CAVEOLIN-1 AND LIPID RAFTS IN MODULATION OF AUTOPHAGY SHI YIN (BSc, Zhejiang University, P.R. China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2014 i Declaration ii Acknowledgements I would like to take this opportunity to express my deepest respect and most sincere gratitude to my supervisor, A/P Shen Han-Ming, for his professional guidance, as well as the enthusiastic encouragement, persistent patience and instructive discussions throughout the whole course of my study here. It has indeed been an enriching experience to learn this exciting biological area and the ropes of scientific research from his enthusiasm and dedication to scientific research. What I have learned from Prof. Shen will benefit my future career after my graduation and be cherished all the time in my life. I would also like to extend my sincere thanks to my TAC members and A/P Tan Shyong Wei, Kevin and A/P Markus Wenk for their excellent suggestions and continuous supports throughout all our TAC meetings. I would also like to express my deep appreciation to the following people for the materials provided for my study: Dr. Miguel A del Pozo (Centro Nacional de Investigaciones Cardiovasculares) for the Cav-1 WT and KO MEFs; Dr. Michelle M. Hill (The University of Queensland) for the PTRF WT and KO MEFs; Dr. Robert G. Parton (The University of Queensland) for the EGFP and EGFP-Cav-1 re-constituted MCF7 cells; Dr. N Mizushima (Tokyo Medical and Dental University) for the Atg5 KO MEFs, Atg5 Tet-off inducible MEFs (m5-7) with stably expressing GFP-LC3 and HeLa cells with stable expression of GFP-LC3; Dr. Peden (University of Cambridge) for the HeLa cells with stable expression of HA-VAMP7; iii Dr. D. J. Kwiatkowski (Harvard Medical School) for the TSC2 WT and TSC2 KO MEFs; Dr. T. Yoshimori (Osaka University) for the mRFP-GFP tandem fluorescencetagged LC3 construct (tfLC3) and Stawberry-Atg16L; Dr. Galli (University Denis Diderot) for the VAMP7-mRFP vector. And, it has been very fortunate of me and my honour to study in such a warm and harmonious family of our lab throughout these four years. Special thanks go out to Mr. Ong Yeong Bing and Miss Su Jin for their logistical help. You guys always ensure a superb and efficient lab environment which helps me a lot through the length of my study. And I would like to specially thank Dr. Ng Shukie for all the useful techniques I have learnt from you, and also Dr. Tan Shihao for his ever present suggestions and criticisms. All the other members of our lab have also provided most kindly help and support which have made the duration of my stay very enjoyable. I would like to express my gratitude to the following people: Dr. Zhou Jing, Dr. Cui Jianzhou, Dr. Chen Bo, Dr. Yang Naidi, Ms Mo Xiaofan and Mr. Zhang Jianbin. Also, special thanks go out to all the staffs in Saw Swee Hock School of Public Health and Department of Physiology, Yong Loo Lin School of Medicine, as well as to NUS for the Research Scholarship granted to me. Finally, I would like to extend my deep appreciation to my family members for their continuing love, understanding and support. iv List of Publications 1. Shi Y, Tan SH, Ng S, Yang ND, Zhou J, McMahon KA, del Pozo MA, Hill MM, Parton RG, Kim YS, Shen HM. Critical role of caveolin-1 and lipid rafts in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy. Autophagy. 