Table Of ContentMethods in Cell Biology
VOLUME 80
Mitochondria, 2nd Edition
Edited by
Liza A. Pon
Department of Anatomy and Cell Biology
College of Physicians and Surgeons
Columbia University
New York, New York
Eric A. Schon
Department of Neurology and Department of
Genetics and Development
Columbia University
New York, New York
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Cover Photo Credit: Laser scanning confocal image of human fibroblasts
stained for mitochondria using Mito Tracker (red) and microtubules (green)
using a monoclonal anti-tubulin antibody and indirect immunofluorescence.
Image provided by Istvan Boldogh (Columbia University) and
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CONTENTS
Contributors xvii
Preface xxiii
PART I Isolation and Subfractionation of Mitochondria
1. Isolation and Subfractionation of Mitochondria from
Animal Cells and Tissue Culture Lines
Francesco Pallotti and Giorgio Lenaz
I. Introduction 4
II. General Features of Mitochondrial Preparations 5
III. Mitochondria from Beef Heart 8
IV. Mitochondria from Rat Liver 17
V. Mitochondria from Skeletal Muscle 23
VI. Synaptic and Nonsynaptic Mitochondria from DiVerent Rat Brain Regions 26
VII. Mitochondria from Kidney and Testis 29
VIII. Mitochondria from Hamster Brown Adipose Tissue 29
IX. Mitochondria from Insect Flight Muscle 30
X. Mitochondria from Porcine Adrenal Cortex 30
XI. Mitochondria from Human Platelets 31
XII. Mitochondria from Fish Liver 32
XIII. Mitochondria from Sea Urchin Eggs 33
XIV. Mitochondria and Kinetoplasts from Protozoa 33
XV. Mitochondria from Fish Erythrocytes 35
XVI. Mitochondria from Caenorhabditis elegans 35
XVII. Mitochondria from Drosophila 35
XVIII. Mitochondria and Mitoplasts from Cultured Cells 36
References 39
2. Purification and Subfractionation of Mitochondria from the
Yeast Saccharomyces cerevisiae
Istvan R. Boldogh and Liza A. Pon
I. Introduction 45
II. Isolation of Mitochondria 46
III. Analysis of Isolated Mitochondria 53
v
vi Contents
IV. Isolation of Mitochondrial Outer Membranes, Contact Sites,
and Inner Membranes 61
References 63
3. Isolation and Subfractionation of Mitochondria from Plants
A. H. Millar, A. Liddell, and C. J. Leaver
I. Introduction 66
II. Growth and Preparation of Plant Material 66
III. Isolation of Mitochondria 67
IV. Density Gradient Purification of Mitochondria 69
V. Mitochondrial Yield, Purity, Integrity, Storage, and Function 73
VI. Subfractionation of Mitochondrial Compartments 77
VII. In Organello Translation Analysis 83
VIII. Proteome Analysis 85
IX. Conclusion 87
References 87
PART II Biochemical Assays of Mitochondrial Activity
4. Biochemical Assays of Respiratory Chain Complex Activity
Denise M. Kirby, David R. Thorburn, Douglass M. Turnbull,
and Robert W. Taylor
I. Introduction 93
II. Materials and Methods 96
III. Interpreting Results of RC Enzymology 114
References 116
5. Polarographic Assays of Respiratory Chain Complex Activity
Gaetano Villani and Giuseppe Attardi
I. Introduction 121
II. Measurements of Endogenous Respiration in Intact Cells 122
III. KCN Titration of COX Activity in Intact Cells 124
IV. In Situ Analysis of Mitochondrial OXPHOS 128
References 132
6. Optical Imaging Techniques (Histochemical, Immunohistochemical, and
In Situ Hybridization Staining Methods) to Visualize Mitochondria
Kurenai Tanji and Eduardo Bonilla
I. Introduction 135
II. Histochemistry 137
Contents vii
III. Immunohistochemistry 142
IV. In Situ Hybridization to mtDNA 148
References 151
7. Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues
Cristofol Vives-Bauza, Lichuan Yang, and Giovanni Manfredi
I. Introduction 155
II. ATP Synthesis Assays 156
