Centrosomes and Spindle Pole Bodies
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Table of contents
- Cover
- Contentsv
- Contributorsxi
- Prefacexv
- Chapter 1. Purification and Reconstitution of Drosophila y-Tubulin Complexes1
- I. Introduction2
- II. Isolation of γ -Tubulin-Containing Complexes from Drosophila Embryo Extracts3
- III. γTuSC Reconstitution in Sf9 Cells12
- IV. Functional Assays for Isolated γTuSC or γTuRC18
- V. Concluding Remarks23
- References24
- Chapter 2. Using Rapid Freeze and Freeze-Substitution for the Preparation of Yeast Cells for Electro27
- I. Introduction28
- II. How Rapid Freezing and Freeze-Substitution Work30
- III. Rapid Freezing Techniques31
- IV. Freeze-Substitution Protocols33
- V. Embedding Protocols34
- VI. Sectioning and Imaging34
- VII. Immunolabeling for Electron Microscopy35
- VIII. Three-Dimensional Modeling of Spindle Pole Bodies and Other Organelles37
- IX. Conclusion40
- References41
- Chapter 3. Digital Fluorescence Microscopy of Cell Cytoplasts with and without the Centrosome43
- I. Introduction43
- II. Experimental Procedures44
- References51
- Chapter 4. Methods for the Study of Pericentrin in Centrosome Assembly and Function53
- I. Introduction54
- II. Pericentrin Localization to Centrosomes54
- III. Pericentrin Recruitment to Centrosomes58
- IV. Obtaining Enriched Fractions of Pericentrin63
- V. Artificial Elevation of Pericentrin Levels66
- VI. Pericentrin Staining to Detect Centrosome Defects in Tumor Tissues and Cell Lines67
- References68
- Chapter 5. Molecular Dissection of Yeast Spindle Pole Bodies by Two Hybrid, in Vitro Binding, and Co71
- I. Introduction71
- II. The Two-Hybrid Approach73
- III. Immuno- and Affinity Precipitation of Spindle Pole Body Components82
- IV. In Vitro Binding of Spindle Pole Body Proteins89
- References92
- Chapter 6. Genetic Analysis of Yeast Spindle Pole Bodies95
- I. Introduction95
- II. Dosage-Dependent Suppressor Screen97
- III. Extragenic Suppressor Screen100
- IV. Synthetic Lethal Screen103
- V. Synthetic Dosage Lethality108
- VI. Conclusions108
- References109
- Chapter 7. Methods for the Study of Centrosomes in Drosophila during Embryogenesis113
- I. Introduction113
- II. Materials114
- III. Methods115
- References123
- Chapter 8. Methods for Identification of Centrosome-Associated Proteins125
- I. Introduction125
- II. Preparation of Centrosomes and Anticentrosomal Antibodies126
- III. Molecular Cloning of cDNA Encoding Centrosome-Associated Proteins129
- IV. Evaluation of Clone Specificity132
- V. Identification of a Novel 135-kDa Centrosomal Protein, Cep135135
- References139
- Chapter 9. Reconstitution of Centrosome Microtubule Nucleation in Drosophila141
- I. Introduction141
- II. Preparation of Extracts and Centrosomes from Drosophila Embryos143
- III. In Vitro Reconstitution of Microtubule Nucleation by Salt-Inactivated Centrosomes144
- IV. Conclusions147
- References148
- Chapter 10. Reconstitution of Centrosome Microtubule Nucleation in Spisula149
- I. Introduction149
- II. Preparations151
- III. Disassembly and Reassembly of Microtubule Nucleation Potential154
- IV. Fractionation of Extracts159
- V. Summary163
- References164
- Chapter 11. In Vitro Approaches for the Study of Microtubule Nucleation at the Fission Yeast Spindle167
- I. Introduction168
- II. Protocol for Permeabilization169
- III. Protocol for Microtubule Nucleation171
- IV. Results173
- References176
- Chapter 12. Gamma Tubulin and Microtubule Nucleation in Mammalian Cells179
- I. Introduction179
- II. Interference with γ -Tubulin Function in Mammalian Cells Abrogates Microtubule Nucleation181
- III. Overexpression of γ -Tubulin in Mammalian Cells Causes Ectopic Microtubule Nucleation188
- IV. Conclusion191
- References192
- Chapter 13. Gamma Tubulin in Plant Cells195
- I. Introduction195
