Mitochondria (eBook)

Liza A. Pon, Eric A. Schon (Herausgeber)

eBook Download: PDF

2001 | 1. Auflage
519 Seiten
Elsevier Science (Verlag)
978-0-08-052253-1 (ISBN)

Infos

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

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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 "e;how to"e; 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. 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

Inhalt

Cover 1
CONTENTS 6
Contributors 14
Preface 18
Chapter 1. Isolation and Subfractionation of Mitochondria from Animal Cells and Tissue Culture Lines 20
I. Introduction 21
II. General Properties of Mitochondrial Preparations 21
III. Mitochondria from Beef Heart 24
IV. Mitochondria from Rat Liver 32
V. Mitochondria from Muscle 38
VI. Synaptic and Nonsynaptic Mitochondria from Different Rat Brain Regions 39
VII. Mitochondria from Hamster Brown Adipose Tissue 42
VIII. Mitochondria from Insect Flight Muscle 43
IX. Mitochondria from Porcine Adrenal Cortex 43
X. Mitochondria from Human Platelets 44
XI. Mitochondria from Fish Liver 47
XII. Mitochondria from Sea Urchin Egg 47
XIII. Mitochondria and Kinetoplasts from Protozoa 47
XIV. Mitochondria and Mitoplasts from Cultured Cells 49
References 50
Chapter 2. Isolation and Subfractionation of Mitochondria from the Yeast Saccharomyces cerevisiae 56
I. Introduction 57
II. Isolation of Yeast Mitochondria of Different Purity 58
III. Analysis of Mitochondria Preparations 63
IV. Purification of Outer Membrane Vesicles from Yeast Mitochondria 67
References 68
Chapter 3. Isolation and Subfractionation of Mitochondria from Plants 72
I. Introduction 73
II. Growth and Preparation of Plant Material 73
III. Isolation of Mitochondria by Differential Centrifugation 74
IV. Density Gradient Purification of Mitochondria 76
V. Mitochondrial Yield, Purity, Integrity, Storage, and Function 80
VI. Subfractionation of Mitochondrial Compartments 83
VII. Proteome Analysis 87
VIII. Analysis of in Organello Translation Products 89
IX. Conclusion 91
References 91
Chapter 4. Assessing Functional Integrity of Mitochondria in Vitro and in Vivo 94
I. Introduction 94
II. In Vitro Assays with Mitochondrial Preparations 95
III. Mitochondrial Functions within Living Cells 106
References 112
Chapter 5. Assaying Mitochondrial Respiratory Complex Activity in Mitochondria Isolated from Human Cells and Tissues 116
I. Introduction 116
II. Preparation of Mitochondrial Fractions 117
III. Spectrophotometric Measurement of the Activities of Individual Complexes I–IV 120
IV. Linked Assays 131
V. Applications of Techniques to Diagnostic Investigations 134
References 135
Chapter 6. In Vivo Measurements of Respiration Control by Cytochrome c Oxidase and in Situ Analysis of Oxidative Phosphorylation 138
I. Introduction 139
II. Measurements of Endogenous Respiration in Intact Cells 139
III. KCN Titration of COX Activity in Intact Cells 141
IV. KCN Titration of Integrated COX Activity 145
V. KCN Titration of Isolated COX Activity 144
VI. In Situ Analysis of Mitochondrial OXPHOS 145
References 149
Chapter 7. Assay of Mitochondrial ATP Synthesis in Animal Cells 152
I. Introduction 152
II. Methodological Considerations 154
III. Experimental Procedures 155
IV. ATP Synthesis in Cellular Models of Mitochondrial Diseases 160
References 162
Chapter 8. Measurement of Membrane Permeability and Permeability Transition of Mitochondria 166
I. Introduction 166
II. Procedures 167
III. Anticipated Results and Pitfalls 175
References 176
Chapter 9. Assaying Actin-Binding Activity of Mitochondria in Yeast 178
I. Introduction 178
II. Purification of Yeast Actin 179
III. Sedimentation Assay for Binding of Actin to Mitochondria 184
IV. Analysis of Mitochondria-Actin Binding 189
References 191
Chapter 10. Analysis and Prediction of Mitochondrial Targeting Peptides 194
I. Introduction 194
II. What Mitochondrial Targeting Peptides (mTPs) Look Like 195
III. Automatic Classification of Known mTPs 196
IV Automatic Identification of Unknown mTPs 197
V Conclusions 202
References 203
Chapter 11. Assaying Protein Import into Mitochondria 208
I. Introduction 209
II. Synthesis of Preproteins 209
III. Standard Protocol for Protein Import into Isolated Mitochondria 215
IV. Assaying Mitochondrial Localization of Imported Preproteins 218
V. Energetic Requirements for Preprotein Import 223
VI. Assaying Import Pathways 224
VII. Assaying Protein Assembly by Blue-Native Electrophoresis 227
VIII. Import of Purified Recombinant Proteins into Mitochondria 228
References 232
Chapter 12. Analysis of Protein–Protein Interactions in Mitochondria by Coimmunoprecipitation and Chemical Cross-Linking 236
I. Introduction 236
II. General Considerations 237
III. Immunoprecipitation 238
IV. Coimmunoprecipitation 241
V. Cross-Linking 244
VI. Summary 247
References 248
Chapter 13. Blue-Native Gels to Isolate Protein Complexes from Mitochondria 250
