Mitochondria
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Table of contents
- Cover
- CONTENTSv
- Contributorsxiii
- Prefacexvii
- Chapter 1. Isolation and Subfractionation of Mitochondria from Animal Cells and Tissue Culture Lines1
- I. Introduction2
- II. General Properties of Mitochondrial Preparations2
- III. Mitochondria from Beef Heart5
- IV. Mitochondria from Rat Liver13
- V. Mitochondria from Muscle19
- VI. Synaptic and Nonsynaptic Mitochondria from Different Rat Brain Regions20
- VII. Mitochondria from Hamster Brown Adipose Tissue23
- VIII. Mitochondria from Insect Flight Muscle24
- IX. Mitochondria from Porcine Adrenal Cortex24
- X. Mitochondria from Human Platelets25
- XI. Mitochondria from Fish Liver28
- XII. Mitochondria from Sea Urchin Egg28
- XIII. Mitochondria and Kinetoplasts from Protozoa28
- XIV. Mitochondria and Mitoplasts from Cultured Cells30
- References31
- Chapter 2. Isolation and Subfractionation of Mitochondria from the Yeast Saccharomyces cerevisiae37
- I. Introduction38
- II. Isolation of Yeast Mitochondria of Different Purity39
- III. Analysis of Mitochondria Preparations44
- IV. Purification of Outer Membrane Vesicles from Yeast Mitochondria48
- References49
- Chapter 3. Isolation and Subfractionation of Mitochondria from Plants53
- I. Introduction54
- II. Growth and Preparation of Plant Material54
- III. Isolation of Mitochondria by Differential Centrifugation55
- IV. Density Gradient Purification of Mitochondria57
- V. Mitochondrial Yield, Purity, Integrity, Storage, and Function61
- VI. Subfractionation of Mitochondrial Compartments64
- VII. Proteome Analysis68
- VIII. Analysis of in Organello Translation Products70
- IX. Conclusion72
- References72
- Chapter 4. Assessing Functional Integrity of Mitochondria in Vitro and in Vivo75
- I. Introduction75
- II. In Vitro Assays with Mitochondrial Preparations76
- III. Mitochondrial Functions within Living Cells87
- References93
- Chapter 5. Assaying Mitochondrial Respiratory Complex Activity in Mitochondria Isolated from Human C97
- I. Introduction97
- II. Preparation of Mitochondrial Fractions98
- III. Spectrophotometric Measurement of the Activities of Individual Complexes I–IV101
- IV. Linked Assays112
- V. Applications of Techniques to Diagnostic Investigations115
- References116
- Chapter 6. In Vivo Measurements of Respiration Control by Cytochrome c Oxidase and in Situ Analysis119
- I. Introduction120
- II. Measurements of Endogenous Respiration in Intact Cells120
- III. KCN Titration of COX Activity in Intact Cells122
- IV. KCN Titration of Integrated COX Activity126
- V. KCN Titration of Isolated COX Activity125
- VI. In Situ Analysis of Mitochondrial OXPHOS126
- References130
- Chapter 7. Assay of Mitochondrial ATP Synthesis in Animal Cells133
- I. Introduction133
- II. Methodological Considerations135
- III. Experimental Procedures136
- IV. ATP Synthesis in Cellular Models of Mitochondrial Diseases141
- References143
- Chapter 8. Measurement of Membrane Permeability and Permeability Transition of Mitochondria147
- I. Introduction147
- II. Procedures148
- III. Anticipated Results and Pitfalls156
- References157
- Chapter 9. Assaying Actin-Binding Activity of Mitochondria in Yeast159
- I. Introduction159
- II. Purification of Yeast Actin160
- III. Sedimentation Assay for Binding of Actin to Mitochondria165
- IV. Analysis of Mitochondria-Actin Binding170
- References172
- Chapter 10. Analysis and Prediction of Mitochondrial Targeting Peptides175
- I. Introduction175
- II. What Mitochondrial Targeting Peptides (mTPs) Look Like176
- III. Automatic Classification of Known mTPs177
