Molecular Characterization of Autophagic Responses Part A

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. Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation 2. Quantifying Autophagic Structures in Mammalian Cells Using Confocal Microscopy 3. The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo 4. Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells 5. High-Throughput Quantification of GFP-LC3⁺ Dots by Automated Fluorescence Microscopy 6. Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses 7. Production of Human ATG Proteins for Lipidation Assays 8. Investigating Structure and Dynamics of Atg8 Family Proteins 9. Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins 10. Assessment of Posttranslational Modifications of ATG proteins 11. Tagged ATG8-Coding Constructs for the In Vitro and In Vivo Assessment of ATG4 Activity 12. Measurement of the Activity of the Atg4 Cysteine Proteases 13. Crystallographic Characterization of ATG Proteins and Their Interacting Partners 14. Dynamics of Atg5–Atg12–Atg16L1 Aggregation and Deaggregation 15. Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy 16. Quantification of Phosphatidylinositol Phosphate Species in Purified Membranes 17. Mass Assays to Quantify Bioactive PtdIns3P and PtdIns5P During Autophagic Responses 18. Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy 19. Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples 20. A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells 21. In Vitro Reconstitution of Autophagosome–Lysosome Fusion 22. In Vitro Reconstitution of Atg8 Conjugation and Deconjugation 23. Study of ULK1 Catalytic Activity and Its Regulation 24. Evaluating the mTOR Pathway in Physiological and Pharmacological Settings 25. Methods to Study the BECN1 Interactome in the Course of Autophagic Responses 26. In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules 27. Methods to Study Lysosomal AMPK Activation 28. Allosteric Modulation of AMPK Enzymatic Activity: In Vitro Characterization 29. Assessing the Catalytic Activity of Transglutaminases in the Context of Autophagic Responses