This is the companion volume to Daniel Klionsky’s Autophagy: Lower Eukaryotes, which features the basic methods in autophagy covering yeasts and alternative fungi (aspergillus, podospora, magnaporthe). Klionsky is one of the leading authorities in the field. He is the editor-in-chief of Autophagy. The November 2007 issue of Nature Reviews highlighted his article, “Autophagy: From phenomenology to molecular understanding in less than a decade. He is currently editing guidelines for the field, with 230 contributing authors, that will publish in Autophagy.
Particularly in times of stress, like starvation and disease, higher organisms have an internal mechanism in their cells for chewing up and recycling parts of themselves. The process of internal “house cleaning in the cell is called autophagy – literally self-eating. Breakthroughs in understanding the molecular basis of autophagy came after the cloning of ATG1 (autophagy-related gene 1) in yeast. (To date, 30 additional yeast genes have been identified.) These ATG genes in yeast were the stepping stones to the explosion of research into the molecular analysis of autophagy in higher eukaryotes. In the future, this research will help to design clinical approaches that can turn on autophagy and halt tumor growth.
1. 1. Introduction 2. 2. Estimation of Autophagy Induction by LC3 Puncta Formation 3. 3. The tfLC3 Assay 4. 4. Determination of LC3 Turnover by Western Blotting 5. 5. Concluding Remarks 6. Chapter 2 Methods for Monitoring Autophagy Using GFP‐LC3 Transgenic Mice 7. 1. Introduction 8. 2. Genetic Features of GFP‐LC3 Mice 9. 3. Mouse Maintenance 10. 4. Genotyping of GFP‐LC3 Mice 11. 5. Sample Preparation 12. 6. Fluorescence Microscopy 13. 7. Precautions 14. Chapter 3 Using Photoactivatable Proteins to Monitor Autophagosome Lifetime 15. 1. Introduction 16. 2. Photoactivatable Fluorescent Protein Labeling 17. 3. Experimental Example: Setting up a Sample and Optimizing Photoactivation 18. 4. Photobleaching 19. 5. Carrying Out the Photochase Assay 20. 6. Pulse‐Labeling Induced Autophagosomes 21. 7. Determining the Half‐Life of Pulse‐Labeled Autophagosomes 22. 8. Controls 23. 9. Conclusions 24. Chapter 4 Assaying of Autophagic Protein Degradation 25. 1. Introduction 26. 2. Assaying the Degradation of Long‐Lived Proteins in Cultured Cell Lines 27. 3. Assay of the Degradation of Long‐Lived Protein in Isolated Rat Hepatocytes 28. 4. Other Methods for Measuring Autophagic Flux 29. 5. Pitfalls 30. Chapter 5 Sequestration Assays for Mammalian Autophagy 31. 1. Introduction 32. 2. Membrane‐Impermeant Autophagy Probes: Introduction into Cytosol by Reversible Electropermeabilization (Electroporation) of the Plasma Membrane 33. 3. Damage‐Induced Cell Permeabilization 34. 4. Electrodisruption: A Simple Method for the Separation of Sedimentable from Soluble Cell Components 35. 5. Electroinjected Sugars as Autophagic Sequestration Probes 36. 6. Cytosolic Proteins as Autophagic Sequestration Probes 37. 7. Autophagic Fragment Generation: An Autophagic Cargo Assay Applicable to Whole Cells 38. 8. Concluding Remarks 39. Chapter 6 Assays to Assess Autophagy Induction and Fusion of Autophagic Vacuoles with a Degradative Compartment, Using Monodansylcadaverine (MDC) and DQ‐BSA 40. 1. Overview 41. 2. Assessing Autophagy Induction by Fluorescence Microscopy and Fluorometry 42. 3. Monitoring the Formation of an Autolysosome 43. 4. Concluding Remarks 44. Acknowledgments 45. Chapter 7 The GST‐BHMT Assay and Related Assays for Autophagy 46. 1. Introduction 47. 2. Measurement of Macroautophagy Using the GST‐BHMT Assay 48. 3. Measurement of Autophagy Using a Linker‐Specific Cleavage Site (LSCS) 49. 