I. Introduction 250
II. Materials and Methods 251
III. Applications 257
IV. Outlook 261
References 261
Chapter 14. Application of Electron Tomography to Mitochondrial Research 264
I. Introduction 264
II. Preliminary Considerations for Application of Electron Tomography 267
III. Methodology 268
References 274
Chapter 15. Epitope Tagging and Visualization of Nuclear-Encoded Mitochondrial Proteins in Yeast 276
I. Introduction 276
II. Single-step Modification of Nuclear-Encoded Mitochondrial Genes in Yeast: An Overview 277
III. Generation and Analysis of the Modified Gene Product: Technical Considerations 277
IV. Visualizing Yeast Mitochondria by Immunostaining 284
References 293
Chapter 16. Targeting of Green Fluorescent Protein to Mitochondria 296
I. Introduction 296
II. Mitochondrial Green Fluorescent Protein (GFP) Fusion Markers 297
III. General Concerns about Targeting of GFP Markers into Mitochondria 300
IV. Conclusions 301
References 302
Chapter 17. Assessment of Mitochondrial Membrane Potential in Situ Using Single Potentiometric Dyes and a Novel Fluorescence Resonance Energy Transfer Technique 304
I. Introduction 304
II. Single Potentiometric Dyes 307
III. Novel Fluorescence Resonance Energy Transfer Assay for ??m 313
References 325
Chapter 18. Optical Imaging Techniques (Histochemical, Immunohistochemical, and in Situ Hybridization Staining Methods) to Visualize Mitochondria 330
I. Introduction 331
II. Histochemistry 332
III. Immunohistochemistry 336
IV. In Situ Hybridization and Single-Fiber Polymerase Chain Reaction (PCR) 340
V. Histochemical Methods 345
VI. Immunohistochemical Methods 345
VII. Molecular Genetic Methods 347
References 348
Chapter 19. Visualization of Mitochondrial Movement in Yeast 352
I. Introduction 352
II. Yeast Cell Growth and Synchronization 354
III. Vital Staining of Yeast Mitochondria 356
IV. Preparation of Growth Chamber 362
V. Image Acquisition 363
VI. Analysis and Quantitation of Movement 365
References 368
Chapter 20. Targeting of Reporter Molecules to Mitochondria to Measure Calcium, ATP, and pH 372
I. Introduction 373
II. Calcium 376
III. ATP 384
IV. pH 391
V. Conclusion 395
References 396
Chapter 21. Genetic Transformation of Saccharomyces cerevisiae Mitochondria 400
I. Introduction 401
II. Important Features of Saccharomyces cerevisiae Mitochondrial (MT) Genetics 401
III. Delivery of DNA to the Mitochondrial Compartment of rho0 Cells and Detection of Mitochondrial Transformants 403
IV. Strategies for Gene replacement in S. cerevisiae mtDNA 407
V. Transformation of rho+ Cells with Plasmids or Linear DNA Fragments 411
VI. Concluding Remarks 413
References 413
Chapter 22. Transmitochondrial Technology in Animal Cells 416
I. Introduction 416
II. Generation of p0 Cells 418
III. Generation of Transmitochondrial Cybrids Using p0 Cells as Nuclear Donors 420
IV. Generation of Transmitochondrial Cybrids Using p+ Cells Treated with Rhodamine 6G as Nuclear Donors 425
V. Generation of Transmitochondrial Hybrid Cells by Microcell-Mediated Chromosome and mtDNA Transfer 426
VI. Manipulating Heteroplasmy 428
References 429
Chapter 23. Diagnostic Assays for Defects in Mitochondrial DNA Replication and Transcription in Yeast and Human Cells 432
I. Introduction 432
II. Diagnosis of Mitochondrial (mt)DNA Replication Defects in Yeast and Human Cells 433
III. Analyzing Mitochondrial Transcripts in Vivo 438
IV. Additional Considerations 442
References 444
Chapter 24. Analysis of Mitochondrial Translation Products in Vivo and in Organello in Yeast 448
I. Introduction 448
II. Labeling of Mitochondrial Translation Products in Vivo 450
III. Labeling of Mitochondrial Translation Products in Organello 450
IV. Use of Mitochondrial Translation for the Study of Various Aspects of Mitochondrial Biogenesis 451
V. Analysis of Mitochondrial Translation Products by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis 454
VI. Discussion 455
References 456
Chapter 25. Numerical Methods for Handling Uncertainty in Microarray Data: An Example Analyzing Perturbed Mitochondrial Function in Yeast 458
I. Introduction 458
II. Quantitative Methods in Microarray Research 459
III. Application of Microarrays to the Study of Cellular Responses to Perturbed Mitochondrial Function 465
References 470
APPENDICES 472
APPENDIX 1. Basic Properties of Mitochondria 472
APPENDIX 2. Linearize Maps of Circular Mitochndrial Genomes from 476
APPENDIX 3. Mitochondrial Genetic Codes in Various Organisms 480
APPENDIX 4. Gene Products Present in Mitochndria of Yeast and Animal Cells 482
APPENDIX 5. Direct and Indirect Inhibitors of Mitochndrial ATP Synthesis 502
Index 516
Volumes in Series 532

Produkt-Details

• Erscheint lt. Verlag: 23.5.2001
• Mitarbeit: Herausgeber (Serie): Paul T. Matsudaira, Leslie Wilson
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Technik
• ISBN-10: 0-08-052253-X / 008052253X
• ISBN-13: 978-0-08-052253-1 / 9780080522531