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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊維元(Wei Yuan Yang) | |
| dc.contributor.author | Huei-Jiun Yang | en |
| dc.contributor.author | 楊惠君 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:09:46Z | - |
| dc.date.copyright | 2014-09-03 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18524 | - |
| dc.description.abstract | 細胞自噬(Autophagy) 是生物體用來降解大分子物質並將之基本組成循環再利用的一種方式。它藉由形成雙層脂質膜結構的自噬小體(autophagosome) 來包圍細胞內老舊胞器或摺疊錯誤的蛋白質。自噬小體與溶小體(lysosome) 融合後，溶小體內的酵素會分解代謝這些物質，將組成這些物質的基本單位回歸於細胞質給細胞重新使用。因此，在細胞自噬的過程中最重要的步驟就是自噬小體的形成。先前研究發現，未成熟的自噬小體(phagophore) 上的LC3B蛋白的多寡會影響自噬小體的形成。LC3B蛋白會藉由共價鍵鍵結與自噬小體上的脂質PE結合，在與溶小體融合前，自噬小體外膜上的LC3B蛋白必須要先被Atg4B蛋白從自噬小體上移除下來至細胞質(去脂化作用，LC3B-II delipidation)，否則將會影響自噬小體與溶小體融合的效力。由於自噬小體的形成是相當動態快速的，且我們缺乏可在活體細胞內偵測LC3B蛋白被去脂化作用的分析方式，於是現今我們並不確定未成熟的自噬小體膜上的LC3B蛋白是否也可被Atg4B蛋白作用移除？如果可以，對生物體而言可能的功用是什麼？在本篇研究，我們建立了一個活體單細胞影像分析方法來研究單一活體細胞內LC3B蛋白被去脂化的活性強弱。藉由阻擋自噬小體形成完全，我們發現未成熟的自噬小體膜上的LC3B蛋白是可以被Atg4B蛋白作用移除的！如果利用顯微注射Atg4B抗體來抑制Atg4B蛋白的去脂作用，更可以加速活細胞內自噬小體的形成！另外，在細胞飢餓(starvation) 狀態下我們發現LC3B蛋白被去脂化的活性是隨著時間而有所改變的！在LC3B蛋白被去脂化的活性較弱時，自噬小體形成的速度較快！這個LC3B蛋白被去脂化的活性改變和細胞內的氧化強度(ROS level) 呈現負相關的關係：氧化強度越高，LC3B蛋白被去脂化的活性越慢。而氧化強度似乎是藉由開關粒線體通透移轉通道(mPTP) 來調控。 | zh_TW |
| dc.description.abstract | Autophagy, an intracellular degradation system, forms double membrane structures, called autophagosomes, to engulf and deliver cytoplasmic constituents to the lysosomes for recycling. A key step of autophagosome formation involves the lipidation of LC3B to phosphatidylethanolamine (PE) on the autophagic membranes. The PE-bound LC3B, termed LC3B-II, can be further delipidated by a cysteine protease Atg4B to cytosol for reusing. Previous studies showed that the delipidation of LC3B-II is necessary for autophagosome to fuse with lysosome. However, it is uncertain whether Atg4B could also process LC3B-II on phagophore, the immature membrane structure of autophagosome, and what the effect of this delipidation is during autophagy. The lack of live-cell assay for LC3B-II delipidation limits the investigations of these questions. Herein, we established an imaging-based methodology that enabled us to explore how LC3B-II delipidation activity varies during autophagy inside living cells. Our live-cell assay provided a novel insight into autophagy induction, and presented direct evidence verifying that phagophore-resident LC3B-II can be delipidated. The access abrogation of Atg4B by anti-Atg4B antibody accelerated the autophagosome formation. During starvation, slowed LC3B-II delipidation activity coupled with accelerated autophagosome formation, which might be regulated via mPTP-dependent ROS burst. The mPTP blockade by CsA was sufficient to abolish starvation-induced ROS burst and slowed autophagosome formation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:09:46Z (GMT). No. of bitstreams: 1 ntu-103-D97B46003-1.pdf: 3134536 bytes, checksum: f6fb24c5f5f9b34b20025d754f8c8539 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | Contents
Preface………………………………………………………………. i Abstract (in Chinese) ………………………………………………. ii Abstract …………………………………………………………..... iii I. Introduction ……………………………………………………. 1 I.1. An overview of autophagy……………………………………………….... 1 I.1.1. Microautophagy……………………………………………………… 1 I.1.2. Chaperone-mediated autophagy (CMA)……………………………… 2 I.1.3. Macroautophagy………………………………………………………. 2 I.2. The nucleation of autophagosome formation on omegasome………………. 3 I.3. The membrane expansion of autophagosome formation ……………………. 4 I.3.1. LC3B lipidation ……………………………………………………….. 4 I.3.2. The direction of LC3B lipidation ……………………………………… 4 I.3.3. Distinguishing phagophores from autophagosomes …………………... 5 I.4. The maturation of autophagosome formation ……………………………….. 6 I.4.1. The lysosomal fusion of autophagosome ……………………………… 6 I.4.2. Lysosome regeneration………………………………………………... 