4. Measurement of Reticulophagy and Mitophagy Using LSCS‐Based Reporters 50. 5. Concluding Remarks 51. Acknowledgments 52. Chapter 8 Monitoring Starvation‐Induced Reactive Oxygen Species Formation 53. 1. Introduction 54. 2. Detection of ROS in Living Cells Using Fluorescent Dyes 55. 3. Interfering with ROS Formation: The Use of Antioxidants to Inhibit Autophagy 56. 4. Concluding Remarks 57. Acknowledgments 58. Chapter 9 Flow Cytometric Analysis of Autophagy in Living Mammalian Cells 59. 1. Introduction 60. 2. Qualitative Analysis of LC3 Levels in Cells 61. 3. Establishing Flow Cytometry and FACS Analysis to Quantify GFP‐LC3 Levels During Amino Acid Deprivation 62. 4. Quantification of Decline in GFP‐LC3 Using Flow Cytometry and FACS Analysis 63. 5. Using FACS to Quantify Autophagy in Response to Specific Treatments 64. 6. Concluding Remarks 65. Acknowledgments 66. Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells 67. 1. Introduction 68. 2. Methods 69. 3. Results and Discussion 70. 4. Concluding Remarks 71. Acknowledgments 72. Chapter 11 Monitoring Mammalian Target of Rapamycin (mTOR) Activity 73. 1. Introduction 74. 2. Methods 75. 3. Concluding Remarks 76. Acknowledgment 77. Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1 78. 1. Introduction 79. 2. Monitoring Autophagy‐Mediated Degradation of Endogenous p62/SQSTM1 80. 3. Monitoring the Autophagic Degradation of p62/SQSTM1 by Live Cell Imaging 81. 4. Concluding Remarks 82. Acknowledgments 83. Chapter 13 Cytosolic LC3 Ratio as a Quantitative Index of Macroautophagy 84. 1. Introduction 85. 2. Measurement of Proteolysis 86. 3. Measurement of the Cytosolic LC3 Ratio 87. 4. Concluding Remarks 88. Chapter 14 Method for Monitoring Pexophagy in Mammalian Cells 89. 1. Introduction 90. 2. Experimental Models for the Study of Pexophagy in Mammals 91. 3. Monitoring Degradation of Excess Peroxisomes 92. 4. Concluding Remarks 93. Acknowledgment 94. Chapter 15 Mitophagy in Mammalian Cells 95. 1. Introduction 96. 2. Reticulocyte Production and Maturation 97. 3. Methods 98. 4. Mouse Models of Mitophagy 99. 5. Conclusions 100. Acknowledgments 101. Chapter 16 Assessing Mammalian Autophagy by WIPI‐1/Atg18 Puncta Formation 102. 1. The Human WIPI Protein Family 103. 2. WIPI‐1 Puncta‐Formation Assay 104. 3. Indirect Immunofluorescence to Visualize Endogenous WIPI‐1 by Confocal Microscopy 105. 4. Direct Fluorescence of Transiently Expressed GFP‐WIPI‐1 106. 5. Generation of Stable GFP‐WIPI‐1 Cell Lines 107. 6. Live‐Cell Imaging of GFP‐WIPI‐1 108. 7. WIPI‐1 Protein‐Lipid Overlay Assay 109. 8. Concluding Remarks 110. Acknowledgments 111. Chapter 17 Correlative Light and Electron Microscopy 112. 1. Introduction 113. 2. CLEM 114. 3. Cell Culture 115. 4. Light Microscopy 116. 5. Electron Microscopy 117. 6. Results, Interpretation, and Presentation 118. 7. Tomography and CLEM 119. 8. Cryoimmunogold EM and CLEM—the Future? 120. 9. Conclusions 121. Chapter 18 Semiconductor Nanocrystals in Autophagy Research 122. 1. Introduction 123. 2. Autophagy Probing in Living Cells Using Different‐Sized Fluorescent NP 124. 3. Application of Nanotechnology‐Based Products for Other Methods in Autophagy Research. The Perspectives 125. 4. Conclusion and Future Perspectives 126. Acknowledgments 127. Chapter 19 Methods to Monitor Chaperone‐Mediated Autophagy 128. 1. Introduction 129. 2. Experimental Models for the Study of CMA 130. 3. Properties of CMA Substrates 131. 4. Methods to Measure CMA 132. 5. Measurement of Protein Degradation Rates 133. 6. Measurement of Levels of Key CMA Components 134. 7. Analysis of the Subcellular Location of CMA‐Active Lysosomes 135. 8. In Vitro Assay to Measure Translocation of CMA Substrates 136. 