Title Page
Contents
ABSTRACT 13
Overview: Aim of the studies 17
Chapter 1. Autophagosome-lysosome fusion is facilitated by plectin-stabilized actin and keratin 8 during macroautophagic process 20
1.1. Introduction 21
1.2. Materials and methods 24
1.2.1. Reagents and antibodies 24
1.2.2. Cell culture and transfection 24
1.2.3. Autophagy assays; mCherry-EGFP-LC3B and GFP-LC3B analysis 25
1.2.4. Western blot analysis 26
1.2.5. Analysis of KRT8 and actin filaments morphology 26
1.2.6. Co-immunoprecipitation 27
1.2.7. Statistical analysis 27
1.3. Results 28
1.3.1 KRT8 facilitates fusion between autophagosomes and lysosomes during ROS-induced autophagy process 28
1.3.2. Disruption of KRT8 filamentous structure attenuates autophagosome-lysosome fusion under ROS-induced autophagy process 31
1.3.3. Unphosphorylated KRT8 is associated with actin filaments through the cytolinker, PLEC 36
1.3.4. PLEC is required for autophagosome-lysosome fusion by supporting the cytoskeletal architecture 40
1.3.5. KRT8 and PLEC facilitates autophagosome-lysosome fusion in macroautophagy process 43
1.3.6. Impairment of actin polymerization diminishes autophagosome-lysosome fusion 47
1.3.7. RAB11-FIP2 is necessary for facilitation of autophagosome-lysosome fusion 50
1.4. Discussion 53
Chapter 2. Accumulation of senescent mitochondria through impaired autophagic lysosome reformation induces degeneration in retinal pigment epithelium 57
2.1. Introduction 58
2.2. Materials and methods 61
2.2.1. Cell culture and reagents 61
2.2.2. Antibodies 61
2.2.3. Intracellular reactive oxygen species (ROS) detection 62
2.2.4. Cytosol calcium ion detection 62
2.2.5. Oxygen consumption rate (OCR) assay 62
2.2.6. Analysis of mitochondrial morphology 63
2.2.7. Transmission electron microscopy (TEM) 63
2.2.8. Flow cytometric analysis; mitochondrial damage detection 64
2.2.9. Mitophagy assay; GFP-LC3B analysis 64
2.2.10. Observation of mitochondrial turnover 64
2.2.11. Western blot analysis 65
2.2.12. Autophagy assay; mCherry-EGFP-LC3B analysis 65
2.2.13. Immunocytochemistry 66
2.2.14. Lysosomal enzymatic activity assay 66
2.2.15. Analysis of lysosome morphology 67
2.2.16. Analysis of Protein Kinase C (PKC) activity 67
2.2.17. Statistical analysis 67
2.3. Results 68
2.3.1. Impaired ALR induces the accumulation of senescent lysosome with reduced autophagic flux 68
2.3.2. Disruption of the ALR promotes PKC activation and induces PKC translocation into mitochondria 73
2.3.3. Accumulation of senescent mitochondria induced by ALR inhibition promotes EMT in RPE cells 80
2.4. Discussion 86
Chapter 3. AMPK activation attenuates degeneration of the retinal pigment epithelium through the maintenance of lysosomal homeostasis 88
3.1. Introduction 89
3.2. Materials and methods 91
3.2.1. Cell culture and reagents 91
3.2.2. Antibodies 91
3.2.3. Animal studies 92
3.2.4. Immunohistochemistry 92
3.2.5. Intracellular reactive oxygen species (ROS) detection 92
3.2.6. Cytosol calcium ion detection 93
3.2.7. Oxygen consumption rate (OCR) assay 93
3.2.8. Analysis of mitochondrial morphology 93
3.2.9. Transmission electron microscopy (TEM) 94
3.2.10. Flow cytometric analysis; mitochondrial damage detection 94
3.2.11. Mitophagy assay; GFP-LC3B analysis 95
3.2.12. Observation of mitochondrial turnover 95
3.2.13. Western blot analysis 95
3.2.14. Autophagy assay; mCherry-EGFP-LC3B analysis 96
3.2.15. Immunocytochemistry 96
3.2.16. Lysosomal enzymatic activity assay 97
3.2.17. Analysis of lysosome morphology 97
3.2.18. Analysis of Protein Kinase C (PKC) activity 98
3.2.19. Statistical analysis 98
3.3. Results 99
3.3.1. AMPK agonist promotes autophagy flux by facilitating lysosome biogenesis 99
3.3.2. Metformin suppresses PKC activation and mitochondrial translocation 105
3.3.3. Metformin reduces accumulation of senescent mitochondria by promoting mitophagy and inhibiting PKC activation 113
3.3.4. Metformin prevents structural collapse of RPE by promoting lysosome biogenesis 116
3.4. Discussion 122