- II. Microtubule Arrays in Plant Cells196
- III. Methods for Cloning Plant γ -Tubulin cDNAs and Genes198
- IV. Methods for Localizing γ -Tubulin in Plant Cells202
- V. Functional Studies of γ -Tubulin in Plants209
- VI. Future Prospects210
- References210
- Chapter 14. Centrosomes and Parthenogenesis213
- I. Introduction213
- II. Parthenogenetic Test in Xenopus214
- III. Coupling between Egg Cell Cycle and Centrosome Duplication Pathway216
- IV. Species Barrier Specificity219
- V. Active or Inactive Centrosomes from the Same Species220
- VI. Prospects221
- References222
- Chapter 15. In Vitro Approaches for the Study of Molecular Motors in Aster Formation225
- I. Introduction225
- II. Microtubule Aster Formation228
- III. Examination of Microtubule Motor Function in Aster Assembly231
- IV. Concluding Remarks236
- References236
- Chapter 16. Methods for the Study of Centrosome-Independent Spindle Assembly in Xenopus Extracts241
- I. Introduction241
- II. Preparation of Xenopus Egg Extract and Necessary Reagents243
- III. Microtubule Polymerization Assays in Extract243
- IV. Spindle Assembly Assays245
- V. Studying Proteins Involved in Spindle Assembly249
- VI. Methods to Observe Spindle Assembly253
- VII. Conclusions254
- References254
- Chapter 17. Methods for the Study of Centrosome Reproduction in Mammalian Cells257
- I. Introduction257
- II. Induction of Centrosome Overproduction in Cultured Mammalian Cells259
- III. Preparation of Extracts from Hydroxyurea-Arrested Chinese Hamster Ovary Cells262
- IV. Analysis of Centrosome Protein Function Using Mammalian Cell Extracts and Salt-Stripped Centroso263
- V. Conclusions and Prospects266
- References266
- Chapter 18. Centrosome Reproduction in Xenopus Lysates269
- I. Introduction269
- II. Reagents and Equipment272
- III. Methods278
- References286
- Chapter 19. Centrosome Reproduction in Vitro: Mammalian Centrosomes in Xenopus Lysates289
- I. Introduction289
- II. Logic of the Assay291
- III. Duplication Assay292
- IV. Major Pitfalls297
- V. Discussion297
- VI. Prospects302
- References303
- Chapter 20. Identification of Centrosome Kinases305
- I. Introduction305
- II. Subcellular Localization of Previously Characterized Protein Kinases307
- III. Isolation of Novel Centrosome Kinases313
- IV. Functional Analysis of Centrosome Kinases315
- V. Identifying Substrates for Centrosome Kinases320
- VI. Perspectives321
- References321
- Chapter 21. Methods for the Analysis of Centrosome Reproduction in Cancer Cells325
- I. Introduction325
- II. Tissue Procurement and Storage326
- III. Preparation and Storage of Xenopus Egg Extract327
- IV. Reaction Mixture Composition and Use328
- V. Experimental Design and Data Analysis330
- VI. Summary332
- VII. Appendices333
- References335
- Chapter 22. Isolation of Centrosomes from Dictyostelium337
- I. Introduction337
- II. Isolation of Dictyostelium Centrosomes340
- III. Analysis of Isolated Dictyostelium Centrosomes344
- IV. Generation of Monoclonal Antibodies against Dictyostelium Centrosomes349
- V. Conclusions354
- References355
- Index359
- Volumes in Series369
Containing a comprehensive collection of convenient and quantitative methods for studying centrosomes, spindle pole bodies and related organelles, this text is a valuable resource for researchers and others interested in studying the role of these organelles in cell replication. Chapters outlining the role of these organelles in other cell functions are also included, and a wide variety of experimental systems for analyzing these organelles are presented. Detailed protocols for experiments are contained in each chapter for researchers to perform in their own labs. This volume outlines key methodologies used to analyze centrosomes and spindle pole bodies, their replication, and reproduction in the clear, well-illustrated style of the Methods in Cell Biology series.