9. Concluding Remarks 137. Acknowledgments 138. Chapter 20 Methods to Monitor Autophagy of Salmonella enterica serovar Typhimurium 139. 1. Introduction 140. 2. Characteristics of Autophagy of S. Typhimurium 141. 3. S. Typhimurium Infection and Autophagy 142. 4. Controls for Autophagy of Bacteria 143. 5. Concluding Remarks 144. Chapter 21 Monitoring Autophagy during Mycobacterium tuberculosis Infection 145. 1. Introduction 146. 2. Methods 147. 2.2. siRNA‐mediated protein knockdown supplying the autophagic killing 148. 2.3. LysoTracker colocalization analysis to monitor the acidification of the M. tuberculosis phagosome 149. 2.4. Monitoring lysosomal protease delivery to the M. tuberculosis phagosome 150. Acknowledgments 151. Chapter 22 Streptococcus‐, Shigella‐, and Listeria‐Induced Autophagy 152. 1. Introduction 153. 2. GAS Infection and Autophagy 154. 3. Shigella Infection and Autophagy 155. 4. Listeria Infection and Autophagy 156. 5. Concluding Remarks 157. Acknowledgments 158. Chapter 23 Kinetic Analysis of Autophagosome Formation and Turnover in Primary Mouse Macrophages 159. 1. Introduction 160. 2. Monitoring Flux Through the Autophagy Pathway 161. 3. Assessing the Impact of Infection on the Autophagy Pathway 162. 4. LC3 localization in Primary Mouse Macrophages 163. 5. Isolation of Bone Marrow‐Derived Macrophages 164. 6. Conclusion 165. Chapter 24 Monitoring Macroautophagy by Major Histocompatibility Complex Class II Presentation of Targeted Antigens 166. 1. Introduction 167. 2. Antigen Presentation on MHC Class II Molecules After Macroautophagy 168. 3. Substrates of Endogenous Antigen Processing for MHC Class II Presentation via Macroautophagy 169. 4. Methods to Measure Antigen Processing for MHC Class II Presentation After Macroautophagy 170. 5. Concluding Remarks 171. Acknowledgments 172. Chapter 25 Detachment‐Induced Autophagy In Three‐Dimensional Epithelial Cell Cultures 173. 1. Introduction 174. 2. Monitoring and Manipulating Autophagy In Mammalian Epithelial Cells 175. 3. Autophagy During Lumen Formation In 3D Epithelial Cultures 176. 4. Measuring Autophagy In Traditional Anoikis Assays 177. 5. Incubation of Cells With Integrin Function‐Blocking Antibodies 178. 6. Conclusions 179. Acknowledgments 180. Chapter 26 Methods for Inducing and Monitoring Liver Autophagy Relative to Aging and Antiaging Caloric Restriction in Rats 181. 1. Overview 182. 2. Models of Caloric Restriction 183. 3. Methods to Induce and Monitor Liver Autophagy 184. 4. A Minimally Invasive Procedure for the Rapid Evaluation of the Induced Intensification of Autophagy 185. 5. Autophagic Protein Degradation in Isolated Liver Cells 186. 6. Concluding Remarks 187. Chapter 27 Physiological Autophagy in the Syrian Hamster Harderian Gland 188. 1. Introduction 189. 2. Methods to Detect Autophagy by Morphological Techniques 190. 3. Methods to Detect Autophagy by Biochemical and Molecular Biology Techniques 191. 4. Measurement of Cell Viability: Ratio of Cathepsins B:D 192. 5. Analysis of the Lysosomal Pathway 193. 6. Western Blot Analysis of Autophagy‐Related Proteins 194. 7. Methods to Detect Chaperone‐Mediated Autophagy 195. 8. Concluding Remarks 196. Acknowledgments
This is the companion volume to Daniel Klionsky’s Autophagy: Lower Eukaryotes, which features the basic methods in autophagy covering yeasts and alternative fungi (aspergillus, podospora, magnaporthe). Klionsky is one of the leading authorities in the field. He is the editor-in-chief of Autophagy. The November 2007 issue of Nature Reviews highlighted his article, “Autophagy: From phenomenology to molecular understanding in less than a decade. He is currently editing guidelines for the field, with 230 contributing authors, that will publish in Autophagy.
