Molecular Characterization of Autophagic Responses Part A

Preface

• 1 Introduction
• Acknowledgments

Chapter One: Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation

• Abstract
• 1 Introduction
• 2 Live Cell Imaging
• 3 Correlative Super Resolution Imaging of Autophagosome Formation
• Acknowledgments

Chapter Two: Quantifying Autophagic Structures in Mammalian Cells Using Confocal Microscopy

• Abstract
• 1 Introduction
• 2 Detection and Quantification of Autophagic Puncta in Fixed Mammalian Cells
• 3 Quantifying Starvation-Induced ATG9 Redistribution by Indirect Immunofluorescence and Confocal Microscopy
• 4 Quantification of ATG9 Compartment/Autophagosome Contact in Live Cells
• Acknowledgments

Chapter Three: The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo

• Abstract
• 1 Introduction
• 2 Materials
• 3 Establishment of Polarized Epithelial Cell Cultures
• 4 Incorporation of DQ™-BSA Conjugates
• 5 Monitoring Autolysosome Formation
• 6 Monitoring LC3-Associated Phagolysosome Formation
• 7 Immunofluorescence Analysis
• 8 Summary
• Acknowledgments

Chapter Four: Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells

• Abstract
• 1 Introduction
• 2 Materials
• 3 Cell Culture, Treatments, and Sample Collection
• 4 Electrophoresis, Western Blot, and Data Analysis
• 5 Notes
• Acknowledgments

Chapter Five: High-Throughput Quantification of GFP-LC3⁺ Dots by Automated Fluorescence Microscopy

• Abstract
• 1 Introduction
• 2 Cell Culture
• 3 Generation of Stable GFP-LC3-Expressing Cells
• 4 Treatments
• 5 Image Analysis
• 6 Concluding Remarks
• 7 Notes
• Acknowledgments

Chapter Six: Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses

• Abstract
• 1 Introduction
• 2 Estimation of Autophagic Process Using pHlurorin-mKate2-human LC3
• 3 Concluding Remarks
• Acknowledgments

Chapter Seven: Production of Human ATG Proteins for Lipidation Assays

• Abstract
• 1 Introduction
• 2 Expression of Human LC3B, ATG7, ATG3, ATG12~ATG5, and ATG16L1
• 3 Protein Purification
• 4 Storing Purified Proteins
• 5 Concluding Remarks
• Acknowledgments

Chapter Eight: Investigating Structure and Dynamics of Atg8 Family Proteins

• Abstract
• 1 Introduction
• 2 X-Ray Crystallography
• 3 NMR Spectroscopy
• 4 MD Simulations
• 5 Applications
• 6 Future Prospects

Chapter Nine: Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins

• Abstract
• 1 Introduction
• 2 Discovering LIR-Containing Proteins and Defining LIRs
• 3 Characterization of Interactions Between Atg8/LC3/GABARAP and LIR-Containing Peptides
• 4 Summary and Future Outlook
• Acknowledgments

Chapter Ten: Assessment of Posttranslational Modifications of ATG proteins

• Abstract
• 1 Introduction
• 2 Monitoring PTMs of ATG Proteins Using Western Blotting
• 3 Monitoring PTMs of ATG Proteins Using Simple Western™ Assays
• 4 Monitoring PTMs of ATG Proteins Using Immunoprecipitation Assays
• 5 Monitoring PTMs of ATG Proteins Using Immunofluorescence Assays
• 6 Concluding Remarks
• Acknowledgments

Chapter Eleven: Tagged ATG8-Coding Constructs for the In Vitro and In Vivo Assessment of ATG4 Activity

• Abstract
• 1 Introduction
• 2 The Atg4–Atg8 System and Its Unexpected Evolutionarily Acquired Complexity
• 3 Monitoring Atg8-Like Proteins Processing in the Context of Autophagic Flux Analysis
• 4 Construction of Mammalian Expression Vectors Including Tagged Atg8 Proteins for Cleavage Assays
• 5 Transfection of Cultured Cells for the Analysis of Atg4-Like Activity
• 6 Hydrodynamic Delivery-Based Expression of Atg8-Like Tagged Constructs for the Analysis of Atg4-Like Activity in Live Animals
• 7 Concluding Remarks
• Acknowledgments

Chapter Twelve: Measurement of the Activity of the Atg4 Cysteine Proteases

• Abstract
• 1 Introduction
• 2 The Structure and Regulatory Machinery of Atg4
• 3 Overview of the Methods to Detect the Atg4 Activity In Vitro and Ex Vivo
• 4 Expression and Purification of Atg4 and Atg8 Proteins for In Vitro Assays
• 5 Measurement of Atg4 Activity In Vitro and Ex Vivo
• 6 Kinetics Analysis of Atg4 Enzymes
• 7 Summary
• Acknowledgments

Chapter Thirteen: Crystallographic Characterization of ATG Proteins and Their Interacting Partners

• Abstract
• 1 Introduction
• 2 Domain Structures of Atg Proteins in the UBL Conjugation Pathway
• 3 Proteins That Have Been Crystallized in the UBL Conjugation System in Autophagy
• 4 Crystallization of Saccharomyces cerevisiae Atg7 Alone or in Complex With Atg3 or Atg10
• 5 Crystallization of Human ATG5–ATG16L1 (1–69) E122D Disease-Associated Mutant
• Acknowledgment

