereas re-expression of MFN1 restored the γ-H2AX, suggesting that mitochondria dynamics affect DNA damage response signaling. Notably, targeted cytosolic irradiation using micro-irradiation laser promotes γ-H2AX accumulation in nucleus. Mechanistically, I found that the JNK activation was significantly reduced in MFN1 knockout cells and γ-H2AX accumulation can be mediated by JNK activation associated ATM phosphorylation under DNA damage response. Accordingly, mitochondrial fission induced by MFN1 depletion or hFis1 overexpression attenuates the homologous recombination and NHEJ of DNA repair mechanisms. In the second part of this research, the mitochondrial elongation mechanism under hypoxic condition was studied. I revealed that mitochondria elongation under hypoxic condition is regulated through SIRT1-mediated MFN1 deacetylation and accumulation. I further observed MFN1 as a direct target of SIRT1 and TIP60. MFN1 is hyper-acetylated by TIP60 and this modification promotes the degradation of MFN1 through a proteasome dependent pathway. Moreover, upon hypoxic condition, SIRT1 de-acetylates MFN1 and this de-acetylation modulates its stabilization. Thus, these findings provide a novel function of how mitonuclear communication safeguards cellular and organismal fitness and regulates lifespan.
more목차
PART I 1
I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
A. Antibodies and reagents 4
B. Cell culture and transfections 5
C. Plasmid construction 5
D. RNA interference 5
E. Isolation of mitochondria 6
F. Immunoblot analysis and immunoprecipitation assay 6
G. Immunofluorescence microscopy 6
H. Laser micro-irradiation 7
I. Survival assay 7
J. Homologous recombination repair assay 7
K. Chromatin fractionation 8
III. RESULTS 8
A. Mitochondrial dynamics prime DNA damage signaling 8
B. Mitochondrial dynamics prime DNA damage signaling independent of mitochondrial dysfunction 13
C. MFN1 depletion effects DNA damage response signaling through ATM dependence pathway 17
D. MFN1 depletion mediated mitochondrial fission attenuates γ-H2AX accumulation in nucleus by targeted cytosolic micro-irradiation 21
E. The inhibition of JNK activation in MFN1 depeletion attenuates DNA damage response 24
F. JNK associates with the phosphorylation of ATM 29
G. Deletion of MFN1 attenuates homologous recombination repair and non-homologous end-joining 33
IV. DISCUSSION 37
V. CONCLUSION 39
PART II 40
I. INTRODUCTION 40
II. MATERIALS AND METHODS 41
A. Cell lines 41
B. Antibodies and reagents 41
C. Plasmid construction 42
D. Western blotting 42
E. Co-immunoprecipitation assay 43
F. Isolation of mitochondria 43
G. Immunostaining 43
H. In vitro acetylation assay 44
I. Ubiquitination assay 44
J. RNA extraction, reverse transcription and polymerase chain reaction (PCR) analysis 44
III. RESULTS 45
A. Nicotinamide (NAM) promotes ubiquitin-proteasome mediated degradation of MFN1 45
B. TIP60 accelerates MFN1 degradation by the ubiquitin-proteasome system 49
C. SIRT1 deacetylases and stabilizes MFN1 53
D. Mitochondria elongation under hypoxic condition is mediated through SIRT1-mediated MFN1 stabilization 58
IV. DISCUSSION 65
V. CONCLUSION 66
REFERENCES 67
국문요약 74