6 I.5. The role and regulations of mTORC1 under starvation-induced autophagy.... 7 I.5.1. The signaling regulations for mTORC1 activation ……………………. 7 I.5.2. The effects of mTORC1 inactivation during starvation ………………. 8 I.6. LC3 family…………………………………………………………………... 9 I.6.1. Gene expression and localization of LC3 family……………………… 9 I.6.2. The functions of LC3 family…………………………………………... 10 I.7. Atg4 family and functions …………………………………………………… 11 I.8. Current methods for analyzing LC3s cleavage……………………………… 12 II. Materials and Methods ………………………………………….. 14 II.1. Plasmid Construction ……………………………………………………….. 14 II.2. Cell culture, Transfection, and Starvation ………………………………….. 14 II.3. Live cell imaging for monitoring LC3B-II delipidation by Atg4B………… 14 II.4. The calculations of LC3B-II delipidation activity and autophagic spots’ area 15 II.5. The detection of autophagosome formation time…………………………… 16 II.6. Microinjection………………………………………………………………. 17 II.7. Western blotting …………………………………………………………….. 17 II.8. ROS detection………………………………………………………………. 17 III. Results …………………………………………………………. 18 III.1. The imaging-based methodology for quantifying LC3B-II delipidation from autophagosomes in a single living-cell ………………………………. 18 III.2. LC3B-II can be delipidation from phagophores …………………………… 20 III.3. Atg4B inhibition can accelerate autophagosome formation ………………. 21 III.4. The temporal regulation of LC3B-II delipidation activities during starvation in living cells ……………………………………………………. 22 III.5. Possible mechanisms of regulating LC3B-II delipidation activity………… 23 IV. Discussions ……………………………………………………... 25 V. Figures ………………………………………………………….. 28 VI. Tables ………………………………………………………….. 52 VII. References ……………………………………………………. 53 VIII. Appendices ………………………………………………….. 62 VIII.1. The matlab code for calculating the fluorescent intensities of the images generated by LC3B-II delipidation assay……………………………….. 62 VIII.2. The matlab code for analyzing the autophagic spots’ area………………. 67 VIII.3. Abbreviations …………………………………………………………….. 69 VIII.4. The thesis of MDH……………………………………………………….. ( Monodansylpentane as a Blue-Fluorescent Lipid-Droplet Marker for Multi-Color Live-Cell Imaging ) 71 List of Figures Figure 1. An overview of autophagy……………………………………………… 28 Figure 2. Autophagosome formation on omegasome……………………………… 30 Figure 3. The crystal structures of LC3 family members and ubiquitin …………… 31 Figure 4. The Roles of LC3 family in intracellular trafficking processes …………. 32 Figure 5. Current methods for analyzing LC3 cleavage…………………………… 33 Figure 6. The methodology for quantifying the LC3B-II delipidation in living cells 35 Figure 7. LC3B-II can be delipidated from phagophores …………………………. 40 Figure 8. Atg4B inhibition accelerated the autophagosome formation …………… 43 Figure 9. The temporal regulation of LC3B-II delipidation activities during starvation in living cells …………………………………………………. 46 Figure 10. Possible mechanisms capable of regulating LC3B-II delipidation activity………………………………………………………………… 48 List of Tables Table 1. The Atg proteins participating in the core machinery of autophagosome formation ……………………………………………… 52 Table 2. Comparisons of all methods for analyzing LC3 cleavage……………. 52 | |
| dc.language.iso | en | |
| dc.title | 活體單細胞光學影像分析細胞自噬時LC3的切除作用 | zh_TW |
| dc.title | Single cell analysis of LC3 delipidation during autophagy | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳瑞華(Ruey-Hwa Chen),陳光超(Guang-Chao Chen),孟子青(Tzu-Ching  Meng),廖仲麒(Jung-Chi Liao) | |
| dc.subject.keyword | 細胞自噬,未成熟的自噬小體,LC3B蛋白被去脂化作用,細胞飢餓,活體單細胞影像分析, | zh_TW |
| dc.subject.keyword | autophagy,phagophore,LC3B-II delipidation,Atg4B,starvation,live-cell assay, | en |
| dc.relation.page | 88 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2014-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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