- IV Automatic Identification of Unknown mTPs178
- V Conclusions183
- References184
- Chapter 11. Assaying Protein Import into Mitochondria189
- I. Introduction190
- II. Synthesis of Preproteins190
- III. Standard Protocol for Protein Import into Isolated Mitochondria196
- IV. Assaying Mitochondrial Localization of Imported Preproteins199
- V. Energetic Requirements for Preprotein Import204
- VI. Assaying Import Pathways205
- VII. Assaying Protein Assembly by Blue-Native Electrophoresis208
- VIII. Import of Purified Recombinant Proteins into Mitochondria209
- References213
- Chapter 12. Analysis of Protein–Protein Interactions in Mitochondria by Coimmunoprecipitation and217
- I. Introduction217
- II. General Considerations218
- III. Immunoprecipitation219
- IV. Coimmunoprecipitation222
- V. Cross-Linking225
- VI. Summary228
- References229
- Chapter 13. Blue-Native Gels to Isolate Protein Complexes from Mitochondria231
- I. Introduction231
- II. Materials and Methods232
- III. Applications238
- IV. Outlook242
- References242
- Chapter 14. Application of Electron Tomography to Mitochondrial Research245
- I. Introduction245
- II. Preliminary Considerations for Application of Electron Tomography248
- III. Methodology249
- References255
- Chapter 15. Epitope Tagging and Visualization of Nuclear-Encoded Mitochondrial Proteins in Yeast257
- I. Introduction257
- II. Single-step Modification of Nuclear-Encoded Mitochondrial Genes in Yeast: An Overview258
- III. Generation and Analysis of the Modified Gene Product: Technical Considerations258
- IV. Visualizing Yeast Mitochondria by Immunostaining265
- References274
- Chapter 16. Targeting of Green Fluorescent Protein to Mitochondria277
- I. Introduction277
- II. Mitochondrial Green Fluorescent Protein (GFP) Fusion Markers278
- III. General Concerns about Targeting of GFP Markers into Mitochondria281
- IV. Conclusions282
- References283
- Chapter 17. Assessment of Mitochondrial Membrane Potential in Situ Using Single Potentiometric Dyes285
- I. Introduction285
- II. Single Potentiometric Dyes288
- III. Novel Fluorescence Resonance Energy Transfer Assay for ∆Ψm294
- References306
- Chapter 18. Optical Imaging Techniques (Histochemical, Immunohistochemical, and in Situ Hybridizatio311
- I. Introduction312
- II. Histochemistry313
- III. Immunohistochemistry317
- IV. In Situ Hybridization and Single-Fiber Polymerase Chain Reaction (PCR)321
- V. Histochemical Methods326
- VI. Immunohistochemical Methods326
- VII. Molecular Genetic Methods328
- References329
- Chapter 19. Visualization of Mitochondrial Movement in Yeast333
- I. Introduction333
- II. Yeast Cell Growth and Synchronization335
- III. Vital Staining of Yeast Mitochondria337
- IV. Preparation of Growth Chamber343
- V. Image Acquisition344
- VI. Analysis and Quantitation of Movement346
- References349
- Chapter 20. Targeting of Reporter Molecules to Mitochondria to Measure Calcium, ATP, and pH353
- I. Introduction354
- II. Calcium357
- III. ATP365
- IV. pH372
- V. Conclusion376
- References377
- Chapter 21. Genetic Transformation of Saccharomyces cerevisiae Mitochondria381
- I. Introduction382
- II. Important Features of Saccharomyces cerevisiae Mitochondrial (MT) Genetics382
- III. Delivery of DNA to the Mitochondrial Compartment of rho0 Cells and Detection of Mitochondrial T384
- IV. Strategies for Gene replacement in S. cerevisiae mtDNA388
- V. Transformation of rho+ Cells with Plasmids or Linear DNA Fragments392
- VI. Concluding Remarks394
- References394
- Chapter 22. Transmitochondrial Technology in Animal Cells397
- I. Introduction397
- II. Generation of p0 Cells399