2015 (in press) 2. Yang ND, Tan SH, Ng S, Shi Y, Zhou J, Tan K SW, Wong WS F, Shen HM. Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin. J Biol Chem. 2014 Nov 28;289(48):33425-41. 3. Cui JZ, Lu KH, Wang Y, Shi Y, Tan SH, Lee CG, Gong ZY, Shen HM. Integrated and comparative miRNA analysis of starvation-induced autophagy in mouse embryonic fibroblasts. Gene. 2015 (under revision) 4. Tan SH, Shui G, Zhou J, Shi Y, Huang J, Xia D, Wenk MR, Shen HM. Critical role of SCD1 in autophagy regulation via lipogenesis and lipid rafts-coupled AKT-FOXO1 signaling pathway. Autophagy. 2014 Feb 1;10(2):226-42. 5. Zhang Y, Yang ND, Zhou F, Shen T, Duan T, Zhou J, Shi Y, Zhu XQ, Shen HM. (-)-Epigallocatechin-3-gallate induces non-apoptotic cell death in human cancer cells via ROS-mediated lysosomal membrane permeabilization. PLoS One. 2012;7(10):e46749. Presentation at scientific conferences: 1. Shi Y, Tan SH, Ng S, Zhou J, Yang ND and Shen HM. Lipid rafts deficiency promotes autophagy and cell survival of breast cancer cells under metabolic stress."Autophagy in Stress, Development & Disease" v Gordon Research Conference, Lucca (Barga), Italy. 2014 2. Shi Y, Tan SH, Ng S, Zhou J, Yang ND and Shen HM. Lipid rafts deficiency promotes autophagy and cell survival of breast cancer cells under metabolic stress, 7th APOCB Congress and ASCB Workshops, Singapore, Singapore. 2014 3. Shi Y, Tan SH, Ng S, Zhou J, Yang ND and Shen HM. Regulatory Role of Caveolin-1 and Lipid Rafts in Lysosomal Function and Autophagy, "Autophagy: Molecular mechanism, physiology and pathology" EMBO conference. Hurtigruten MS Trollfjord, Norway. 2013 4. Shi Y, Tan SH, Ng S, Zhou J, Yang ND and Shen HM. Regulation of autophagy by lipid rafts, 3rd Xiamen winter symposium, Xiamen, China. 2012 5. Shi Y, Tan SH, Ng S, Zhou J, Yang ND and Shen HM. The novel regulatory function of Lipid raft in autophagy, YLLSOM 2th Annual Graduate Scientific Congress, Singapore, Singapore. 2012 (Best Poster Award) vi Table of Contents CAVEOLIN-1 AND LIPID RAFTS IN MODULATION OF AUTOPHAGY . i Declaration .ii Acknowledgements iii List of Publications v Summary xii List of Figure . xiv List of Abbreviations .xvii Chapter 1. 1.1. Introduction . AUTOPHAGY 1.1.1. Overview of autophagy 1.1.2. The process of autophagy 1.1.3. Autophagy machinery 1.1.4. Lysosome . 10 1.1.5. Regulatory pathways of autophagy 12 1.1.6. Biological functions of autophagy . 15 1.1.7. Implication of autophagy in human diseases . 20 1.2. LIPID RAFTS AND CAV-1 27 1.2.1 Lipid rafts 27 1.2.2 Caveolin-1 . 33 1.3. LIPID RAFTS AND CAV-1 IN AUTOPHAGY 35 1.3.1. Lipid rafts in autophagy . 35 1.3.2. Cav-1 in autophagy 38 1.4. LIPID RAFTS AND CAV-1 IN CANCER 39 1.4.1. Lipid rafts in cancer cell death and progression 39 1.4.2. Cav-1 in cancer development . 40 vii 1.5. SCOPE OF STUDY 41 Chapter 2. Materials and Methods 44 2.1. CELL LINES AND CELL CULTURE . 45 2.2. REAGENTS AND ANTIBODIES 45 2.3. MEASUREMENTS OF LYSOSOMAL FUNCTION 46 2.3.1. LysoTracker staining . 46 2.3.2. Cathepsin activity assay . 46 2.3.3. Proteolysis activity assay . 47 2.4. LIPID RAFTS DETECTION . 47 2.4.1. CTxB staining 47 2.4.2. Filipin staining . 47 2.5. CELL FRACTIONATION 48 2.5.1. Lipid rafts fractionation . 