Molecular Characterization of Autophagic Responses Part B - 1st Edition - ISBN: 9780128096741, 9780128097946

Molecular Characterization of Autophagic Responses Part B, Volume 588

1st Edition

Serial Volume Editors: Lorenzo Galluzzi Guido Kroemer Jose Manuel Bravo-San Pedro
eBook ISBN: 9780128097946
Hardcover ISBN: 9780128096741
Imprint: Academic Press
Published Date: 22nd February 2017
Page Count: 608
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Table of Contents

Chapter One: Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux

  • Abstract
  • 1 Introduction
  • 2 The Rluc-LC3 Assay
  • 3 Designing the Rluc-LC3wt and Rluc-LC3G120A Fusion Proteins
  • 4 Establishing Rluc-LC3wt- and Rluc-LC3G120A-Expressing Cells
  • 5 The Rluc-LC3 Assay Performed on Cell Lysates
  • 6 The Rluc-LC3 Assay Performed on Live Cells
  • Acknowledgments

Chapter Two: Measurement of Autolysosomal pH by Dual-Wavelength Ratio Imaging

  • Abstract
  • 1 Introduction
  • 2 Cell Preparation
  • 3 Equipment Setup and Software Requirements
  • 4 Image Acquisition
  • 5 In Situ pH Calibration
  • 6 Image Analysis and Determination of Autolysosomal pH
  • 7 Conclusion
  • Acknowledgments

Chapter Three: Long-Lived Protein Degradation During Autophagy

  • Abstract
  • 1 Introduction
  • 2 Proteolysis
  • 3 Other Methods for Measuring Autophagic Flux
  • 4 Conclusion
  • Acknowledgments

Chapter Four: Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging

  • Abstract
  • 1 Introduction
  • 2 Materials, Equipment, and Solutions
  • 3 Fundamentals: AHA Labeling Combined With the iTRAQ Approach for Identification of De Novo Protein Synthesis
  • 4 Protocol
  • 5 Limitations

Chapter Five: Methods to Monitor and Manipulate TFEB Activity During Autophagy

  • Abstract
  • 1 Introduction
  • 2 Methods to Monitor TFEB/TFE3 Activation
  • Acknowledgments

Chapter Six: Application of CRISPR/Cas9 to Autophagy Research

  • Abstract
  • 1 Introduction
  • 2 Establishment of a Constitutively Active CRISPR/Cas9 System for Deletion of Autophagy in Human and Murine Cells
  • 3 Regulated Disruption of Autophagy Using Tetracycline-Inducible CRISPR/Cas9 Systems
  • 4 Discussion
  • Acknowledgments

Chapter Seven: A Molecular Reporter for Monitoring Autophagic Flux in Nervous System In Vivo

  • Abstract
  • 1 Introduction
  • 2 Assessing Autophagy Flux In Vitro
  • 3 In Vivo Measurements of Autophagic Flux
  • 4 Pharmacological Induction of Autophagy
  • 5 Tissue Processing and Histology
  • 6 Quantification of Fluorescent Puncta
  • 7 Ex Vivo Analysis of LC3 Vesicle Trafficking
  • 8 Concluding Remarks
  • Acknowledgments

Chapter Eight: Magnetic Resonance Spectroscopy to Study Glycolytic Metabolism During Autophagy

  • Abstract
  • 1 Introduction
  • 2 Measurement of Intracellular Lactate and Glucose Levels in Autophagic Cells or Tumor Extracts
  • 3 Measurements of the Rate of Lactate Secretion and Glucose Uptake in Cultured Cells
  • 4 Hyperpolarization Methods for Dissolution DNP Using Pyruvic Acid
  • 5 Discussion
  • Acknowledgments

Chapter Nine: Assessment of Glycolytic Flux and Mitochondrial Respiration in the Course of Autophagic Responses

  • Abstract
  • 1 Introduction
  • 2 Cell Culture and Treatments
  • 3 Common Procedures
  • 4 Assessment of Glycolytic Flux: Principles
  • 5 Assessment of Glycolytic Flux: Protocol
  • 6 Assessment of Mitochondrial Respiration: Principles
  • 7 Assessment of Mitochondrial Respiration: Protocol
  • 8 Calibration and Run
  • 9 Concluding Remarks
  • 10 Notes
  • Acknowledgments

