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Autophagy : lower eukaryotes and non-mammalian systems. Part A / edited by Daniel J. Klionsky. — Amsterdam : Elsevier, c2008. – (58.17435/C719/v.451) |
Contents
CONTENTS
1. Biochemical Methods to Monitor Autophagy-Related Processes in Yeast
1. Introduction
2. Assays to Monitor the Cvt Pathway
3. Assays to Monitor Pexophagy
4. Assays to Monitor Nonspecific Autophagy
5. Additional Methods
References
2. Viability Assays to Monitor Yeast Autophagy
1. Overview
2. Estimation of Yeast Viability with Phloxine B
3. Direct Measurement of Cellular Viability by the Colony-Formation Assay
References
3. The Quantitative Pho8Δ60 Assay of Nonspecific Autophagy 33
1. Overview
2. The Pho8A6O Assay
3. Interpretation of the Results
References
4. Fluorescence Microscopy-Based Assays for Monitoring Yeast Atg Protein Trafficking
1. Introduction
2. Ape1 and Atg8 Transport into the Vacuole
3. Construction of the prApel and Atg8 Fluorescent Fusions
4. Visualization of the Fluorescence Signals
5. Atg9 Trafficking and the TAKA Assay
6. Creation of the Strains for the Analysis of Atg9 Trafficking
Acknowledgments
References
5. Measuring Macroautophagy in S. cerevisiae: Autophagic Body Accumulation and Total Protein Turnover 57
1. Introduction
2. Qualitative Measurement of Macroautophagy in S. cerevisiae
3. Quantitative Measurement of Macroautophagy in .5. cerevisiae
4. Conclusion
References
6. Aminopeptidase I Enzymatic Activity
1. Introduction
2. Methods to Measure Leucine-aminopeptidase Activity
3. Concluding Remarks
References
7. Monitoring Autophagy in Yeast using FM 4-64 Fluorescence 79
1. Overview
2. Molecular Properties and Endosomal-pathway Tracking Behavior of the FM 4-64 Fluorophore
References
8. Monitoring Mitophagy in Yeast
1. Introduction
2. Visualization of Mitochondrial Sequestration within the Vacuole by Fluorescence Microscopy
3. Mitochondrially Targeted Alkaline Phosphatase
4. Degradation of Mitochondrial Proteins
5. Visualization of Mitophagy by Electron Microscopy
Acknowledgments
References
9. Monitoring Organelle Turnover in Yeast Using Fluorescent Protein Tags 109
1. Selective Organelle Degradation by Autophagy in Yeast
2. Principle of the Assay and General Considerations
3. Targeting of Fluorescent Proteins (FPs) to Different Organelles
4. Materials and Methods
5. Analysis and Typical Images of Organellar Turnover
6. Summary and Perspectives
Acknowledgments
References
10. Electron Microscopy in Yeast
1. Introduction
2. Morphological Examination of Membrane Dynamics in Autophagy and the Cvt Pathway
3. Characterization of the Autophagosome and Autophagic Body Membrane in Starving Cells
4. Subcellular Localization Analysis of ATG Proteins in Autophagy and the Cvt Pathway
5. Morphometric Analysis of Autophagy-Related Structures
Acknowledgments
References
11. Cell-Free Reconstitution of Microautophagy in Yeast
1. Introduction: Microautophagy in Yeast
2. Why Reconstitute Microautophagy In Vitro with Yeast Vacuoles?
3. Methods for Reconstituting Microautophagy with Isolated Vacuoles
References
12. Autophagy in Wine Making
1. Introduction
2. Detection of Autophagy Using Laboratory Yeast Strains Under Enological Conditions
3. Detection of Autophagy in Wine-making Using Industrial Yeast Strains 169
4. Conclusions
References
13. Purification and In Vitro Analysis of Yeast Vacuoles
1. Introduction
2. Methods
3. Discussion
Acknowledgments
References
14. Pexophagy in Honsenulo polymorpho
1. Introduction
2. H. polymorpha as a Model System for Peroxisome Degradation
3. Cultivation of H. polymorpha and Induction of Pexophagy
4. Analysis of Peroxisome Degradation
5. Concluding Remarks
Acknowledgments
References
15. Pexophagy in Pichia pastoris
1. Introduction: Use of Pichia pastoris as a Model Organism to Study Pexophagy
2. Culture Methods to Induce Micropexophagy or Macropexophagy in P. pastoris
3. Immunoblot Analysis to Monitor Pexophagy Progression
4. Microscopy Methods to Follow Pexophagy in P. pastoris
