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Mitochondrial Function
1st Edition - November 10, 2014
Editors: Anne Murphy, David Chan
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 0 1 4 1 5 - 8
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 0 1 6 1 5 - 2
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. Methods to assess mitochondrial… Read more
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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. Methods to assess mitochondrial function is of great interest to neuroscientists studying chronic forms of neurodegeneration, including Parkinson's, Alzheimer's, ALS, Huntington's and other triplet repeat diseases, but also to those working on acute conditions such as stroke and traumatic brain injury. This volume covers research methods on how to assess the life cycle of mitochondria including trafficking, fusion, fission, and degradation. Multiple perspectives on the complex and difficult problem of measurement of mitochondrial reactive oxygen species production with fluorescent indicators and techniques ranging in scope from measurements on isolated mitochondria to non-invasive imaging of metabolic function.
- Continues the legacy of this premier serial with quality chapters authored by leaders in the field
- Covers research methods in biomineralization science
- Provides invaluable details on state-of-the-art methods to assess a broad array of mitochondrial functions
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.
- List of Videos
- Series Page
- Preface
- Chapter One. High-Content Functional Genomic Screening to Identify Novel Regulators of the PINK1–Parkin Pathway
- Abstract
- 1 Introduction and Theory
- 2 General Screen Design Strategy
- 3 RNAi Screen for Genes Involved Required for PARK2 Translocation
- 4 Equipment
- 5 Materials
- 6 Genome Screen Protocol
- Acknowledgments
- References
- Chapter Two. Measurement of Mitochondrial Turnover and Life Cycle Using MitoTimer
- Abstract
- 1 Introduction
- 2 Expressing MitoTimer in Cells
- 3 Imaging MitoTimer
- 4 Interpreting MitoTimer Readout
- 5 Summary
- References
- Chapter Three. Monitoring Mitophagy in Mammalian Cells
- Abstract
- 1 Introduction
- 2 Transmission Electron Microscopy
- 3 Western Blot Analysis of Mitophagy
- 4 Fluorescence Methods for Analyzing Mitophagy
- 5 Analyzing Mitophagy Using Mitochondria Mass
- 6 Mitochondria-Targeting Probes for Mitophagy Assay
- 7 Mitophagy Inducer and Inhibitors
- 8 Future Perspectives
- Acknowledgments
- References
- Chapter Four. Photoactivatable Green Fluorescent Protein-Based Visualization and Quantification of Mitochondrial Fusion and Mitochondrial Network Complexity in Living Cells
- Abstract
- 1 Introduction
- 2 Visualization and Quantification of Mitochondrial Fusion in Living Cells
- 3 Estimation of Relative Size of Mitochondrial Units and Mitochondrial Network Complexity
- 4 Time-Lapse Imaging of Mitochondria in Living Cells: General Considerations
- 5 Concluding Remarks: Other Potential Applications of Mito-PAGFP
- Acknowledgments
- References
- Chapter Five. Characterization of Mitochondrial Transport in Neurons
- Abstract
- Abbreviation
- 1 Introduction
- 2 Analyses of Mitochondrial Transport in Embryonic Neuron Cultures
- 3 Analyses of Mitochondrial Transport in Adult Neuron Cultures
- 4 Characterization of Mitochondrial Transport at Synapses
- 5 Summary
- Acknowledgments
- References
- Chapter Six. Imaging of Mitochondrial Dynamics in Motor and Sensory Axons of Living Mice
- Abstract
- 1 Introduction
- 2 Protocols to Image Mitochondrial Transport in the Peripheral Nerves of Living Mice