- III. Generation of Transmitochondrial Cybrids Using p0 Cells as Nuclear Donors401
- IV. Generation of Transmitochondrial Cybrids Using p+ Cells Treated with Rhodamine 6G as Nuclear Do406
- V. Generation of Transmitochondrial Hybrid Cells by Microcell-Mediated Chromosome and mtDNA Transfer407
- VI. Manipulating Heteroplasmy409
- References410
- Chapter 23. Diagnostic Assays for Defects in Mitochondrial DNA Replication and Transcription in Yeas413
- I. Introduction413
- II. Diagnosis of Mitochondrial (mt)DNA Replication Defects in Yeast and Human Cells414
- III. Analyzing Mitochondrial Transcripts in Vivo419
- IV. Additional Considerations423
- References425
- Chapter 24. Analysis of Mitochondrial Translation Products in Vivo and in Organello in Yeast429
- I. Introduction429
- II. Labeling of Mitochondrial Translation Products in Vivo431
- III. Labeling of Mitochondrial Translation Products in Organello431
- IV. Use of Mitochondrial Translation for the Study of Various Aspects of Mitochondrial Biogenesis432
- V. Analysis of Mitochondrial Translation Products by Sodium Dodecyl Sulfate–Polyacrylamide Gel Ele435
- VI. Discussion436
- References437
- Chapter 25. Numerical Methods for Handling Uncertainty in Microarray Data: An Example Analyzing Pert439
- I. Introduction439
- II. Quantitative Methods in Microarray Research440
- III. Application of Microarrays to the Study of Cellular Responses to Perturbed Mitochondrial Functi446
- References451
- APPENDICES453
- APPENDIX 1. Basic Properties of Mitochondria453
- APPENDIX 2. Linearize Maps of Circular Mitochndrial Genomes from457
- APPENDIX 3. Mitochondrial Genetic Codes in Various Organisms461
- APPENDIX 4. Gene Products Present in Mitochndria of Yeast and Animal Cells463
- APPENDIX 5. Direct and Indirect Inhibitors of Mitochndrial ATP Synthesis483
- Index497
- Volumes in Series513
This title employs biochemical, cell biological, and genetic approaches to study mitochondrial structure, function, and biogenesis. Also of interest are the consequences of impaired mitochondrial function on cells, tissues, and organs.
The book is full of step-by-step "how to" methods with sample results, interpretations, and pitfalls. There is a unique set of appendices that include gene catalogs, mtDNA maps, and reagents for probing respiratory chain function. Finally, there are applications of state-of-the art microarray and gene chip technologies.
Key Features
* Isolation of mitochondria from commonly used cells and tissues
* Assays for mitochondrial activities, including respiration, ATP production, permeability, protein import, and interactions with the cytoskeleton
* Biochemical and optical methods for studying protein-protein interactions in mitochondria
* Approaches to studying mitochondrial replication, transcription, and translation
* Transmitochondrial technologies
* Methods in microassay data analysis
The book is full of step-by-step "how to" methods with sample results, interpretations, and pitfalls. There is a unique set of appendices that include gene catalogs, mtDNA maps, and reagents for probing respiratory chain function. Finally, there are applications of state-of-the art microarray and gene chip technologies.
Key Features
* Isolation of mitochondria from commonly used cells and tissues
* Assays for mitochondrial activities, including respiration, ATP production, permeability, protein import, and interactions with the cytoskeleton
* Biochemical and optical methods for studying protein-protein interactions in mitochondria
* Approaches to studying mitochondrial replication, transcription, and translation
* Transmitochondrial technologies
* Methods in microassay data analysis