Particularly in times of stress, like starvation and disease, higher organisms have an internal mechanism in their cells for chewing up and recycling parts of themselves. The process of internal “house cleaning in the cell is called autophagy – literally self-eating. Breakthroughs in understanding the molecular basis of autophagy came after the cloning of ATG1 (autophagy-related gene 1) in yeast. (To date, 30 additional yeast genes have been identified.) These ATG genes in yeast were the stepping stones to the explosion of research into the molecular analysis of autophagy in higher eukaryotes. In the future, this research will help to design clinical approaches that can turn on autophagy and halt tumor growth.
1. 1. Introduction 2. 2. Estimation of Autophagy Induction by LC3 Puncta Formation 3. 3. The tfLC3 Assay 4. 4. Determination of LC3 Turnover by Western Blotting 5. 5. Concluding Remarks 6. Chapter 2 Methods for Monitoring Autophagy Using GFP‐LC3 Transgenic Mice 7. 1. Introduction 8. 2. Genetic Features of GFP‐LC3 Mice 9. 3. Mouse Maintenance 10. 4. Genotyping of GFP‐LC3 Mice 11. 5. Sample Preparation 12. 6. Fluorescence Microscopy 13. 7. Precautions 14. Chapter 3 Using Photoactivatable Proteins to Monitor Autophagosome Lifetime 15. 1. Introduction 16. 2. Photoactivatable Fluorescent Protein Labeling 17. 3. Experimental Example: Setting up a Sample and Optimizing Photoactivation 18. 4. Photobleaching 19. 5. Carrying Out the Photochase Assay 20. 6. Pulse‐Labeling Induced Autophagosomes 21. 7. Determining the Half‐Life of Pulse‐Labeled Autophagosomes 22. 8. Controls 23. 9. Conclusions 24. Chapter 4 Assaying of Autophagic Protein Degradation 25. 1. Introduction 26. 2. Assaying the Degradation of Long‐Lived Proteins in Cultured Cell Lines 27. 3. Assay of the Degradation of Long‐Lived Protein in Isolated Rat Hepatocytes 28. 4. Other Methods for Measuring Autophagic Flux 29. 5. Pitfalls 30. Chapter 5 Sequestration Assays for Mammalian Autophagy 31. 1. Introduction 32. 2. Membrane‐Impermeant Autophagy Probes: Introduction into Cytosol by Reversible Electropermeabilization (Electroporation) of the Plasma Membrane 33. 3. Damage‐Induced Cell Permeabilization 34. 4. Electrodisruption: A Simple Method for the Separation of Sedimentable from Soluble Cell Components 35. 5. Electroinjected Sugars as Autophagic Sequestration Probes 36. 6. Cytosolic Proteins as Autophagic Sequestration Probes 37. 7. Autophagic Fragment Generation: An Autophagic Cargo Assay Applicable to Whole Cells 38. 8. Concluding Remarks 39. Chapter 6 Assays to Assess Autophagy Induction and Fusion of Autophagic Vacuoles with a Degradative Compartment, Using Monodansylcadaverine (MDC) and DQ‐BSA 40. 1. Overview 41. 2. Assessing Autophagy Induction by Fluorescence Microscopy and Fluorometry 42. 3. Monitoring the Formation of an Autolysosome 43. 4. Concluding Remarks 44. Acknowledgments 45. Chapter 7 The GST‐BHMT Assay and Related Assays for Autophagy 46. 1. Introduction 47. 2. Measurement of Macroautophagy Using the GST‐BHMT Assay 48. 3. Measurement of Autophagy Using a Linker‐Specific Cleavage Site (LSCS) 49. 4. Measurement of Reticulophagy and Mitophagy Using LSCS‐Based Reporters 50. 5. Concluding Remarks 51. Acknowledgments 52. Chapter 8 Monitoring Starvation‐Induced Reactive Oxygen Species Formation 53. 1. Introduction 54. 