III. Methodological Considerations 157
IV. Experimental Procedures 160
V. Measurement of High-Energy Phosphates in Animal Tissue
and Cultured Cells by HPLC 165
References 169
8. Measurement of the Ratio of Lactate to Pyruvate in Skin Fibroblast Cultures
Nevi Mackay and Brian H. Robinson
I. Introduction 173
II. Principle 174
III. Procedure 175
IV. Results 177
References 178
9. Assays of Fatty Acid-Oxidation Activity
Michael J. Bennett
I. Introduction 179
II. Metabolite Measurements 183
III. Enzyme and Transporter Assays 186
IV. Metabolic Flux Studies 193
V. Discussion 195
References 195
10. Biochemical Assays for Mitochondrial Activity: Assays of
TCA Cycle Enzymes and PDHc
Ann Saada Reisch and Orly Elpeleg
I. Introduction 199
II. Pyruvate Dehydrogenase Complex 202
III. Citrate Synthase 207
IV. Aconitase 209
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V. Isocitrate Dehydrogenase 209
VI.-Ketoglutarate Dehydrogenase Complex 210
VII. Succinyl CoA Synthetase 212
VIII. Succinate Dehydrogenase 213
IX. Fumarase 214
X. Malate Dehydrogenase 217
XI. Conclusion 217
References 217
11. Assays of Cardiolipin Levels
Michael Schlame
I. Introduction 223
II. Molecular Species of Cardiolipin 225
III. Cardiolipin Analysis by TLC 226
IV. Cardiolipin Analysis by HPLC 230
V. Cardiolipin Analysis by MS 234
VI. Applications of Cardiolipin Measurements 236
References 238
12. Measurement of VDAC Permeability in Intact Mitochondria
and in Reconstituted Systems
Marco Colombini
I. Introduction 241
II. Determining the Permeability of the Mitochondrial Outer
Membrane to Metabolites 243
III. VDAC Activity After Reconstitution into Phospholipid Membranes 253
IV. Summary 259
References 259
13. Methods for Studying Iron Metabolism in Yeast Mitochondria
Sabine Molik, Roland Lill, and Ulrich Mu¨hlenhoff
I. Introduction 261
II. Determination of Cellular Fe/S Protein Formation and Heme Synthesis 263
III. Determination of Mitochondrial Iron Contents 272
IV. Reporter Assays for Analysis of Iron-Dependent Gene Expression
in S. cerevisiae 274
References 279
Contents ix
PART III Assays for Mitochondrial Respiratory Activity and
Permeability in Living Cells
14. Imaging of Mitochondrial Polarization and Depolarization with
Cationic Fluorophores
John J. Lemasters and Venkat K. Ramshesh
I. Introduction 283
II. Quantitative Imaging of C with Fluorescent Cations 284
III. Visualization of Depolarized Mitochondria 289
IV. Conclusion 293
References 294
15. Biosensors for the Detection of Calcium and pH
Paolo Pinton, Alessandro Rimessi, Anna Romagnoli, Andrea Prandini,
and Rosario Rizzuto
I. Introduction 297
II. Targeting Strategies and Transfection 298
III. Calcium Reporters 300
IV. pH Reporters 316
V. Conclusions 321
References 322
16. Measurement of Membrane Permeability and the Permeability
Transition of Mitochondria
Naoufal Zamzami, Carine Maisse, Didier Me´tivier, and Guido Kroemer
I. Introduction 327
II. Procedures 328
References 338
17. Luciferase Expression for ATP Imaging: Application to Cardiac Myocytes
Christopher J. Bell, Giovanni Manfredi, Elinor J. Griffiths, and Guy A. Rutter
I. Introduction 341
II. ATP Homeostasis in the Heart 343
III. Measurement of Mitochondrial and Cytosolic ATP in Cardiac Myocytes 343
IV. Measurement of ATP in Subcellular Compartments by Luminometery 346
References 351
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PART IV Oxidative Stress Measurements
18. Measurement of Reactive Oxygen Species in Cells and Mitochondria
J. S. Armstrong and M. Whiteman
I. Introduction 355
II. Measurement of ROS in Cells 357
III. Measurement of ROS in Mitochondria 369
