Format:
1 Online-Ressource (xxix, 561 Seiten)
,
Illustrationen, Diagramme
Edition:
First edition
ISBN:
9780128097953
Series Statement:
Methods in Enzymology volume 587
Content:
Front Cover -- Molecular Characterization of Autophagic Responses, Part A -- Copyright -- Contents -- Contributors -- Preface -- 1. Introduction -- Acknowledgments -- References -- Chapter One: Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation -- 1. Introduction -- 2. Live Cell Imaging -- 2.1. Brief Overview of Protocol -- 3. Correlative Super Resolution Imaging of Autophagosome Formation -- 3.1. Fixation of Samples on the Microscope Stage -- 3.2. Labeling of Samples -- 3.3. Prepare the Microscope for Structured Illumination Imaging -- 3.4. Relocate Cell(s) of Interest -- 3.5. Acquire 3D Structured Illumination Raw Image Data -- 3.6. Reconstruct 3D Structured Illumination Images and Assess for Artifacts -- 3.7. Acquire Raw dSTORM Image Data -- Acknowledgments -- References -- Note added in proof -- Chapter Two: Quantifying Autophagic Structures in Mammalian Cells Using Confocal Microscopy -- 1. Introduction -- 2. Detection and Quantification of Autophagic Puncta in Fixed Mammalian Cells -- 2.1. Preparation of Coverslips for Confocal Immunofluorescence Microscopy -- 2.1.1. Cell Culture and Autophagy Induction -- 2.1.2. Permeablization and Antigen Staining -- 2.1.3. Direct Fluorescence -- 2.1.3. Confocal Imaging -- 2.2. Identifying Autophagic Puncta Using Imaris -- 2.2.1. Segmenting Cells, Cytosol, and Nuclei Using Imaris -- 3. Quantifying Starvation-Induced ATG9 Redistribution by Indirect Immunofluorescence and Confocal Microscopy -- 3.1. Background -- 3.2. Cell Culture and Indirect Immunofluorescence Labeling -- 3.2.1. Cell Culture and Induction of Autophagy -- 3.2.2. Indirect Immunofluorescence Labeling -- 3.2.3. Confocal Imaging -- 3.3. Image Analysis -- 4. Quantification of ATG9 Compartment/Autophagosome Contact in Live Cells -- 4.1. Preparation of Cell Cultures for Live Cell Confocal Microscopy
Content:
4.2. Analysis of Proximity Between mRFP-ATG9 and GFP-LC3B Structures Using Imaris -- 4.2.1. Manually Creating Cell Surfaces in Imaris -- 4.2.2. Measuring Distance Between Two Vesicle Populations Using the Distance Transformation Function -- Acknowledgments -- References -- Chapter Three: The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo -- 1. Introduction -- 2. Materials -- 3. Establishment of Polarized Epithelial Cell Cultures -- 4. Incorporation of DQ™-BSA Conjugates -- 5. Monitoring Autolysosome Formation -- 5.1. Rapamycin Stimulation -- 5.2. Serum Starvation -- 5.3. Evaluation of Autolysosome Formation -- 6. Monitoring LC3-Associated Phagolysosome Formation -- 7. Immunofluorescence Analysis -- 7.1. Labeling Endogenous LC3 -- 7.2. Confocal Imaging -- 7.3. Important Considerations in Experimental Design -- 8. Summary -- Acknowledgments -- References -- Chapter Four: Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells -- 1. Introduction -- 2. Materials -- 2.1. Cell Lines -- 2.2. Reagents -- 2.3. Antibodies -- 2.4. Solutions -- 2.5. Equipment -- 2.6. Labware -- 3. Cell Culture, Treatments, and Sample Collection -- 3.1. Cell Culture -- 3.2. Treatments -- 3.3. Sample Preparation -- 4. Electrophoresis, Western Blot, and Data Analysis -- 4.1. Electrophoresis -- 4.2. Western Blotting -- 4.3. Interpretation -- 5. Notes -- Acknowledgments -- References -- Chapter Five: High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy -- 1. Introduction -- 2. Cell Culture -- 3. Generation of Stable GFP-LC3-Expressing Cells -- 4. Treatments -- 5. Image Analysis -- 6. Concluding Remarks -- 7. Notes -- Acknowledgments -- References -- Chapter Six: Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses -- 1. Introduction -- 2. Estimation of Autophagic Process Using pHlurorin-mKate2-human LC3
Content:
2.1. Plasmids for an Expression of pHlurorin-mKate2-hLC3 and Its Negative Control -- 2.2. Monitoring Autophagic Responses Using pHluorin-mKate2-hLC3 in Mammalian Cells -- 2.3. Interpretation of the Results -- 3. Concluding Remarks -- Acknowledgments -- References -- Chapter Seven: Production of Human ATG Proteins for Lipidation Assays -- 1. Introduction -- 2. Expression of Human LC3B, ATG7, ATG3, ATG12~ATG5, and ATG16L1 -- 2.1. General Overview of Protein Expression and Purification -- 2.2. Expression Constructs -- 2.3. Protein Expression -- 2.3.1. Expression in E. coli -- 2.3.2. Expression in Insect Cells -- 3. Protein Purification -- 3.1. Preparation of Cell Lysate -- 3.2. Glutathione-Affinity Chromatography for LC3B, ATG7, ATG3, and