Molecular Characterization of Autophagic Responses Part A, Volume 587

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

Molecular Characterization of Autophagic Responses, Part A, presents a collection of methods for the qualitative and quantitative evaluation of virtually all the morphological, biochemical, and functional manifestations of autophagy, in vitro, ex vivo and in vivo, in organisms as distant as yeast and man.

Autophagy is an evolutionarily conserved mechanism for the lysosomal degradation of superfluous or dangerous cytoplasmic entities, and plays a critical role in the preservation of cellular and organismal homeostasis. Monitoring the biochemical processes that accompany autophagy is fundamental for understanding whether autophagic responses are efficient or dysfunctional.

• Offers a detailed overview of the protocols used to study autophagy and various aspects of autophagic responses
• Written in an accessible style by renowned experts in the field

Students and entry-level scientists who are for the first time approaching the study of autophagy as well as experienced researchers