- 3 Imaging of Mitochondrial Dynamics in the Peripheral Nerves of Living Mice
- 4 Conclusions
- Acknowledgments
- References
- Chapter Seven. Analyzing Mitochondrial Dynamics in Mouse Organotypic Slice Cultures
- Abstract
- 1 Parasagittal Slice Cultures of the Basal Ganglia
- 2 Cerebellar Slice Cultures
- 3 Monitoring Mitochondrial Dynamics in Slice Cultures
- References
- Chapter Eight. Analysis of Mitochondrial Traffic in Drosophila
- Abstract
- 1 Introduction
- 2 Dissection Methods for Drosophila Larvae
- 3 Imaging
- 4 Analysis
- 5 Conclusions
- Acknowledgment
- References
- Chapter Nine. In Vivo Imaging of Mitochondria in Intact Zebrafish Larvae
- Abstract
- 1 Introduction
- 2 Preparation of Transgenic MitoFish for Live Imaging
- 3 Visualizing Axonal Transport of Mitochondria in Zebrafish Sensory Neurons
- 4 Processing and Quantification of Imaging Files
- 5 Conclusions
- Acknowledgments
- References
- Chapter Ten. The Use of miniSOG in the Localization of Mitochondrial Proteins
- Abstract
- 1 Introduction
- 2 Requirements for CLEM Labeling
- 3 miniSOG Features
- 4 Resolution
- 5 Photooxidation Protocol for a Monolayer of Cultured Cells
- 6 Photooxidation Protocol for Tissues
- 7 Example of miniSOG Use with MCU
- 8 Conclusions and Future Work
- Acknowledgments
- References
- Chapter Eleven. Assessing the Function of Mitochondria-Associated ER Membranes
- Abstract
- 1 Introduction
- 2 Isolation of MAM
- 3 Assaying MAM Activity
- References
- Chapter Twelve. Measurement of ROS Homeostasis in Isolated Mitochondria
- Abstract
- 1 Importance of Quantifying Mitochondrial ROS Homeostasis
- 2 Detection of Mitochondrial ROS Formation
- 3 Mitochondrial Elimination of H2O2
- 4 Methods for the Detection of Oxidative Stress
- Acknowledgments
- References
- Chapter Thirteen. Use of Potentiometric Fluorophores in the Measurement of Mitochondrial Reactive Oxygen Species
- Abstract
- 1 Introduction
- 2 ROS Detection Using MitoSOX—Subcellular Localization and Fluorescence Yield
- 3 Validating MitoSOX Using a Negative Control
- 4 Choosing a Correct MitoSOX Loading Paradigm—Additional Considerations
- 5 Is MitoSOX Imaging Useful?
- 6 Preparation of Primary Rat Cortical Neurons for Imaging
- 7 Optimizing MitoSOX Concentration and Establishing Mitochondrial Localization
- 8 Imaging Using MitoSOX
- Acknowledgment
- References
- Chapter Fourteen. Spatial, Temporal, and Quantitative Manipulation of Intracellular Hydrogen Peroxide in Cultured Cells
- Abstract
- 1 Introduction
- 2 Production of H2O2 Using DAAO
- 3 Two-Photon Fluorescence Imaging of H2O2
- 4 Summary
- Acknowledgments
- References
- Chapter Fifteen. Biochemical and Biophysical Methods for Studying Mitochondrial Iron Metabolism
- Abstract
- 1 Introduction
- 2 Measurement of Total Iron Concentration
- 3 In Situ Analysis of Iron in the Mitochondria
- 4 Biophysical Methods for Studying Iron in Isolated Mitochondria
- 5 Conclusions
- Acknowledgments
- References
- Chapter Sixteen. Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data
- Abstract
- 1 Introduction
- 2 The Oxygen Consumption Rate
- 3 The Extracellular Acidification Rate
- Summary
- Acknowledgments
- References
- Chapter Seventeen. Imaging Changes in the Cytosolic ATP-to-ADP Ratio
- Abstract
- 1 Introduction
- 2 Methods
- 3 Expected Results
- 4 Summary
- References
- Chapter Eighteen. The Use of Mitochondria-Targeted Endonucleases to Manipulate mtDNA
- Abstract
- 1 Mitochondrial DNA
- 2 Mitochondria-Targeted Restriction Nucleases to Cleave mtDNA and Model OXPHOS Diseases