References 126
Abstract (in Korean) 147
Chapter 1 9
Table 1.1. List of proteins found to associate with KRT8 through immunoprecipitation and subsequent LC-MS/MS analysis 38
Chapter 1 10
Figure 1.1. KRT8 expression level is associated with autophagy progression in RPE cells under oxidative stress condition. (A) Confocal microscopic images in mCherry-... 30
Figure 1.2. Treatment of RPE cells with okadaic acid suppresses autophagosome- lysosome fusion. ARPE-19 cells were treated with paraquat in the presence or absence... 34
Figure 1.3. Knockdown of the PP2A expression level diminishes autophagosome and lysosome fusion. (A) Confocal microscopic images of anti-PP2A siRNA-transfected... 35
Figure 1.4. Inhibition of KRT8 dephosphorylation attenuates the interaction between actin filaments, PLEC, and KRT8. (A) Immunoblots of KRT8 and actin in anti-PLEC... 39
Figure 1.5. PLEC expression is correlated with autophagy progression in RPE cells under oxidative stress. (A) Immunoblots of PLEC and autophagic markers (SQSTM1,... 41
Figure 1.6. KRT8 and PLEC is required for autolysosome formation in macroautophagy caused by MTOR inhibition condition. (A) Confocal microscopic... 45
Figure 1.7. Disruption of actin filaments inhibits autophagosome-lysosome fusion. ARPE-19 cells were treated with Torin1 in the presence or absence of cytochalasin D... 49
Figure 1.8. Expression level of RAB11-FIP2 is associated with autophagy progression in RPE cells under oxidative stress. (A) Immunoblots of RAB11-FIP2... 52
Chapter 2 11
Figure 2.1. Impairment of autophagic lysosome reformation (ALR) promotes the accumulation of senescent lysosomes. (A) Confocal microscopy images of... 71
Figure 2.2. Inhibition of autophagic lysosome reformation (ALR) attenuates the progression of autophagy. (A) ALR-inhibited ARPE-19 cells were transfected with... 72
Figure 2.3. Inhibition of autophagic lysosome reformation (ALR) elevates cytosolic calcium and reactive oxygen species (ROS) levels. (A) Fluorescence microscopy... 77
Figure 2.4. Suppression of autophagic lysosome reformation (ALR) promotes protein kinase C (PKC) activation and facilitates the translocation of activated PKC into the... 78
Figure 2.5. Impairment of autophagic lysosome reformation (ALR) promotes the accumulation of senescent mitochondria. (A) Confocal fluorescence microscopy... 83
Figure 2.6. Inhibition of autophagic lysosome reformation (ALR) facilitates epithelial-mesenchymal transition (EMT) by promoting the phosphorylation of... 85
Chapter 3 12
Figure 3.1. Metformin maintains lysosomal homeostasis by promoting lysosome biogenesis. (A) Confocal microscopic images of transcription factor EB (TFEB)-... 103
Figure 3.2. Metformin enhances autophagy flux by promoting the biogenesis of lysosomes. (A) Confocal microscopy images of ARPE-19 cells expressing mCherry-... 104
Figure 3.3. Metformin attenuates the activation of classical protein kinase C (PKC) isoforms induced by the inhibition of autophagic lysosome reformation. (A)... 109
Figure 3.4. Metformin mitigates the activation of novel protein kinase C (PKC) isoforms resulting from the inhibition of autophagic lysosome reformation. (A)... 111
Figure 3.5. Metformin attenuates the protein kinase C (PKC) activation under the inhibition of autophagic lysosome reformation. ARPE-19 cells were incubated with... 112
Figure 3.6. Treatment with metformin reduced the accumulation of senescent mitochondria under autophagic lysosome reformation attenuation condition. (A)... 115
Figure 3.7. Metformin suppresses the progression of epithelial-mesenchymal transition (EMT) by inhibiting the phosphorylation of glycogen synthase kinase 3 beta... 120
Figure 3.8. Metformin protects retinal epithelial cells from structural collapse induced by the inhibition of autophagic lysosome reformation. (C) Experimental timeline of... 121
Figure 3.9. Graphical representation of the relationship between slowed autophagic flux caused by dysfunctional lysosomal accumulation and the... 125