IV. General Problems Associated with the Measurement of ROS 372
V. Conclusions 373
References 374
19. Measurements of the Antioxidant Enzyme Activities of Superoxide Dismutase,
Catalase, and Glutathione Peroxidase
Cristofol Vives-Bauza, Anatoly Starkov, and Elena Garcia-Arumi
I. Introduction 379
II. Experimental Procedures 380
References 391
20. Methods for Measuring the Regulation of Respiration by Nitric Oxide
Sruti Shiva, Paul S. Brookes, and Victor M. Darley-Usmar
I. Introduction 396
II. Apparatus 398
III. Calibration of the NO Electrode 401
IV. NO Threshold Measurement 406
V. Open-Flow Respirometry 411
VI. An In Vitro Model of Ischemia/Reperfusion Injury 413
VII. Conclusion 414
References 415
21. Methods for Determining the Modification of Protein Thiols by Reactive Lipids
JooYeun Oh, Michelle S. Johnson, and Aimee Landar
I. Introduction 417
II. Rationale 419
III. Methods 421
IV. Discussion 432
References 433
Contents xi
PART V Mitochondrial Genes and Gene Expression
22. Detection of Mutations in mtDNA
Ali Naini and Sara Shanske
I. Introduction 438
II. Large-Scale Rearrangements of mtDNA 439
III. Detecting mtDNA Rearrangements 440
IV. Details of Southern Blot Hybridization Analysis 443
V. Depletion of mtDNA 447
VI. Point Mutations in mtDNA 449
References 460
23. Diagnostic Assays for Defects in mtDNA Replication and Transcription
in Yeast and Humans
Gerald S. Shadel and Bonnie L. Seidel-Rogol
I. Introduction 465
II. Diagnosis of mtDNA Replication Defects in Yeast and Human Cells 466
III. Analyzing Mitochondrial Transcripts In Vivo 472
IV. Additional Considerations 477
References 477
24. Microdissection and Analytical PCR for the Investigation of mtDNA Lesions
Sion L. Williams and Carlos T. Moraes
I. Introduction 481
II. Microdissection and DNA Purification 482
III. Analytical PCR for Investigation of mtDNA in Microdissected Samples 487
IV. Concluding Remarks 498
References 499
25. Transmitochondrial Technology in Animal Cells
Sandra R. Bacman and Carlos T. Moraes
I. Introduction 504
II. Generation of Nuclear Donors 504
III. Generation of mtDNA Donors 510
IV. Generation of Transmitochondrial Cybrids 514
V. Manipulating Heteroplasmy 519
References 520
xii Contents
26. Genetic Transformation of Saccharomyces cerevisiae and
Chlamydomonas reinhardtii Mitochondria
Nathalie Bonnefoy, Claire Remacle, and Thomas D. Fox
I. Introduction 526
II. Important Features of S. cerevisiae and C. reinhardtii Mitochondrial Genetics 527
0
III. Delivery of DNA to the Mitochondrial Compartment of rho
Cells and Detection of Mitochondrial Transformants 532
IV. Strategies for Gene Replacement in S. cerevisiae mtDNA 538
þ
V. Transformation of rho Cells with Plasmids or Linear DNA Fragments 542
VI. Concluding Remarks 544
References 544
27. Generation of Transmitochondrial Mice: Development of Xenomitochondrial
Mice to Model Neurodegenerative Diseases
Carl A. Pinkert and Ian A. Trounce
I. Introduction 549
II. Spontaneous and Induced Models of Mitochondrial Disease 550
III. Transgenic Models 552
IV. Introduction of Mutant mtDNA into Mitochondria 552
V. The First Transmitochondrial Mice 555
VI. Summary/Future Directions 564
References 565
28. In Vivo and In Organello Analyses of Mitochondrial Translation
P. Ferna´ndez-Silva, R. Ac´ın-Pe´rez, E. Ferna´ndez-Vizarra, A. Pe´rez-Martos,
and J. A. Enriquez
I. Introduction 571
II. Rationale 573
III. Methods 576
IV. Materials 583
V. Discussion 584
VI. Summary 585
References 585
PART VI Assays for Mitochondrial Morphology and Motility
29. Visualization of Mitochondria in Budding Yeast
Theresa C. Swayne, Anna C. Gay, and Liza A. Pon
I. Introduction 592
II. Use of Vital Dyes to Stain Yeast Mitochondria 592
