The role of F-actin binding protein Cotl1 in regulation of mitochondrial dynamics
- 주제(키워드) mitochondria , mitochondrial dynamics , morphology , fusion , fission , mitochondrial-shaping protein , Cotl1 (coactosine-like protein 1) , F-actin , cytoskeleton , Drp1 (dynamic related protein 1) , gene knockdown , gene knockout
- 발행기관 아주대학교
- 지도교수 Seon-Yong Jeong
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029961
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
– Abstract – The role of F-actin binding protein Cotl1 in regulation of mitochondrial dynamics Mitochondrial fission and fusion are important in maintaining normal cell physiology, and regulated by mitochondrial-shaping proteins, including Drp1, Fis1, Mfn1, Mfn2, and Opa1. Dynamic interaction between mitochondria and cytoskeleton is important for maintaining mitochondrial function and structure. F-actin, a major component of cytoskeleton, is essential in cell stability and morphogenesis. In this study, I investigated whether Coactosine-like protein 1 (Cotl1), an F-actin-binding protein, is involved in mitochondrial morphology regulation. The Cotl1 was selected as a candidate for elucidating mitochondria and cytoskeleton network regulatory molecule via the differential display method of whole gene expression among three types of cells with different mitochondrial morphology. Cellular localization analysis revealed predominant, endogenous Cotl1 overexpression in the cytosol. However, in Drp1-depleted cells, mitochondria were elongated owing to fission inhibition and mitochondrial co-localization of Cotl1 significantly increased. Knockdown of Cotl1 resulted in significantly elongated mitochondria, abnormal F-actin morphology, and increased mitochondrial F-actin accumulation. Furthermore, confocal microscopy and transmission electron microscopy analyses demonstrated that mitochondria of primary cultured heart cells derived from Cotl1-knockout mouse were significantly elongated compared to those from wild-type mouse. Moreover, Cotl1-knockdown cells and Cotl1-knockout heart cells showed increased Mfn1 and Mfn2 expression, and decreased phosphorylation of Drp1 at Serine 616 residue. These results suggest a possible role of Cotl1 in mitochondrial fission and/or fusion process mediated by binding with F-actin.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 10
A. Annealing control primer (ACP)-based screening of differentially expressed genes 10
B. Plasmids, shRNA constructs and oligonucleotide sequence for siRNA
11
C. Cell culture and drugs treatment 12
D. Antibodies 12
E. Immunofluorescence 13
F. Confocal microscopy 13
G. Transmission electron microscopy 14
H. Primary cell culture 15
I. Quantification of mitochondrial length 16
J. Western blotting 16
K. Cell fractionation 17
L. Statistical analysis 17
III. RESULTS 18
A. Identification of differentially expressed genes among three types of HeLa cells with different mitochondrial morphology 18
B. Cellular localization of endogenous Cotl1 and overexpressed GFP-Cotl1 in HeLa, C2C12, and IMR90 cells 25
C. Changes in Cotl1 distribution in elongated mitochondria in shDrp1 cells 29
D. Changes in mitochondrial and F-actin morphologies in Cotl1-knock-down cells 32
E. Alterations in the expression of mitochondrial-shaping proteins in Cotl1-knockdown cells…………………………………………………….. 37
F. Alterations in mitochondrial morphology and mitochondrial-shaping protein expression in primary cultured heart cells derived from Cotl1-knockout mouse 39
IV. DISCUSSION 42
V. CONCLUSION 46
REFERENCES 47
국문요약 55
