Mitosis and Meiosis (eBook)

Front Cover 1
Methods in Cell Biology, Volume 61 4
Copyright Page 5
Contents 6
Contributors 12
Preface 16
Chapter 1. Isolation of Centrosomes from Drosophila Embryos 18
I. Introduction 18
II. Isolation of Centrosomes from Drosophila Embryos 19
III. Assays for Microtubule Nucleation by Isolated Centrosomes 22
IV. Conclusions 28
References 28
Chapter 2. Studying the Composition and Function of Centrosomes in Vertebrates 30
I. Introduction 31
II. Isolation of Centrosomes from Animal Cells 32
III. Preparation of Immunological Probes from Isolated Centrosomes 35
IV. Ultrastructural Analysis of Isolated Centrosomes 37
V. Biochemical Composition of Centrosomes 41
VI. Functional Assays of Isolated Centrosomes 44
VII. Prospects 46
References 48
Chapter 3. Isolation of Centrosomes from Spisulu solidissima Oocytes 52
I. Introduction 53
II. Obtaining Organisms 54
III. Isolation and Activation of Spisulu solidissima Oocytes 54
IV. Preparation of Oocyte Lysates 58
V. Preparation of Microtubule Protein 60
VI. Isolation of Centrosomes from Activated Oocyte Lysates 62
VII. lmmunofluorescence of Centrosomes and Asters 66
VIII. Electron Microscopy of Asters and Centrosomes 69
IX. Summary 70
References 73
Chapter 4. Methods for in Situ Localization of Proteins and DNA in the Centromere–Kinetochore Complex 74
I. Introduction 74
II. In Situ Localization of Proteins: Indirect Immunofluorescence 76
III. In Situ Localization of Proteins: Immunogold EM 80
IV. Fluorescent in Situ Hybridization Using DNA Satellite Probes 83
V. Combination Staining: DNA/Protein 86
VI. Specialized Techniques 89
References 95
Chapter 5. Three-Dimensional Transmission Electron Microscopy and Its Application to Mitosis Research 98
I. Introduction 99
II. Resolution and Choosing between Tomography and Serial Sections 101
III. Electron Tomography 104
IV. Serial Section Reconstruction 115
V. Analysis and Display of 3D Reconstructions 116
VI. Software Packages 122
VII. Summary and Conclusions 123
References 125
Chapter 6. Enlightening Mitosis: Construction and Expression of Green Fluorescent Protein Fusion Proteins 130
I. Introduction: Visualizing the Molecular Anatomy of the Spindle 131
II. Fluorescence Properties of GFP 131
III. Strategies for Constructing Fusion Proteins 135
IV. Expression in Mammalian Cells 140
References 150
Chapter 7. Recombinant p50/Dynamitin as a Tool to Examine the Role of Dynactin in Intracellular Processes 154
I. Introduction 154
II. Production of Recombinant p50/Dynamitin 155
III. Disruption of the Dynactin Complex by p50/Dynamitin in Xenopus Egg Extracts 157
IV. Disruption of Spindle Poles Using p50/Dynamitin 159
References 159
Chapter 8. In Vitro Assays for Studying Saccharomyces cerevisiae Kinetochore Activity 162
I. Introduction 162
II. Microtubule-Binding Assays for S. cerevisiae Kinetochores 163
Ill. Band Shift Assay for the Kinetochore Complex 168
References 170
Chapter 9. Fluorescent Speckle Microscopy of Spindle Microtubule Assembly and Motility in Living Cells 172
I. lntroduction 173
II. Principles of the Fluorescence Speckle Method for Microtubules 173
III. Specimen Methods 175
IV. Microscopy and Image Acquisition 178
V. Image Processing and Analysis 181
VI. Examples 184
VII. Future Considerations 187
References 189
Chapter 10. Polarized Light Microscopy of Spindles 192
I. Introduction 192
II. Polarized Light Microscopy 195
III. Analysis of Spindle Birefringence 207
IV. Optimum Cell Types for Polarized Light Microscopy of Spindles 214
References 223
Chapter 11. Micromanipulation of Chromosomes and Spindles in Insect Spermatocytes 226
I. Introduction 226
II. Preparing for Micromanipulation 227
