Methods in Cell Biology: Mitosis and Meiosis

, by Rieder, Conly L.; Matsudaira, Paul; Wilson, Leslie

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ISBN: 9780125441636 | 0125441630
Cover: Hardcover
Copyright: 1/1/1999

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Mitosis and Meiosis details the wide variety of methods currently used to study how cells divide as yeast and insect spermatocytes, higher plants, and sea urchin zygotes. With chapters covering micromanipulation of chromosomes and making, expressing, and imaging GFP-fusion proteins, this volume contains state-of-the-art "how to" secrets that allow researchers to obtain novel information on the biology of centrosomes and kinetochores and how these organelles interact to form the spindle. Chapters Contain Information On: * How to generate, screen, and study mutants of mitosis in yeast, fungi, and flies * Techniques to best image fluorescent and nonfluorescent tagged dividing cells * The use and action of mitoclastic drugs * How to generate antibodies to mitotic components and inject them into cells * Methods that can also be used to obtain information on cellular processes in nondividing cells

Contributors

(4)

Preface

1. Isolation of Centrosomes from Drosophila Embryos

(13)

Michelle Moritz

Bruce M. Alberts

I. Introduction

(1)

II. Isolation of Centrosomes from Drosophila Embryos

(3)

III. Assays for Microtubule Nucleation by Isolated Centrosomes

(6)

IV. Conclusions

(1)

References

(3)

2. Studying the Composition and Function of Centrosomes in Vertebrates

(22)

Michel Bomens

Mohammed Moudjou

I. Introduction

(1)

II. Isolation of Centrosomes from Animal Cells

(3)

III. Preparation of Immunological Probes from Isolated Centrosomes

(2)

IV. Ultrastructural Analysis of Isolated Centrosomes

(4)

V. Biochemical Composition of Centrosomes

(3)

VI. Functional Assays of Isolated Centrosomes

(2)

VII. Prospects

(2)

References

(5)

3. Isolation of Centrosomes from Spisula solidissima Oocytes

(21)

Robert E. Palazzo

Jacalyn M. Vogel

I. Introduction

(1)

II. Obtaining Organisms

(1)

III. Isolation and Activation of Spisula solidissima Oocytes

(4)

IV. Preparation of Oocyte Lysates

(2)

V. Preparation of Microtubule Protein

(2)

VI. Isolation of Centrosomes from Activated Oocyte Lysates

(4)

VII. Immunofluorescence of Centrosomes and Asters

(3)

VIII. Electron Microscopy of Asters and Centrosomes

(1)

IX. Summary

(3)

References

(1)

4. Methods for in Situ Localization of Proteins and DNA in the Centromere-Kinetochore Complex

(25)

A. Van Hooser

B.R. Brinkley

I. Introduction

(2)

II. In Situ Localization of Proteins: Indirect Immunofluorescence

(4)

III. In Situ Localization of Proteins: Immunogold EM

(3)

IV. Fluorescent in Situ Hybridization Using DNA Satellite Probes

(3)

V. Combination Staining: DNA/Protein

(3)

VI. Specialized Techniques

(6)

References

(4)

5. Three-Dimensional Transmission Electron Microscopy and Its Application to Mitosis Research

(32)

Bruce F. McEwen

Michael Marko

I. Introduction

(2)

II. Resolution and Choosing between Tomography and Serial Sections

(3)

III. Electron Tomography

(11)

IV. Serial Section Reconstruction

(1)

V. Analysis and Display of 3D Reconstructions

(6)

VI. Software Packages

105

(1)

VII. Summary and Conclusions

106

(2)

References

108

(6)

6. Enlightening Mitosis: Construction and Expression of Green Fluorescent Protein Fusion Proteins

114

(23)

Kevin F. Sullivan

I. Introduction: Visualizing the Molecular Anatomy of the Spindle

114

(1)

II. Fluorescence Properties of GFP

114

(4)

III. Strategies for Constructing Fusion Proteins

118

(5)

IV. Expression in Mammalian Cells

123

(10)

References

133

(4)

7. Recombinant p50/Dynamitin as a Tool to Examine the Role of Dynactin in Intracellular Processes

137

(8)

Torsten Wittmann

Tony Hyman

I. Introduction

137

(1)

II. Production of Recombinant p50/Dynamitin

138

(2)

III. Disruption of the Dynactin Complex by p50/Dynamitin in Xenopus Egg Extracts

140

(2)

IV. Disruption of Spindle Poles Using p50/Dynamitin

142

(1)

References

142

(3)

8. In Vitro Assays for Studying Saccharomyces cerevisiae Kinetochore Activity

145

(11)