48 2.5.2. Lysosome fractionation 49 2.6. PROXIMITY LIGATION ASSAY (PLA) 49 2.7. CAV-1 IMMUNOHISTOCHEMISTRY 50 2.8. DETECTION OF CELL DEATH . 51 2.9. TRANSIENT SIRNA TRANSFECTION . 51 2.10. DNA EXTRACTION . 51 2.11. RNA EXTRACTION . 52 2.12. REVERSE TRANSCRIPTASE AND QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION . 52 2.13. PLASMIDS AND TRANSIENT TRANSFECTION 52 2.14. WESTERN BLOTTING 53 2.15. IMMUNOPRECIPITATION . 53 viii 2.16. IMAGE ANALYSIS . 54 2.17. ANALYSIS OF AUTOPHAGIC FLUX BY LC3-II LEVELS USING LYSOSOME INHIBITORS 54 2.18. ANALYSIS OF AUTOPHAGOSOME-LYSOSOME FUSION WITH MRFP- GFP-LC3 REPORTER . 55 2.19. STATISTICAL ANALYSES 56 Chapter 3. Cav-1 deficiency and lipid rafts disruption enhance autophagy at early stage via promotion of autophagosome biogenesis 57 3.1. INTRODUCTION 58 3.2. RESULTS 61 3.2.1. Cav-1 deficiency and lipid rafts disruption induces autophagy flux . 61 3.2.2. Cav-1 deficiency and lipid rafts disruption promote autophagosome formation via engaging VAMP7 73 3.3. DISCUSSION . 82 3.3.1. Autophagy induction by lipid rafts disruption . 82 3.3.2. Lipid rafts disruption promotes autophagosome formation via VAMP7 84 Chapter 4. Cav-1 deficiency and lipid rafts disruption enhance autophagy via promoting lysosomal function at late stage 86 4.1. INTRODUCTION 87 4.2. RESULTS 88 4.2.1. Cav-1 deficiency and lipid rafts disruption enhance lysosomal function via V-ATPase assembly . 88 ix 4.2.2. Cav-1 deficiency and lipid rafts disruption promote autophagosome-lysosome fusion . 100 4.3. DISCUSSION . 104 4.3.1. The regulatory role of Cav-1 and lipid rafts on lysosome . 104 4.3.2. Lipid rafts disruption enhances V-ATPase assembly 105 4.3.3. Lipid rafts disruption promotes autophagosome-lysosome fusion 106 Chapter 5. Autophagy mediated by Cav-1 deficiency and lipid rafts disruption plays a pro-survival role and supports breast cancer development 108 5.1. 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Nat Rev Mol Cell Biol 12, 21-35. 167 [...]... microdomains (defined as lipid rafts) are known to play a crucial role in the assembly of signaling molecules, proteins trafficking and the balance of membrane fluidity There are mainly two types of lipid rafts: the planar lipid rafts and Caveolae Caveolin- 1 (Cav -1) is one of the key membrane proteins in maintaining the structure and function of both types of lipid rafts Currently, the functional role and. ..6 .1 CAV -1 AND LIPID RAFTS IN EARLY STAGE OF AUTOPHAGY 12 9 6.2 CAV -1 AND LIPID RAFTS IN LATE STAGE OF AUTOPHAGY 13 2 6.3 CAV -1 AND LIPID RAFTS IN BREAST CANCER REGULATION 13 4 6.4 CONCLUSIONS AND FUTURE RESEARCH 13 6 References 14 0 xi Summary Autophagy refers to an evolutionarily conserved process in which intracellular protein aggregates and damaged organelles are engulfed in. .. inhibit mTORC1 activity and induce autophagic response (Laplante and Sabatini, 2 012 ; Rabinowitz and White, 2 010 ) What's more, there are recent reports showing a direct role of AMPK in autophagy modulation through a direct phosphorylation of ULK1 and Beclin1 (Egan et al., 2 011 ; Kim et al., 2 013 a; Kim et al., 2 011 ) Figure 1. 6 Regulatory pathways of autophagy 14 1. 