Molecular Characterization of Autophagic Responses Part A, Volume 587
1st Edition
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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
Description
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.
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
Students and entry-level scientists who are for the first time approaching the study of autophagy as well as experienced researchers
Details
- No. of pages:
- 598
- Language:
- English
- Copyright:
- © Academic Press 2017
- Published:
- 20th February 2017
- Imprint:
- Academic Press
- Hardcover ISBN:
- 9780128096758
- eBook ISBN:
- 9780128097953
Reviews
Praise for the Series:
"Should be on the shelves of all libraries in the world as a whole collection." --Chemistry in Industry
"The work most often consulted in the lab." --Enzymologia
"The Methods in Enzymology series represents the gold-standard." --Neuroscience
Ratings and Reviews
About the Serial Volume Editors

Lorenzo Galluzzi Serial Volume Editor
Lorenzo Galluzzi (born 1980) is currently Assistant Professor of Cell Biology in Radiation Oncology at the Department of Radiation Oncology of the Weill Cornell Medical College (New York, USA), and Honorary Associate Professor at the Faculty of Medicine of the Paris Descartes University (Paris, France). Prior to joining Weill Cornell Medical College (2017), Lorenzo Galluzzi was a Junior Scientist of the Research Team “Apoptosis, Cancer and Immunity” at the Cordeliers Research Center (Paris, France; 2012-2016). Lorenzo Galluzzi did his post-doctoral training at the Gustave Roussy Cancer Center (Villejuif, France; 2009-2011), after receiving his PhD from the Paris Sud University (Le Kremlin-Bicetre, France; 2005-2008). He is also Associate Director of the European Academy for Tumor Immunology (EATI), and Founding Member of the European Research Institute for Integrated Cellular Pathology (ERI-ICP). Lorenzo Galluzzi is best known for major experimental and conceptual contributions to the fields of cell death, autophagy, tumor metabolism and tumor immunology. In particular, he provided profound insights into the links between adaptive stress responses in cancer cells and the activation of a clinically relevant tumor-targeting immune response in the context of chemotherapy and radiation therapy. Lorenzo Galluzzi has published more than 350 scientific articles in international peer-reviewed journals. According to a survey published by Lab Times, he is currently the 6th and the youngest of the 30 most-cited European cell biologists (relative to the period 2007–2013). Lorenzo Galluzzi currently operates as Editor-in-Chief of three journals: OncoImmunology (which he co-founded in 2011), International Review of Cell and Molecular Biology, and Molecular and Cellular Oncology (which he co-founded in 2013). In addition, Lorenzo Galluzzi currently serves as Founding Editor for Microbial Cell and Cell Stress, and Associate Editor for Cell Death and Disease.
Affiliations and Expertise
Weill Cornell Medical College, USA

Guido Kroemer Serial Volume Editor
Guido Kroemer got his M.D. in 1985 from the University of Innsbruck, Austria, and his Ph.D. in molecular biology in 1992 from the Autonomous University of Madrid, Spain. He is currently Professor at the Faculty of Medicine of the University of Paris Descartes/Paris V, Director of the INSERM research team ‘Apoptosis, Cancer and Immunity’, Director of the Metabolomics and Cell Biology platforms of the Gustave Roussy Cancer Campus, and Practitioner at the Hôpital Européen George Pompidou (Paris, France). He is also the Director of the Paris Alliance of Cancer Research Institutes (PACRI) and the Labex 'Immuno-Oncology'. Dr. Kroemer is best known for the discoveries that mitochondrial membrane permeabilization constitutes a decisive step in regulated cell death; that autophagy is a cytoprotective mechanism with lifespan-extending effects; and that anticancer therapies are successful only if they stimulate tumour-targeting immune responses. He is currently the most-cited cell biologist in Europe (relative to the period 2007-2013), and he has received the Descartes Prize of the European Union, the Carus Medal of the Leopoldina, the Dautrebande Prize of the Belgian Royal Academy of Medicine, the Léopold Griffuel Prize of the French Association for Cancer Research, the Mitjavile prize of the French National Academy of Medicine and a European Research Council Advanced Investigator Award.
Affiliations and Expertise
INSERM Cordeliers Research Cancer Paris; Hopital Europeen Georges Pompidou; Universite Paris Descartes, France

Jose Manuel Bravo-San Pedro Serial Volume Editor
José Manuel Bravo-San Pedro graduated from the University of Extremadura (Caceres, Spain) in 2011, and now works as a post-doctoral fellow in the laboratory of Guido Kroemer. His main research interests encompass the molecular cross-talk between autophagy and regulated cell death, and the interconnections between cellular autophagic responses and organismal metabolism.
Affiliations and Expertise
Apoptosis, Cancer & Immunity Laboratory, INSERM Cordeliers Research Center, University of Paris Descartes, Paris, France