5. Concluding Remarks
Acknowledgments
References
16. Methods of Plate Pexophagy Monitoring and Positive Selection for ATG Gene Cloning in Yeasts
1. Introduction
2. Methods of Plate Assays for Peroxisomal Enzymes in Yeast Colonies
3. Enzyme Plate Assays in Selection of Yeast Mutants Defective in Pexophagy or Catabolite Repression
4. Positive Selection of Pexophagy Mutants and Cloning of ATG Genes using Allyl Alcohol as a Selective Agent
5. Concluding Remarks
Acknowledgments
References
17. Autophagy in the Filamentous Fungus Aspergillus fumigotus 241
1. Introduction
2. Analysis of Autophagy-Dependent Processes in A. fumigatus
3. Analysis of Autophagosome Accumulation
4. Conclusion
References
18. Monitoring Autophagy in the Filamentous Fungus Podospora anserina
1. Introduction
2. Podospora anserina
3. Vegetative incompatibility as an Alternative Way of Autophagy Induction
4. Autophagosome and Autophagic Body Examination
5. Phenotypic Traits of Podospora Autophagy Mutants
6. Concluding Remarks
Acknowledgments
References
19. Monitoring Autophagy in Mognoporthe oryzoe
1. Introduction
2. Targeted Gene Replacement of Autophagy-Related Genes in M. oryzae Leading to Nonpathogenicity
3. Construction of Gene Replacement Vectors
4. Complementation of the ~MgAT61 Mutant
5. Analysis of Genomic DNA
6. Plant Infection Assays
7. Electron Microscopy
8. Subcellular Localization of GFP-tagged MgATGI
9. Modified TAKA Assay in M. oryzae
10. Western Blot Analyses of M. oryzae Autophagic Proteins expressed in Pichia pastoris
11. Visualization of Autophagic Protein Interactions in M. oryzae Using Bimolecular Fluorescence Complementation
Acknowledgments
References
20. Methods for Functional Analysis of Macroautophagy in Filamentous Fungi
1. Introduction
2. Methods for the Functional Analysis of Autophagy in Filamentous Fungi
3. Concluding Remarks
Acknowledgments
References
21. Autophagy in Candida albicans
1. Introduction
2. Resistance to Nitrogen Starvation
3. Cytological Methods for Monitoring Autophagy
4. Tracking Autophagy Through Western Blot Analysis
5. Summary
Acknowledgments
References
22. Analysis of Autophagy during Infections of Cryptococcus neoformans
1. Introduction
2. Mouse Models of Infection by C. neoformans
3. Suppression Plasmids for Autophagy-Related Genes
4. Biochemical and Microscopy Methods for Study of Autophagy in C. neoformans
5. Light and Electron Microscopy to Study Autophagy During Macrophage Infection
6. Detection of Autophagy-Related Gene Products During Human Infection
7. Concluding Remarks
References
23. Autophagy and Autophagic Cell Death in Dictyostelium
1. Introduction
2. Induction of Autophagic Cell Death
3. Mutagenesis to Obtain Autophagy and Autophagic Cell Death Mutants
4. Study of Autophagy and Autophagic Cell Death Mutants
Acknowledgments
References
24. Analysis of Autophagy in the Enteric Protozoan Parasite Entamoeba
1. Introduction
2. Unique Features of Autophagy in Entamoeba
3. Analysis of Autophagy in Entamoeba
4. Conclusion
Acknowledgments
References
25. Kinetoplastida: Model Organisms for Simple Autophagic Pathways?
1. Introduction
2. Experimental Procedures to Handle the Different Species of the Order Kinetoplastida
3. Autophagy in Protozoa
4. Analysis of Autophagy in Kinetoplastida
5. Concluding Remarks
References
26. Methods to Investigate Autophagy During Starvation and Regeneration in Hydra
1. The Value of the Hydra Model System for Investigating Autophagy
2. Experimental Paradigms to Follow Autophagy in Hydra
3. Concluding Remarks
Abbreviations
Acknowledgments
References
27. Autophagy in Freshwater Planarians
1. Getting Started
2. With Just a Few Strokes of the Brush: The Essence of Planarian as Model System
3. Planarians: A New Model for the Study of Autophagy
4. What We Know About Autophagy in Planarians
5. Methods Available to Study Autophagy in Planarians
6. Methods Being Developed to Study Autophagy in Planarians
7. Concluding Remarks
Acknowledgments