1.6 Biological functions of autophagy At... Model of the regulatory roles of lipid rafts and Cav -1 in autophagy, cellular stress response and tumorigenesis 13 8 xvi List of Abbreviations 3-MA 3-methyladenine 4EBP1 factor 4E-binding protein AA amino acid AMPK adenosine monophosphate–activated protein kinase Atgs autophagy- related-genes Baf balfilomycin A1 BSA bovine serum albumin CAFs cancer-associated-fibroblasts Cav -1 Caveolin- 1 CK2 Casein kinase... of Cav -1 sensitizes the MCF7 cells to cell death induced by starvation stress 12 1 Figure 5.7 Downregulation of Cav -1 with enhanced autophagy in human breast cancer tissues 12 4 Figure 6 .1 Model of the regulatory role of lipid rafts and Cav -1 in early stage of autophagy 13 0 Figure 6.2 Model of the regulatory role of lipid rafts and Cav -1 in late stage of autophagy 13 4... mechanism of Cav -1 and lipid rafts in autophagy remain largely elusive The hypothesis is that the Cav -1 and lipid rafts modulate autophagy and via which they play important roles in cell stress responses and cancer development In order to test this hypothesis, the following investigations were performed to study: (i) the role of Cav -1 and lipid rafts in autophagy at early stage; (ii) the role of Cav -1 and lipid. .. have provided strong evidence that Cav -1 and lipid rafts are closely implicated in determining cell stress responses via regulation of autophagy Understanding the function of Cav -1 and lipid rafts in autophagy regulation expand the functional scope of Cav -1 and lipid rafts More importantly, our study provides the potential indicator for the suitability of using autophagy suppression as a therapeutic... and Mizushima, 2 014 ) The detailed steps are described below and illustrated in Figure 1. 1 (Rubinsztein et al., 2 012 ) 2 Figure 1. 1 Summary of the different stages of the autophagy process in mammalian cells (Modified based on (Rubinsztein et al., 2 012 )) 1. 1.2 .1 Induction or initiation The initiation of autophagy starts with emergence of phagophore or preautophagosomal structure (PAS), and which process... ULK1/Atg1 complex downstream of mechanistic target of rapamycin complex 1 (mTORC1) The mTORC1 plays a role in the regulation of cell growth and protein synthesis by the phosphorylation of two key translational regulators eukaryote initiation factor 4E-binding protein (4EBP1) and S6 kinase (S6K) (Jewell et al., 2 013 ; Zoncu et al., 2 011 ) mTOR inhibitors such as rapamycin are well known to induce autophagy, ... (Choi et al., 2 013 ) The detailed information of the lysosome will be discussed in the Section 1. 1.4 1. 1.3 Autophagy machinery 1. 1.3 .1 ULK1/2 complex The unc- 51- like kinase (ULK) 1/ 2 complex is responsible for the initiation step of autophagosome formation ULK1/2 complex is composed by three major components: ULK1/2, ATG13, and FIP200 (Mizushima, 2 010 ) ULK1 and ULK2 are mammalian Atg proteins which appear . 27 1. 2.2 Caveolin- 1 33 1. 3. LIPID RAFTS AND CAV -1 IN AUTOPHAGY 35 1. 3 .1. Lipid rafts in autophagy 35 1. 3.2. Cav -1 in autophagy 38 1. 4. LIPID RAFTS AND CAV -1 IN CANCER 39 1. 4 .1. Lipid rafts. 10 1. 1.5. Regulatory pathways of autophagy 12 1. 1.6. Biological functions of autophagy 15 1. 1.7. Implication of autophagy in human diseases 20 1. 2. LIPID RAFTS AND CAV -1 27 1. 2 .1 Lipid rafts. List of Figure xiv List of Abbreviations xvii Chapter 1. Introduction 1 1. 1. AUTOPHAGY 2 1. 1 .1. Overview of autophagy 2 1. 1.2. The process of autophagy 2 1. 1.3. Autophagy machinery 4 1. 1.4.