2. Detection of ROS in Living Cells Using Fluorescent Dyes 55. 3. Interfering with ROS Formation: The Use of Antioxidants to Inhibit Autophagy 56. 4. Concluding Remarks 57. Acknowledgments 58. Chapter 9 Flow Cytometric Analysis of Autophagy in Living Mammalian Cells 59. 1. Introduction 60. 2. Qualitative Analysis of LC3 Levels in Cells 61. 3. Establishing Flow Cytometry and FACS Analysis to Quantify GFP‐LC3 Levels During Amino Acid Deprivation 62. 4. Quantification of Decline in GFP‐LC3 Using Flow Cytometry and FACS Analysis 63. 5. Using FACS to Quantify Autophagy in Response to Specific Treatments 64. 6. Concluding Remarks 65. Acknowledgments 66. Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells 67. 1. Introduction 68. 2. Methods 69. 3. Results and Discussion 70. 4. Concluding Remarks 71. Acknowledgments 72. Chapter 11 Monitoring Mammalian Target of Rapamycin (mTOR) Activity 73. 1. Introduction 74. 2. Methods 75. 3. Concluding Remarks 76. Acknowledgment 77. Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1 78. 1. Introduction 79. 2. Monitoring Autophagy‐Mediated Degradation of Endogenous p62/SQSTM1 80. 3. Monitoring the Autophagic Degradation of p62/SQSTM1 by Live Cell Imaging 81. 4. Concluding Remarks 82. Acknowledgments 83. Chapter 13 Cytosolic LC3 Ratio as a Quantitative Index of Macroautophagy 84. 1. Introduction 85. 2. Measurement of Proteolysis 86. 3. Measurement of the Cytosolic LC3 Ratio 87. 4. Concluding Remarks 88. Chapter 14 Method for Monitoring Pexophagy in Mammalian Cells 89. 1. Introduction 90. 2. Experimental Models for the Study of Pexophagy in Mammals 91. 3. Monitoring Degradation of Excess Peroxisomes 92. 4. Concluding Remarks 93. Acknowledgment 94. Chapter 15 Mitophagy in Mammalian Cells 95. 1. Introduction 96. 2. Reticulocyte Production and Maturation 97. 3. Methods 98. 4. Mouse Models of Mitophagy 99. 5. Conclusions 100. Acknowledgments 101. Chapter 16 Assessing Mammalian Autophagy by WIPI‐1/Atg18 Puncta Formation 102. 1. The Human WIPI Protein Family 103. 2. WIPI‐1 Puncta‐Formation Assay 104. 3. Indirect Immunofluorescence to Visualize Endogenous WIPI‐1 by Confocal Microscopy 105. 4. Direct Fluorescence of Transiently Expressed GFP‐WIPI‐1 106. 5. Generation of Stable GFP‐WIPI‐1 Cell Lines 107. 6. Live‐Cell Imaging of GFP‐WIPI‐1 108. 7. WIPI‐1 Protein‐Lipid Overlay Assay 109. 8. Concluding Remarks 110. Acknowledgments 111. Chapter 17 Correlative Light and Electron Microscopy 112. 1. Introduction 113. 2. CLEM 114. 3. Cell Culture 115. 4. Light Microscopy 116. 5. Electron Microscopy 117. 6. Results, Interpretation, and Presentation 118. 7. Tomography and CLEM 119. 8. Cryoimmunogold EM and CLEM—the Future? 120. 9. Conclusions 121. Chapter 18 Semiconductor Nanocrystals in Autophagy Research 122. 1. Introduction 123. 2. Autophagy Probing in Living Cells Using Different‐Sized Fluorescent NP 124. 3. Application of Nanotechnology‐Based Products for Other Methods in Autophagy Research. The Perspectives 125. 4. Conclusion and Future Perspectives 126. Acknowledgments 127. Chapter 19 Methods to Monitor Chaperone‐Mediated Autophagy 128. 1. Introduction 129. 2. Experimental Models for the Study of CMA 130. 3. Properties of CMA Substrates 131. 4. Methods to Measure CMA 132. 5. Measurement of Protein Degradation Rates 133. 6. Measurement of Levels of Key CMA Components 134. 