- 3 mtDNA Heteroplasmy and Approaches to Alter the Balance Between Wild-Type and Mutant mtDNA
- 4 mtDNA Heteroplasmy Shift Using Restriction Endonucleases
- 5 Designer Endonucleases for the Modulation of mtDNA Heteroplasmy
- 6 mtDNA Heteroplasmy Shift Using Zinc-Finger Nucleases
- 7 Heteroplasmy Shift Using TAL Effector Nucleases
- 8 Single-Strand Annealing Assay to Analyze the Efficacy and Specificity of Designer Nuclease
- 9 The Use of Cybrid Cells to Test Approaches to Change Mitochondrial DNA Heteroplasmy
- 10 Immunodetection and Mitochondrial Localization in Cells and Tissues
- 11 Changing mtDNA Heteroplasmy in Cultured Cells with mito-TALENs
- 12 Evaluation of the mtDNA Content
- 13 Future Perspectives
- Acknowledgments
- References
- Chapter Nineteen. Induced Pluripotent Stem Cell-Derived Models for mtDNA Diseases
- Abstract
- 1 Introduction
- 2 Induced Pluripotent Stem Cells
- 3 Mitochondrial Disease
- 4 Conclusion
- 5 Generation of iPSCs from mtDNA Disease Patients
- Acknowledgment
- References
- Chapter Twenty. The Use of 18F-BCPP-EF as a PET Probe for Complex I Activity in the Brain
- Abstract
- 1 Introduction
- 2 Design and Assessment of Candidate Compounds for MC-I Imaging Probes
- 3 Application for Stroke/Ischemic Damage Imaging
- 4 Application for Imaging of Aging Effects on MC-I Activity
- 5 Conclusion
- References
- Chapter Twenty-One. MR OEF Imaging in MELAS
- Abstract
- 1 Introduction of Magnetic Resonance Imaging Techniques
- 2 Significance of OEF Imaging in MELAS
- 3 Cerebral OEF Changes in Our Study of MELAS
- References
- Author Index
- Subject Index
- Color Plate
- No. of pages: 506
- Language: English
- Published: November 10, 2014
- Imprint: Academic Press
- Hardback ISBN: 9780128014158
- eBook ISBN: 9780128016152
AM
Anne Murphy
Anne N. Murphy, Ph.D. is currently an Associate Professor in the Department of Pharmacology within the medical school at the University of California, San Diego. She obtained her PhD from The George Washington University School of Medicine and Health Sciences in Biochemistry and Molecular Biology. Her post-doctoral studies were conducted at the Johns Hopkins Hospital and at The National Cancer Institute. Her career in both academic and biotechnology settings has focussed on mitochondrial bioenergetic function and the discovery of mitochondrial-targeted therapeutic strategies. She has particular interest in how mitochondrial metabolite and ion transport controls cell metabolism.
Affiliations and expertise
Department of Pharmacology, University of California, USADC
David Chan
David Chan is currently Professor of Biology at the California Institute of Technology. He graduated from Harvard College with a degree in Biochemical Sciences. He then joined the MD-PhD program at Harvard Medical School, completing his graduate studies with Philip Leder. His postdoctoral training was with Peter Kim at the Whitehead Institute for Biomedical Research at MIT.
His lab’s main interest is to understand the role of mitochondrial dynamics in cellular function and human physiology. Mitochondria are dynamic organelles that have many important functions in cells, including energy generation, metabolism, and regulation of cell death. A key feature of mitochondria is that they undergo cycles of fusion and fission, and his lab is trying to understand the role of these processes in controlling their function. In addition, several human diseases arise from a perturbation of these processes, and he hopes to understand the cellular mechanisms involved in disease pathogenesis.
Affiliations and expertise
Division of Biology, California Institute of Technology, USA