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.
1. Cover image - p. iv 2. Title page - p. iv 3. Table of Contents - p. iv 4. Copyright - p. iv 5. Contributors - p. xv 6. Preface - p. xv 7. Chapter One: Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation - p. xxix 8. Chapter Two: Quantifying Autophagic Structures in Mammalian Cells Using Confocal Microscopy - p. 20 9. Chapter Three: The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo - p. 42 10. Chapter Four: Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells - p. 54 11. Chapter Five: High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy - p. 70 12. Chapter Six: Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses - p. 86 13. Chapter Seven: Production of Human ATG Proteins for Lipidation Assays - p. 96 14. Chapter Eight: Investigating Structure and Dynamics of Atg8 Family Proteins - p. 113 15. Chapter Nine: Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins - p. 142 16. Chapter Ten: Assessment of Posttranslational Modifications of ATG proteins - p. 169 17. Chapter Eleven: Tagged ATG8-Coding Constructs for the In Vitro and In Vivo Assessment of ATG4 Activity - p. 188 18. Chapter Twelve: Measurement of the Activity of the Atg4 Cysteine Proteases - p. 205 19. Chapter Thirteen: Crystallographic Characterization of ATG Proteins and Their Interacting Partners - p. 225 20. Chapter Fourteen: Dynamics of Atg5–Atg12–Atg16L1 Aggregation and Deaggregation - p. 246 21. Chapter Fifteen: Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy - p. 255 22. Chapter Sixteen: Quantification of Phosphatidylinositol Phosphate Species in Purified Membranes - p. 269 23. Chapter Seventeen: Mass Assays to Quantify Bioactive PtdIns3P and PtdIns5P During Autophagic Responses - p. 291 24. Chapter Eighteen: Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy - p. 310 25. Chapter Nineteen: Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples - p. 330 26. Chapter Twenty: A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells - p. 349 27. Chapter Twenty-One: In Vitro Reconstitution of Autophagosome–Lysosome Fusion - p. 364 28. Chapter Twenty-Two: In Vitro Reconstitution of Atg8 Conjugation and Deconjugation - p. 376 29. Chapter Twenty-Three: Study of ULK1 Catalytic Activity and Its Regulation - p. 390 30. Chapter Twenty-Four: Evaluating the mTOR Pathway in Physiological and Pharmacological Settings - p. 404 31. Chapter Twenty-Five: Methods to Study the BECN1 Interactome in the Course of Autophagic Responses - p. 428 32. Chapter Twenty-Six: In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules - p. 445 33. Chapter Twenty-Seven: Methods to Study Lysosomal AMPK Activation - p. 464 34. Chapter Twenty-Eight: Allosteric Modulation of AMPK Enzymatic Activity: In Vitro Characterization - p. 480 35. Chapter Twenty-Nine: Assessing the Catalytic Activity of Transglutaminases in the Context of Autophagic Responses - p. 509 36. Author Index - p. 521 37. Subject Index - p. 549
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.
1. Cover image - p. iv 2. Title page - p. iv 3. Table of Contents - p. iv 4. Copyright - p. iv 5. Contributors - p. xv 6. Preface - p. xv 7. Chapter One: Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation - p. xxix 8. Chapter Two: Quantifying Autophagic Structures in Mammalian Cells Using Confocal Microscopy - p. 20 9. Chapter Three: The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo - p. 42 10. Chapter Four: Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells - p. 54 11. Chapter Five: High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy - p. 70 12. Chapter Six: Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses - p. 86 13. Chapter Seven: Production of Human ATG Proteins for Lipidation Assays - p. 96 14. Chapter Eight: Investigating Structure and Dynamics of Atg8 Family Proteins - p. 113 15. Chapter Nine: Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins - p. 142 16. Chapter Ten: Assessment of Posttranslational Modifications of ATG proteins - p. 169 17. Chapter Eleven: Tagged ATG8-Coding Constructs for the In Vitro and In Vivo Assessment of ATG4 Activity - p. 188 18. Chapter Twelve: Measurement of the Activity of the Atg4 Cysteine Proteases - p. 205 19. Chapter Thirteen: Crystallographic Characterization of ATG Proteins and Their Interacting Partners - p. 225 20. Chapter Fourteen: Dynamics of Atg5–Atg12–Atg16L1 Aggregation and Deaggregation - p. 246 21. Chapter Fifteen: Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy - p. 255 22. Chapter Sixteen: Quantification of Phosphatidylinositol Phosphate Species in Purified Membranes - p. 269 23. Chapter Seventeen: Mass Assays to Quantify Bioactive PtdIns3P and PtdIns5P During Autophagic Responses - p. 291 24. Chapter Eighteen: Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy - p. 310 25. Chapter Nineteen: Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples - p. 330 26. Chapter Twenty: A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells - p. 349 27. Chapter Twenty-One: In Vitro Reconstitution of Autophagosome–Lysosome Fusion - p. 364 28. Chapter Twenty-Two: In Vitro Reconstitution of Atg8 Conjugation and Deconjugation - p. 376 29. Chapter Twenty-Three: Study of ULK1 Catalytic Activity and Its Regulation - p. 390 30. Chapter Twenty-Four: Evaluating the mTOR Pathway in Physiological and Pharmacological Settings - p. 404 31. Chapter Twenty-Five: Methods to Study the BECN1 Interactome in the Course of Autophagic Responses - p. 428 32. Chapter Twenty-Six: In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules - p. 445 33. Chapter Twenty-Seven: Methods to Study Lysosomal AMPK Activation - p. 464 34. Chapter Twenty-Eight: Allosteric Modulation of AMPK Enzymatic Activity: In Vitro Characterization - p. 480 35. Chapter Twenty-Nine: Assessing the Catalytic Activity of Transglutaminases in the Context of Autophagic Responses - p. 509 36. Author Index - p. 521 37. Subject Index - p. 549