References
28. Qualitative and Quantitative Characterization of Autophagy in Caenorhobditis elegons by Electron Microscopy
1. Introduction
2. The Challenge of Identifying Autophagic Structures by Electron Microscopy: Overview and General Principles
3. Caenorhabditis as an Object of Autophagy Studies by Electron Microscopy
4. Some Ultrastructural Features of Autophagy-Related Mutations
5. Conclusions and Perspectives
Acknowledgments
References
29. Monitoring the Role of Autophagy in C elegons Aging
1. Introduction to Longevity Pathways in C elegans
2. Examination of Life Span and Visual Detection of Autophagy and Autolysosome Formation
3. Conclusion
Acknowledgments
References
30. Autophagy in Caenorhabditis elegans
1. Introduction
2. Inactivation of Autophagy Genes by RNA Interference
3. Handling Mutants With a Deletion in the Autophagy Pathway
4. Monitoring Autophagy-Related Gene Activities During Development
6. Conclusions and Future Perspectives
Acknowledgments
References
31. Chimeric Fluorescent Fusion Proteins to Monitor Autophagy in Plants
1. Introduction
2. Fluorescent Proteins and Autofluorescence in Plant Cells and Organelles
3. Visual Detection of Autophagosomes and Autophagic Bodies Using Fluorescent Protein-Tagged Atg8
4. Visual Detection of Autophagic Degradation Using Fluorescent Protein-Tagged Synthetic Cargo
5. Quantification of Fluorescent Fusion Proteins After Separation by Gel Electrophoresis
Acknowledgments
References
32. Use of Protease Inhibitors for Detecting Autophagy in Plants 557
1. Introduction
2. Measurement of Protein Degradation and Intracellular Protease in BY-2 Cells
3. Detection of the Accumulation of Autolysosomes in BY-2 Cells with Neutral Red and Quinacrine
4. Staining of Autolysosomes in BY-2 Cells by the Use of Endocytosis Markers
5. Enzyme Cytochemistry for Acid Phosphatase by Light Microscopy
6. Neutral Red and LysoTracker Red Staining to Detect Autolysosomes and Cytoplasmic Inclusions in the Central Vacuole in Plant Root-Tip Cells
7. ImmunoStaining of Lysosomes/Vacuoles in Barley Root-Tip Cells
8. Electron Microscopy of Autolysosomes and Vacuoles Containing Cytoplasmic Inclusions in Plant Cells
9. Enzyme Cytochemistry for Acid Phosphatase by Electron Microscopy
References
33. Lysosomes and Autophagy in Aquatic Animals 581
1. Overview: Autophagic and Lysosomal Responses in Cell Physiology and Pathological Reactions Induced by Environmental Stress
2. Visual Detection of Autophagic Responses
3. Autophagic Protein Degradation
4. Morphometric Methods
5. Autophagy-Related Lysosomal Membrane Stability Methods
6. In vivo Neutral Red Retention Method for Lysosomal Stability (Cellular Dye Retention)
7. Concluding Remarks: Application of Lysosomal-Autophagic Reactions to Evaluation of the Health of the Environment
8. Conclusions
References
34. Autophagy in Ticks
1. Introduction
2. Rearing of the 3-Host Tick Haemaphysalis Iongicornis
3. Autophagy-Related Genes of H. Iongicomis
4. Expression Profiles of HIATG12 from Nymphal to Adult Stages
5. Detection of HIAtg Proteins in Midgut Ceils
6. Ultrastructurai Observation of Autophagosome- and Autolysosome-like Structures in Midgut Cells of Unfed Ticks
7. Conclusion
Acknowledgments
References
35. Quantitative Analysis of Autophagic Activity in Drosophilo Neural Tissues by Measuring the Turnover Rates of Pathway Substrates 639
1. Introduction
2. Detection of Insoluble Ubiquitinated Protein (IUP) Substrates
3. Sequential Detergent Fraction of Drosophila Proteins
4. Detection of Carbonlyated Protein Substrates
5. Conclusions References
36. Genetic Manipulation and Monitoring of Autophagy in Drosophilo 653
1. Introduction
2. Methods
3. Conclusions
References
37. Monitoring Autophagy in Insect Eggs
1. Introduction
2. Overview of Oogenesis in Insects
3. Methods to Study Autophagy in Insect Eggs
4. Concluding Remarks
Acknowledgments
References
38. In Vitro Methods to Monitor Autophagy in Lepidoptera
1. Background
2. Methods
Acknowledgments
References