7. Analysis of the Subcellular Location of CMA‐Active Lysosomes 135. 8. In Vitro Assay to Measure Translocation of CMA Substrates 136. 9. Concluding Remarks 137. Acknowledgments 138. Chapter 20 Methods to Monitor Autophagy of Salmonella enterica serovar Typhimurium 139. 1. Introduction 140. 2. Characteristics of Autophagy of S. Typhimurium 141. 3. S. Typhimurium Infection and Autophagy 142. 4. Controls for Autophagy of Bacteria 143. 5. Concluding Remarks 144. Chapter 21 Monitoring Autophagy during Mycobacterium tuberculosis Infection 145. 1. Introduction 146. 2. Methods 147. 2.2. siRNA‐mediated protein knockdown supplying the autophagic killing 148. 2.3. LysoTracker colocalization analysis to monitor the acidification of the M. tuberculosis phagosome 149. 2.4. Monitoring lysosomal protease delivery to the M. tuberculosis phagosome 150. Acknowledgments 151. Chapter 22 Streptococcus‐, Shigella‐, and Listeria‐Induced Autophagy 152. 1. Introduction 153. 2. GAS Infection and Autophagy 154. 3. Shigella Infection and Autophagy 155. 4. Listeria Infection and Autophagy 156. 5. Concluding Remarks 157. Acknowledgments 158. Chapter 23 Kinetic Analysis of Autophagosome Formation and Turnover in Primary Mouse Macrophages 159. 1. Introduction 160. 2. Monitoring Flux Through the Autophagy Pathway 161. 3. Assessing the Impact of Infection on the Autophagy Pathway 162. 4. LC3 localization in Primary Mouse Macrophages 163. 5. Isolation of Bone Marrow‐Derived Macrophages 164. 6. Conclusion 165. Chapter 24 Monitoring Macroautophagy by Major Histocompatibility Complex Class II Presentation of Targeted Antigens 166. 1. Introduction 167. 2. Antigen Presentation on MHC Class II Molecules After Macroautophagy 168. 3. Substrates of Endogenous Antigen Processing for MHC Class II Presentation via Macroautophagy 169. 4. Methods to Measure Antigen Processing for MHC Class II Presentation After Macroautophagy 170. 5. Concluding Remarks 171. Acknowledgments 172. Chapter 25 Detachment‐Induced Autophagy In Three‐Dimensional Epithelial Cell Cultures 173. 1. Introduction 174. 2. Monitoring and Manipulating Autophagy In Mammalian Epithelial Cells 175. 3. Autophagy During Lumen Formation In 3D Epithelial Cultures 176. 4. Measuring Autophagy In Traditional Anoikis Assays 177. 5. Incubation of Cells With Integrin Function‐Blocking Antibodies 178. 6. Conclusions 179. Acknowledgments 180. Chapter 26 Methods for Inducing and Monitoring Liver Autophagy Relative to Aging and Antiaging Caloric Restriction in Rats 181. 1. Overview 182. 2. Models of Caloric Restriction 183. 3. Methods to Induce and Monitor Liver Autophagy 184. 4. A Minimally Invasive Procedure for the Rapid Evaluation of the Induced Intensification of Autophagy 185. 5. Autophagic Protein Degradation in Isolated Liver Cells 186. 6. Concluding Remarks 187. Chapter 27 Physiological Autophagy in the Syrian Hamster Harderian Gland 188. 1. Introduction 189. 2. Methods to Detect Autophagy by Morphological Techniques 190. 3. Methods to Detect Autophagy by Biochemical and Molecular Biology Techniques 191. 4. Measurement of Cell Viability: Ratio of Cathepsins B:D 192. 5. Analysis of the Lysosomal Pathway 193. 6. Western Blot Analysis of Autophagy‐Related Proteins 194. 7. Methods to Detect Chaperone‐Mediated Autophagy 195. 8. Concluding Remarks 196. Acknowledgments