Chapter Ten: Methods to Assess Mitochondrial Morphology in Mammalian Cells Mounting Autophagic or Mitophagic Responses

  • Abstract
  • 1 Introduction
  • 2 High-Resolution Imaging of Mitochondria in Live Cells Based on Fluorescent Protein Variants
  • 3 High-Resolution Imaging of Mitochondriain Live Cells With Mitochondrial-Specific Fluorescent Dyes
  • 4 Analysis of the Results
  • 5 Assessment of Mitochondrial Morphology Using Electron Microscopy
  • 6 Summary
  • Acknowledgments

Chapter Eleven: Monitoring Mitophagy in Mammalian Cells

  • Abstract
  • 1 Introduction
  • 2 Transmission Electron Microscopy
  • 3 Western Blotting Analysis of Mitophagy
  • 4 Detected Mitophagy by Analysis Mitochondrial Mass
  • 5 Fluorescence Probes
  • 6 Mitophagy Mouse Models
  • 7 MAM Assay
  • 8 Mitophagy Inducers and Inhibitors
  • 9 Future Perspectives
  • Acknowledgments

Chapter Twelve: Cytofluorometric Assessment of Mitophagic Flux in Mammalian Cells and Tissues

  • Abstract
  • 1 Introduction
  • 2 Mitophagy Evaluation in Cell Lines
  • 3 Mitophagy Evaluation in Tissue
  • 4 Assessment of Mitophagic Flux
  • 5 Summary
  • Acknowledgments

Chapter Thirteen: Automated Analysis of Fluorescence Colocalization: Application to Mitophagy

  • Abstract
  • 1 Introduction
  • 2 Cell Culture and Treatments
  • 3 Acquisition Procedure
  • 4 Assessment of Colocalization: Principles
  • 5 The R Environment
  • 6 Assessment of Colocalization: Protocol
  • 7 Concluding Remarks
  • 8 Notes
  • Acknowledgments

Chapter Fourteen: Assays to Monitor Lysophagy

  • Abstract
  • 1 Introduction
  • 2 Known Experimental Triggers for Lysophagy
  • 3 Experimental Protocol for Triggering Lysophagy With Light
  • 4 Detection of Lysophagy
  • 5 Conclusions

Chapter Fifteen: Kinetics of Protein Aggregates Disposal by Aggrephagy

  • Abstract
  • 1 Introduction
  • 2 Experimental Models of Protein Inclusions and Their Characteristics
  • 3 Aggrephagy: Selective Clearance of Protein Aggregates
  • 4 Methods to Measure Aggrephagy
  • 5 Modulation of Autophagic Responses
  • 6 Microscopic Analysis of Protein Inclusion Clearance by Aggrephagy
  • 7 Biochemical Analysis of Protein Inclusion Turnover by Aggrephagy
  • 8 Concluding Remarks
  • Acknowledgments

Chapter Sixteen: Methods to Study Chaperone-Mediated Autophagy

  • Abstract
  • 1 Introduction
  • 2 Methods to Test a Protein as Possible CMA Substrate
  • 3 Methods to Assay CMA Activity
  • 4 Concluding Remarks
  • Acknowledgments

Chapter Seventeen: Quantitative Assay of Macroautophagy Using Pho8△60 Assay and GFP-Cleavage Assay in Yeast

  • Abstract
  • 1 Introduction to Pho8△60 Assay
  • 2 Standard Method of Pho8△60 Assay
  • 3 Genome Wide Pho8△60 Assay
  • 4 GFP-Cleavage Assay for Autophagy

Chapter Eighteen: Monitoring the Formation of Autophagosomal Precursor Structures in Yeast Saccharomyces cerevisiae