ATG12~ATG5-ATG16L1 -- 3.3. Protease-Mediated Elution From Beads for Purification of LC3B, ATG3, and ATG7 -- 3.4. Glutathione-Mediated ATG12~ATG5-ATG16L1 Elution From GS4B -- 3.5. Nickel-Affinity Chromatography, Imidazole-Mediated Elution From Nickel Beads, and TEV Cleavage to Obtain ATG12 (52-1 ... -- 3.6. Ion Exchange Chromatography on ATG3 and ATG12 (52-140)~ATG5-ATG16L1 (1-69) -- 3.7. Gel Filtration Chromatography for LC3B, ATG7, ATG3, and ATG12~ATG5-ATG16L1 -- 4. Storing Purified Proteins -- 5. Concluding Remarks -- Acknowledgments -- References -- Chapter Eight: Investigating Structure and Dynamics of Atg8 Family Proteins -- 1. Introduction -- 2. X-Ray Crystallography -- 2.1. Basics -- 2.2. Structure Determination -- 2.3. Practical Notes -- 3. NMR Spectroscopy -- 3.1. Basics -- 3.2. Structure Determination -- 3.3. Investigating Dynamics -- 3.4. Mapping Interactions -- 4. MD Simulations -- 4.1. Linking Theory and Experiments -- 4.2. Force Fields -- 4.3. Numerical Integration of Newton´s Second Law of Motion -- 4.4. Enhanced Sampling Techniques in MD Simulations -- 5. Applications
Content:
5.1. Determining Structures of Nonlipidated Atg8 Proteins -- 5.2. Investigating Lipidated Atg8 Proteins -- 5.3. Assessing Dynamic Properties -- 6. Future Prospects -- References -- Chapter Nine: Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins -- 1. Introduction -- 2. Discovering LIR-Containing Proteins and Defining LIRs -- 2.1. Identification of Atg8/LC3/GABARAP-Interacting Proteins by Yeast-Two-Hybrid Screening -- 2.1.1. Preparation of Yeast for Transformation -- 2.1.2. Transformation of Yeast With the Bait Construct -- 2.1.3. Testing Expression of the Bait in Yeast Culture -- 2.1.4. Transformation of Yeast With the cDNA Library -- 2.1.5. Identification of Colonies Positive for the Bait:Prey Interaction by β-Galactosidase Assay -- 2.1.6. Determination of Prey Identity -- 2.2. Use of GST Pulldown Assay to Test for Atg8/LC3/GABARAP Binding -- 2.2.1. Preparation of Lysates From Bacteria Expressing GST-Atg8/LC3/GABARAP -- 2.2.2. Purification and Immobilization of GST-Atg8/LC3/GABARAP Proteins on Glutathione-Sepharose Beads -- 2.2.3. Testing the Amount of Immobilized GST-Atg8/LC3/GABARAP on the Beads -- 2.2.4. In Vitro Translation of a Target Protein -- 2.2.5. Preclearing of In Vitro-Translated Target Protein -- 2.2.6. GST Pulldown, Gel Electrophoresis, and Visualization of Binding -- 2.3. Use of Peptide Arrays for Identification and Narrowing of the LIR -- 2.3.1. Synthesis of Peptide Arrays -- 2.3.2. Production and Purification of GST-Atg8/LC3/GABARAPs -- 2.3.3. Peptide Overlay Assay -- 3. Characterization of Interactions Between Atg8/LC3/GABARAP and LIR-Containing Peptides -- 3.1. Expression and Purification of LIR-Containing Peptides -- 3.2. Determination of Affinity and Driving Forces of the Interaction by ITC -- 4. Summary and Future Outlook -- Acknowledgments -- References
Content:
Chapter Ten: Assessment of Posttranslational Modifications of ATG proteins -- 1. Introduction -- 2. Monitoring PTMs of ATG Proteins Using Western Blotting -- 2.1. Overview -- 2.2. Protocol -- 3. Monitoring PTMs of ATG Proteins Using Simple Western™ Assays -- 3.1. Overview -- 3.2. Protocol -- 4. Monitoring PTMs of ATG Proteins Using Immunoprecipitation Assays -- 4.1. Overview -- 4.2. Protocol -- 5. Monitoring PTMs of ATG Proteins Using Immunofluorescence Assays -- 5.1. Overview -- 5.2. Protocol -- 6. Concluding Remarks -- Acknowledgments -- References -- Chapter Eleven: Tagged ATG8-Coding Constructs for the In Vitro and In Vivo Assessment of ATG4 Activity -- 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 A ... -- 7. Concluding Remarks -- Acknowledgments -- References -- Chapter Twelve: Measurement of the Activity of the Atg4 Cysteine Proteases -- 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 -- 4.1. Construct Design for Recombinant Atg4s and Atg8s -- 4.1.1. Rationale -- 4.1.2. Methods -- 4.2. Protein Expression and Purification -- 4.2.1. Methods -- 4.3. Design, Expression, and Purification of FRET Substrates -- 4.3.1. Rationale -- 4.3.2. Methods -- 5. Measurement of Atg4 Activity In Vitro and Ex Vivo
Content:
5.1. Gel-Based Assay Under in vitro Conditions
In:
Part A
Additional Edition:
ISBN 9780128096758
Additional Edition:
Print version Galluzzi, Lorenzo Molecular Characterization of Autophagic Responses Part A Saint Louis : Elsevier Science,c2017 ISBN 9780128096758
Language:
English
Author information:
Kroemer, Guido 1961-
Author information:
Galluzzi, Lorenzo 1980-
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