Centrosomes and Spindle Pole Bodies

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ISBN: 9780125439855 | 0125439857
Cover: Paperback
Copyright: 8/27/2001

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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.

Contributors

Preface

Purification and Reconstitution of Drosophila γ-Tubulin Complexes

Ruwanthi N. Gunawardane

Yixian Zheng

Karen Oegema

Christiane Wiese

Introduction

(1)

Isolation of γ-Tubulin-Containing Complexes from Drosophila Embryo Extracts

(9)

γTuSC Reconstitution in Sf9 Cells

(6)

Functional Assays for Isolated γTuSC or γTuRC

(5)

Concluding Remarks

(5)

References

(4)

Using Rapid Freeze and Freeze-Substitution for the Preparation of Yeast Cells for Electron Microscopy and Three-Dimensional Analysis

Thomas H. Giddings, Jr.

Eileen T. O'Toole

Mary Morphew

David N. Mastronarde

J. Richard McIntosh

Mark Winey

Introduction

(1)

How Rapid Freezing and Freeze-Substitution Work

(2)

Rapid Freezing Techniques

(2)

Freeze-Substitution Protocols

(1)

Embedding Protocols

(1)

Sectioning and Imaging

(1)

Immunolabeling for Electron Microscopy

(2)

Three-Dimensional Modeling of Spindle Pole Bodies and Other Organelles

(3)

Conclusion

(3)

References

(2)

Digital Fluorescence Microscopy of Cell Cytoplasts with and without the Centrosome

Vladimir Rodionov

Elena Nadezhdina

John Peloquin

Gary Borisy

Introduction

(1)

Experimental Procedures

(10)

References

(3)

Methods for the Study of Pericentrin in Centrosome Assembly and Function

Aruna Purohit

German A. Pihan

Stephen J. Doxsey

Introduction

(1)

Pericentrin Localization to Centrosomes

(4)

Pericentrin Recruitment to Centrosomes

(5)

Obtaining Enriched Fractions of Pericentrin

(3)

Artificial Elevation of Pericentrin Levels

(1)

Pericentrin Staining to Detect Centrosome Defects in Tumor Tissues and Cell Lines

(4)

References

(3)

Molecular Dissection of Yeast Spindle Pole Bodies by Two Hybrid, in Vitro Binding, and Co-purification

C. Schramm

C. Janke

E. Schiebel

Introduction

(2)

The Two-Hybrid Approach

(11)

Immuno- and Affinity Precipitation of Spindle Pole Body Components

(5)

In Vitro Binding of Spindle Pole Body Proteins

(6)

References

(3)

Genetic Analysis of Yeast Spindle Pole Bodies

Trisha N. Davis

Introduction

(2)

Dosage-Dependent Suppressor Screen

(3)

Extragenic Suppressor Screen

100

(3)

Synthetic Lethal Screen

103

(5)

Synthetic Dosage Lethality

108

(1)

Conclusions

108

(5)

References

109

(4)

Methods for the Study of Centrosomes in Drosophila during Embryogenesis

Uyen Tram

Blake Riggs

Carol Koyama

Alain Debec

William Sullivan

Introduction

113

(1)

Materials

114

(1)

Methods

115

(10)

References

122

(3)

Methods for Identification of Centrosome-Associated Proteins

Ryoko Kuriyama

Toshiro Ohta

Jacalyn Vogel

Gang Peng

Introduction

125

(3)

Preparation of Centrosomes and Anticentrosomal Antibodies

128

(1)

Molecular Cloning of cDNA Encoding Centrosome-Associated Proteins

129

(3)

Evaluation of Clone Specificity

132

(3)

Identification of a Novel 135-kDa Centrosomal Protein, Cep135

135

(6)

References

139

(2)

Reconstitution of Centrosome Microtubule Nucleation in Drosophila

Michelle Moritz

Michael B. Braunfeld

Bruce M. Alberts

David A. Agard

Introduction

141

(2)

Preparation of Extracts and Centrosomes from Drosophila Embryos

143

(1)

In Vitro Reconstitution of Microtubule Nucleation by Salt-Inactivated Centrosomes

144

(3)

Conclusions

147

(2)

References

148

(1)

Reconstitution of Centrosome Microtubule Nucleation in Spisula

Bradley J. Schnackenberg

Robert E. Palazzo

Introduction

149

(2)

Preparations

151

(3)

Disassembly and Reassembly of Microtubule Nucleation Potential

154

(5)

Fractionation of Extracts

159

(4)

Summary

163

(5)

References

164

(4)

In Vitro Approaches for the Study of Microtubule Nucleation at the Fission Yeast Spindle Pole Body

Hirohisa Masuda

Saeko Takada

Takehiko Shibata

W. Zacheus Cande

Yasushi Hiraoka

Introduction

168

(1)