  • Abstract
  • 1 Introduction
  • 2 Standard Procedures
  • 3 Recruitment of Atg Machinery to the PAS
  • 4 Localization of Atg Proteins on Forming Autophagosomes
  • 5 Bimolecular Fluorescence Complementation (BiFC)
  • 6 Coimmunoprecipitation (Co-IP) Experiments
  • 7 Concluding Remarks
  • Acknowledgments

Chapter Nineteen: Methods to Assess Autophagy and Chronological Aging in Yeast

  • Abstract
  • 1 Introduction
  • 2 Autophagy and Longevity: An Intimate Connection
  • 3 Methods to Assess CLS in Yeast
  • 4 Methods to Measure Nonselective Autophagy in Yeast
  • Acknowledgments

Chapter Twenty: Methods to Measure Lipophagy in Yeast

  • Abstract
  • 1 Introduction
  • 2 Induction of Autophagy by Starvation
  • 3 Visualization of LDs and Vacuoles In Vivo
  • 4 Biochemical Assays to Monitor Lipophagy in Yeast
  • 5 Isolation of Vacuoles From Yeast for Enzymatic Analyses
  • 6 Electron Microscopy
  • 7 Summary
  • Acknowledgments

Chapter Twenty-One: Assays to Monitor Pexophagy in Yeast

  • Abstract
  • 1 Introduction
  • 2 Assays to Monitor Pexophagy
  • 3 Concluding Remarks
  • Acknowledgments

Chapter Twenty-Two: Monitoring Autophagic Responses in Caenorhabditis elegans

  • Abstract
  • 1 Introduction
  • 2 Reporters for Autophagy
  • 3 Autophagy Induction and Suppression
  • 4 Experimental Considerations
  • 5 Experimental Procedures
  • 6 Concluding Remarks
  • Acknowledgments

Chapter Twenty-Three: Characterization of Autophagic Responses in Drosophila melanogaster

  • Abstract
  • 1 Introduction
  • 2 Staging of Animals and Tissue Dissection
  • 3 Morphological Monitoring of Autophagy by Transmission Electron Microscopy
  • 4 Analysis of Autophagy Markers In Vivo
  • 5 Biochemical Methods
  • 6 Genetic Analysis
  • Acknowledgments

Chapter Twenty-Four: Methods to Study Autophagy in Zebrafish

  • Abstract
  • 1 Introduction
  • 2 Monitoring Autophagy in Zebrafish
  • 3 Modulation of Autophagic Responses in Zebrafish
  • 4 Experimental Procedures
  • 5 Concluding Remarks
  • Acknowledgments

Chapter Twenty-Five: Biochemical Methods to Monitor Autophagic Responses in Plants

  • Abstract
  • 1 Introduction
  • 2 Biochemical Assays for Autophagy Monitoring
  • Acknowledgments

Chapter Twenty-Six: Using Photoconvertible and Extractable Fluorescent Proteins to Study Autophagy in Plants

  • Abstract
  • 1 Introduction
  • 2 Expression of KikGR Fusion Proteins in Tobacco BY-2 Cells
  • 3 Photoconversion of KikGR and mKikGR Fusion Proteins in Transformed Tobacco Cells
  • 4 Detection of Photoconverted and Nonconverted KikGR and mKikGR Fusion Proteins After Separation of Proteins by SDS-PAGE
  • 5 Detection of Photoconverted and Nonconverted KikGR and mKikGR Fusion Proteins Using a Microscope
  • Acknowledgments

Description

Molecular Characterization of Autophagic Responses, Part B 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:
608
Copyright:
© Academic Press 2017
Published:
Imprint:
Academic Press
eBook ISBN:
9780128097946
Hardcover ISBN:
9780128096741

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 received his Ph.D. in 2008 from the University of Paris Sud/Paris XI (France), and now works as a research manager in the laboratory of Guido Kroemer. He is particularly fascinated by several aspects of mitochondrial cell death, autophagy, cancer cell metabolism and tumour immunology. He has published more than 270 articles in peer-reviewed scientific journals, and is currently the 6th and youngest of the 30 most-cited European cell biologists (relative to the period 2007-2013).

Affiliations and Expertise

Department of Radiation Oncology, Weill Cornell Medical College, NY, 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