Molecular Characterization of Autophagic Responses Part B

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