Protocol for Permeabilization

169

(2)

Protocol for Microtubule Nucleation

171

(2)

Results

173

(6)

References

176

(3)

Gamma Tubulin and Microtubule Nucleation in Mammalian Cells

Harish C. Joshi

Jun Zhou

Introduction

179

(2)

Interference with γ-Tubulin Function in Mammalian Cells Abrogates Microtubule Nucleation

181

(6)

Overexpression of γ-Tubulin in Mammalian Cells Causes Ectopic Microtubule Nucleation

187

(4)

Conclusion

191

(4)

References

192

(3)

Gamma Tubulin in Plant Cells

Yulia Ovechkina

Berl R. Oakley

Introduction

195

(1)

Microtubule Arrays in Plant Cells

196

(2)

Methods for Cloning Plant γ-Tubulin cDNAs and Genes

198

(4)

Methods for Localizing γ-Tubulin in Plant Cells

202

(7)

Functional Studies of γ-Tubulin in Plants

209

(1)

Future Prospects

210

(3)

References

210

(3)

Centrosomes and Parthenogenesis

Frederic Tournier

Michel Bornens

Introduction

213

(1)

Parthenogenetic Test in Xenopus

214

(2)

Coupling between Egg Cell Cycle and Centrosome Duplication Pathway

216

(3)

Species Barrier Specificity

219

(1)

Active or Inactive Centrosomes from the Same Species

220

(1)

Prospects

221

(4)

References

222

(3)

In Vitro Approaches for the Study of Molecular Motors in Aster Formation

Duane A. Compton

Introduction

225

(3)

Microtubule Aster Formation

228

(3)

Examination of Microtubule Motor Function in Aster Assembly

231

(5)

Concluding Remarks

236

(5)

References

236

(5)

Methods for the Study of Centrosome-Independent Spindle Assembly in Xenopus Extracts

Sarah M. Wignall

Rebecca Heald

Introduction

241

(2)

Preparation of Xenopus Egg Extract and Necessary Reagents

243

(1)

Microtubule Polymerization Assays in Extract

243

(2)

Spindle Assembly Assays

245

(4)

Studying Proteins Involved in Spindle Assembly

249

(4)

Methods to Observe Spindle Assembly

253

(1)

Conclusions

254

(3)

References

254

(3)

Methods for the Study of Centrosome Reproduction in Mammalian Cells

Ron Balczon

Introduction

257

(2)

Induction of Centrosome Overproduction in Cultured Mammalian Cells

259

(3)

Prepartion of Extracts from Hydroxyurea-Arrested Chinese Hamster Ovary Cells

262

(1)

Analysis of Centrosome Protein Function Using Mammalian Cell Extrats and Salt-Stripped Centrosome Remnants

263

(3)

Conclusions and Prospects

266

(3)

References

266

(3)

Centrosome Reproduction in Xenopus Lysates

Edward H. Hinchcliffe

Greenfield Sluder

Introduction

269

(3)

Reagents and Equipment

272

(6)

Methods

278

(11)

References

286

(3)

Centrosome Reproduction in Vitro: Mammalian Centrosomes in Xenopus Lysates

Matthieu Piel

Michel Bornens

Introduction

289

(2)

Logic of the Assay

291

(1)

Duplication Assay

292

(5)

Major Pitfalls

297

(1)

Discussion

297

(5)

Prospects

302

(3)

References

303

(2)

Identification of Centrosome Kinases

Andrew M. Fry

Alison J. Faragher

Introduction

305

(2)

Subcellular Localization of Previously Characterized Protein Kinases

307

(6)

Isolation of Novel Centrosome Kinases

313

(2)

Functional Analysis of Centrosome Kinases

315

(5)

Identifying Substrates for Centrosome Kinases

320

(1)

Perspectives

321

(4)

References

321

(4)

Methods for the Analysis of Centrosome Reproductio in Cancer Cells

Wilma L. Lingle

Jeffrey L. Salisbury

Introduction

325

(1)

Tissue Procurement and Storage

326

(1)

Preparation and Storage of Xenopus Egg Extract

327

(1)

Reaction Mixture Composition and Use

328

(2)

Experimental Design and Data Analysis

330

(2)

Summary

332

(1)

Appendices

333

(4)

References

335

(2)

Isolation of Centrosomes from Dictyostelium

Ralph Graf

Introduction

337

(3)

Isolation of Dictyostelium Centrosomes

340

(4)

Analysis of Isolated Dictyostelium Centrosomes

344

(5)

Generation of Monoclonal Antibodies against Dictyostelium Centrosomes

349

(5)

Conclusions

354

(5)

References

355

(4)

Index

359

(10)

Volumes in Series

369

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Centrosomes and Spindle Pole Bodies

Summary

Table of Contents

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