臺灣博碩士論文加值系統

細胞自噬為細胞吞食自身組成物的機制，其目的在於維持細胞正常功能與健康。細胞飢餓時，細胞自噬體主要形成於內質網與粒線體交界處。目前研究仍不清楚細胞如何即時在該處激發自噬體生成。因此，本篇研究著重於探討內質網與粒線體的鈣離子、活性氧類訊息傳導路徑如何在此處調控細胞自噬體生成。我們使用共軛焦顯微鏡觀察活細胞之細胞自噬活性，並在處理鈣離子、活性氧類抑制物後偵測細胞自噬活性的改變。我們發現細胞自噬活性在飢餓10-15分鐘時有顯著增加。另外，細胞自噬激發發生前，我們觀察到鈣離子湧入粒線體、粒線體活性氧類製造與釋放增加，而抑制這些現象導致細胞自噬激發消失。我們實驗室先前研究中發現同時期之 LC3去連結酶Atg4活性受抑制。此外，我們看到細胞膜損壞所引起之細胞自噬受粒線體活性氧類釋放調控。綜合以上結果，我們提出以下模式：短期飢餓時，鈣離子進入粒線體，引發粒線體製造並釋出活性氧類。釋出之活性氧類透過抑制該處Atg4以活化細胞自噬。此鈣離子活性氧類調控路徑為一普遍細胞自噬控制機制。此路徑不僅參與飢餓與細胞膜損壞引起的細胞自噬，也可能影響其他跟鈣離子擾動相關的細胞自噬活化。

Autophagy, a self-digestive mechanism, is vital for cellular functions and health. During starvation, autophagosomes form mainly at the endoplasmic reticulum (ER)-mitochondria contact sites. However, how cells timely induce autophagy at these sites are unclear. We aimed to elucidate the roles of ER and mitochondrial calcium-reactive oxygen species (ROS) signaling in autophagosome formation at these sites. We used confocal live-cell imaging to monitor autophagy activity in the absence or presence of calcium and ROS inhibitors. We found that autophagy activity burst at 10-15 minutes of starvation. The autophagy burst was preceded by mitochondrial calcium influx, mitochondrial ROS production and release; the inhibition of these phenomena abolished autophagy burst. Previously, our lab saw Atg4, an LC3 de-conjugating enzyme, was inhibited in the same time frame. Additionally, autophagy induced by plasma membrane damage was also controlled by mitochondrial ROS release. Taken together, we propose that during short-term starvation, mitochondrial calcium influx induces ROS production and release. ROS then facilitate autophagy by local Atg4 inhibition. This calcium-ROS signaling pathway may represent a general autophagy regulatory mechanism in starvation, plasma membrane damage and other autophagy stimuli that involve calcium perturbations.

中文摘要 i
ABSTRACT ii
CONTENTS iii
LIST OF FIGURES vi
Chapter 1 Introduction 1
1.1 Autophagy 2
1.2 Spatial regulation of autophagosome formation 2
1.2.1 ER in autophagosome formation 2
1.2.2 Mitochondria in autophagosome formation 3
1.2.3 ER-mitochondria contact sites in autophagosome formation 4
1.3 ROS and autophagosome formation 4
1.3.1 Autophagy activation by ROS 5
1.3.2 ROS signaling in autophagy regulation 7
1.4 Crosstalk between calcium and ROS 7
1.4.1 Calcium stimulation of mitochondrial ROS production 7
1.4.2 Calcium stimulation of mitochondrial ROS release 8
1.5 Autophagy, calcium and plasma membrane damage 9
1.5.1 Calcium increase and plasma membrane damage 9
1.5.2 Calcium and plasma membrane damage repair 10
1.5.3 Autophagy and plasma membrane damage 10
Chapter 2 Materials and methods 12
2.1 Cell culture 12
2.2 Transfection, Starvation and Confocal live-cell imaging 12
2.3 Monitoring autophagosome maturation events 13
2.4 MitoSOX assay 14
2.5 Western Blotting 15
2.6 Mitoflash and autophagosome formation 16
2.7 Mitochondrial calcium and ROS measurement after plasma membrane damage 16
2.8 Statistical analysis 17
Chapter 3 Results 18
3.1 Autophagy burst during short-term starvation mediated by calcium and ROS 18
3.1.1 Autophagy activity surges during short-term starvation 18
3.1.2 Mitochondrial ROS regulate autophagy burst 18
3.1.3 Mitochondrial calcium influx regulates ROS increase and release 19
3.1.4 Atg4 activity not regulated at protein expression levels 20
3.2 Mitoflash and autophagy regulation 20
3.2.1 Co-occurrence of calcium and mitoflash 20
3.2.2 Mitoflash and autophagosome maturation 21
3.3 Plasma membrane damage-induced autophagy mediated by calcium and ROS 21
3.3.1 Mitochondrial calcium increase upon plasma membrane damage 22
3.3.2 Mitochondrial ROS increase upon plasma membrane damage 22
3.3.3 Plasma membrane damage-induced autophagy controlled by
mitochondrial ROS release 23
Chapter 4 Discussion 24
Chapter 5 Figures 31
REFERENCE 44

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