Chapter Fourteen: Dynamics of Atg5–Atg12–Atg16L1 Aggregation and Deaggregation

• Abstract
• 1 Introduction
• 2 Experimental Conditions
• 3 Atg5–Atg12–Atg16L1 Reporter Systems
• 4 Conclusion

Chapter Fifteen: Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy

• Abstract
• 1 Introduction
• 2 Cell Culture Conditions
• 3 Transient Transfection of Plasmid
• 4 Autophagy Assay Conditions
• 5 Detection by Fluorescence Microscopy
• 6 Quantification of PtdIns3P Puncta Formation
• 7 Concluding Remarks
• 8 Notes
• Acknowledgments

Chapter Sixteen: Quantification of Phosphatidylinositol Phosphate Species in Purified Membranes

• Abstract
• 1 Introduction
• 2 Detection and Quantification of PI(3)P and PI(4)P in Lysosomes or Autophagosomes by PIP-Binding Proteins
• 3 Quantification of PIPs in Lysosome Preparations by RP-HPLC-MS
• 4 Results and Discussion
• Acknowledgments

Chapter Seventeen: Mass Assays to Quantify Bioactive PtdIns3P and PtdIns5P During Autophagic Responses

• Abstract
• 1 Introduction
• 2 Preparation of Recombinant Proteins
• 3 Lipid Extraction From Biological Samples
• 4 Purification of Phosphatidylinositol Monophosphates
• 5 Quantification of PtdIns3P by Mass Assay
• 6 Quantification of PtdIns5P by Mass Assay
• Acknowledgments

Chapter Eighteen: Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy

• Abstract
• 1 PI(5)P Role in Autophagy
• 2 Microscopy-Based Detection of PI(5)P
• 3 Manipulations of PI(5)P Levels to Visualize PI(5)P During Autophagy
• 4 Super-Resolution Structured Illumination Microscopy (SR-SIM) to Visualize PI(5)P During Autophagy
• 5 Concluding Remarks
• Acknowledgments

Chapter Nineteen: Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples

• Abstract
• 1 Introduction
• 2 Cryoimmobilization
• 3 Materials and Methods
• 4 Results
• 5 Discussion
• Acknowledgments

Chapter Twenty: A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells

• Abstract
• 1 Introduction
• 2 Measuring Nonselective Autophagic Sequestration of Cytosol in Cultured Cells
• 3 Concluding Remarks
• Acknowledgments

Chapter Twenty-One: In Vitro Reconstitution of Autophagosome–Lysosome Fusion

• Abstract
• 1 Introduction
• 2 SNARE Protein Purification
• 3 Protein Reconstitution
• 4 Fluorescent Measurement
• 5 Single-Vesicle Assay
• 6 Summary
• Acknowledgments

Chapter Twenty-Two: In Vitro Reconstitution of Atg8 Conjugation and Deconjugation

• Abstract
• 1 Introduction
• 2 Methods for In Vitro Atg8 Lipidation

Chapter Twenty-Three: Study of ULK1 Catalytic Activity and Its Regulation

• Abstract
• 1 Introduction
• 2 Detection of Phospho-ULK1 Variants by Immunoblotting
• 3 Analysis of ULK1 Phosphorylation by Mass Spectrometry
• 4 Analysis of ULK1 Inhibitors by In Vitro Kinase Assays
• 5 Summary
• Acknowledgments

Chapter Twenty-Four: Evaluating the mTOR Pathway in Physiological and Pharmacological Settings

• Abstract
• 1 Introduction
• 2 Functional Readouts and Inhibitors for the mTOR Pathway
• 3 Methods
• 4 Concluding Remarks
• Acknowledgments

Chapter Twenty-Five: Methods to Study the BECN1 Interactome in the Course of Autophagic Responses

• Abstract
• 1 Introduction
• 2 Materials
• 3 Methods
• Acknowledgments

Chapter Twenty-Six: In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules

• Abstract
• 1 Introduction
• 2 Purification of Recombinant VPS34 Proteins
• 3 Catalytic Assay
• 4 Binding Assay
• 5 Crystallization
• 6 Cell Assay
• 7 Conclusion
• Acknowledgments

Chapter Twenty-Seven: Methods to Study Lysosomal AMPK Activation

• Abstract
• 1 Introduction
• 2 Analysis of Lysosomal Localization of AXIN/LKB1
• 3 In Vitro Reconstitution of Lysosomal AMPK Activation
• Acknowledgment

Chapter Twenty-Eight: Allosteric Modulation of AMPK Enzymatic Activity: In Vitro Characterization

• Abstract
• 1 Introduction
• 2 Notes About Key Reagents
• 3 Assays for Measuring Allosteric Activation of AMPK
• 4 Steady-State Kinetic Analysis of AMPK Activators
• 5 Assays for Monitoring Phosphorylation at Thr172 of the α-Subunit
• 6 Activation–Protection Assay
• 7 Summary

Chapter Twenty-Nine: Assessing the Catalytic Activity of Transglutaminases in the Context of Autophagic Responses

• Abstract
• 1 Introduction
• 2 TG Transamidating Activity Assay
• 3 Analysis of TG2 Degradation During Autophagy
• 4 Analysis of TG2 Interaction With p62 During Autophagy