
Molecular Characterization of Autophagic Responses Part A
Description
Key Features
- 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
Readership
Table of Contents
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
Product details
- No. of pages: 598
- Language: English
- Copyright: © Academic Press 2017
- Published: February 18, 2017
- Imprint: Academic Press
- Hardcover ISBN: 9780128096758
- eBook ISBN: 9780128097953
About the Serial Volume Editors
Lorenzo Galluzzi

Affiliations and Expertise
Guido Kroemer

Affiliations and Expertise
Jose Manuel Bravo-San Pedro

Affiliations and Expertise
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