Ill. Manipulating Cell Components 233
References 234
Chapter 12. Microinjection of Mitotic Cells 236
I. lntroduction 236
II. Choice of Cells 238
III. Timing of Injection 240
IV. Microinjection Procedure 240
V. Conclusions 245
References 246
Chaspter 13. Obtaining Antibodies to Spindle Components 250
I. Introduction 250
II. Methods 252
III. Discussion 260
References 261
Chapter 14. Using Antisense Technology to Study Mitosis 262
I. Introduction 262
II. Antisense Mechanism of Action 264
III. Choice of Antisense Reagents 265
IV. Assaying Target Protein Levels 271
V. Antisense Reagents Used to Study Cell Division 272
References 278
Chapter 15. The Use and Action of Drugs in Analyzing Mitosis 284
I. Introduction: Why Use Drugs? 284
II. Brief Overview of Microtubule Assembly Dynamics 285
III. Mechanisms of Action of Major Antimitotic Drugs: Binding to Tubulin and Microtubules and Effects on Microtubule Polymerization and Dynamics and on Mitosis 287
IV. Determination of Intracellular Drug Levels 304
V. How to Use Antimitotic Drugs: Practical Guidelines 305
References 306
Chapter 16. Correlative Light and Electron Microscopy of Mitotic Cells in Monolayer Cultures 314
I. Introduction 314
II. Light Microscopy 316
III. Flat Embedding 319
IV. Preparing the Cell for Sectioning 322
V. Obtaining the Required Ultrastructural Information 324
References 330
Chapter 17. Identification and Characterization of Mitotic Mutations in Drosophila 334
I. Introduction 335
II. Maternal-Effect Mutations That Disrupt the Syncytial Mitotic Divisions 339
III. Cytological Analysis of the Syncytial Mitoses 341
IV. Zygotic Mutations That Disrupt Mitosis in Larval Tissues 351
V. Cytological Analysis of Larval Brain and Imaginal Discs 354
References 361
Chapter 18. Methods for Isolating and Analyzing Mitotic Mutants in Aspergillus nidulans 364
I. Introduction 365
II. Characteristics of Aspergillus nidulans 365
III. Strains and Media 370
IV. Harvesting Conidia and Preparing Conidial Suspensions 371
V. Mutagenesis 372
VI. Methods for Isolating Mitotic Mutants in A . nidulans 372
VII. Morphologcal Analysis of Mutants 378
VIII. Genetic Analysis of Mutants 380
IX. Molecular Genetic Methods for Working with A . nidulans 382
References 384
Chapter 19. Using Green Fluorescent Protein Fusion Proteins to Quantitate Microtubule and Spindle Dynamics in Budding Yeast 386
I. Introduction 386
II. Construction of Protein–GFP Fusion and Promoter Selection 387
III. Quantifying Fluorescence in Cell Populations 390
IV. The Imaging System 392
V. Quantitative Solution to the Imaging Problem 393
VI. lmage Acquisition and Processing 395
VII. Applications and Examples: Expression of Dynein–GFP in Vivo 396
References 398
Chapter 20. The Use of Xenopus Egg Extracts to Study Mitotic Spindle Assembly and Function in Vitro 402
I. Introduction 403
II. Preparation of CSF Extracts for Spindle Assembly 404
III. Spindle Assembly Reactions 410
IV. Monitoring Spindle Assembly Reactions 414
V. Manipulation of Extracts 418
VI. Data Analysis and Interpretation 421
VII. Anaphase in Vitro 422
VIII. Conclusions 427
References 428
Chapter 21. Methods for Studying Cell Division in Higher Plants 430
I. Introduction 430
II. Cell Types of the Study of Cell Division 431
III. Microinjection of Plant Cells 445
IV. Conclusions 450
References 451
Chapter 22. Using Sea Urchin Gametes for the Study of Mitosis 456
I. Introduction 457
II. The Experimental System 457
III. Maintenance of the Organisms 458
IV. Obtaining Gametes 460
V. Zygotes 461
VI. Mounting Cells for Observation 465
VII. Other Methods 479
VIII. Annotated List of References 481
References 489
Index 490