Fedor Severin

Ken Kaplan

Peter Sorger

Tony Hyman

I. Introduction

145

(1)

II. Microtubule-Binding Assays for S. cerevisiae Kinetochores

146

(5)

III. Band Shift Assay for the Kinetochore Complex

151

(2)

References

153

(3)

9. Fluorescent Speckle Microscopy of Spindle Microtubule Assembly and Motility in Living Cells

156

(19)

Clare Waterman-Storer

Arshad Desai

E.D. Salmon

I. Introduction

156

(1)

II. Principles of the Fluorescence Speckle Method for Microtubules

156

(2)

III. Specimen Methods

158

(3)

IV. Microscopy and Image Acquisition

161

(3)

V. Image Processing and Analysis

164

(3)

VI. Examples

167

(3)

VII. Future Considerations

170

(2)

References

172

(3)

10. Polarized Light Microscopy of Spindles

175

(34)

Rudolf Oldenbourg

I. Introduction

175

(3)

II. Polarized Light Microscopy

178

(12)

III. Analysis of Spindle Birefringence

190

(7)

IV. Optimum Cell Types for Polarized Light Microscopy of Spindles

197

(9)

References

206

(3)

11. Micromanipulation of Chromosomes and Spindles in Insect Spermatocytes

209

(10)

Dahong Zhang

R. Bruce Nicklas

I. Introduction

209

(1)

II. Preparing for Micromanipulation

210

(6)

III. Manipulating Cell Components

216

(1)

References

217

(2)

12. Microinjection of Mitotic Cells

219

(14)

Patricia Wadsworth

I. Introduction

219

(2)

II. Choice of Cells

221

(2)

III. Timing of Injection

223

(1)

IV. Microinjection Procedure

223

(5)

V. Conclusion

228

(1)

References

229

(4)

13. Obtaining Antibodies to Spindle Components

233

(12)

Ryoko Kuriyama

Kathy Ensrud

I. Introduction

233

(2)

II. Methods

235

(8)

III. Discussion

243

(1)

References

244

(1)

14. Using Antisense Technology to Study Mitosis

245

(22)

Linda Wordeman

Mike Wagenbach

I. Introduction

245

(2)

II. Antisense Mechanism of Action

247

(1)

III. Choice of Antisense Reagents

248

(6)

IV. Assaying Target Protein Levels

254

(1)

V. Antisense Reagents Used to Study Cell Division

255

(6)

References

261

(6)

15. The Use and Action of Drugs in Analyzing Mitosis

267

(30)

Mary Ann Jordan

Leslie Wilson

I. Introduction: Why Use Drugs?

267

(1)

II. Brief Overview of Microtubule Assembly Dynamics

268

(2)

III. Mechanisms of Action of Major Antimitotic Drugs: Binding to Tubulin and Microtubules and Effects on Microtubule Polymerization and Dynamics and on Mitosis

270

(17)

IV. Determination of Intracellular Drug Levels

287

(1)

V. How to Use Antimitotic Drugs: Practical Guidelines

288

(1)

References

289

(8)

16. Correlative Light and Electron Microscopy of Mitotic Cells in Monolayer Cultures

297

(21)

Conly L. Rieder

Grisel Cassels

I. Introduction

297

(2)

II. Light Microscopy

299

(3)

III. Flat Embedding

302

(3)

IV. Preparing the Cell for Sectioning

305

(2)

V. Obtaining the Required Ultrastructural Information

307

(6)

References

313

(5)

17. Identification and Characterization of Mitotic Mutations in Drosophila

318

(30)

William E. Theurkauf

Margarete M. S. Heck

I. Introduction

318

(4)

II. Maternal-Effect Mutations That Disrupt the Syncytial Mitotic Divisions

322

(2)

III. Cytological Analysis of the Syncytial Mitoses

324

(10)

IV. Zygotic Mutations That Disrupt Mitosis in Larval Tissues

334

(3)

V. Cytological Analysis of Larval Brain and Imaginal Discs

337

(7)

References

344

(4)

18. Methods for Isolating and Analyzing Mitotic Mutants in Aspergillus nidulans

348

(21)

Berl R. Oakley

I. Introduction

348

(1)

II. Characteristics of Aspergillus nidulans

348

(5)

III. Strains and Media

353

(1)

IV. Harvesting Conidia and Preparing Conidial Suspensions

354

(1)

V. Mutagenesis

355

(1)

VI. Methods for Isolating Mitotic Mutants in A. nidulans

355

(6)

VII. Morphological Analysis of Mutants

361

(2)

VIII. Genetic Analysis of Mutants

363

(2)

IX. Molecular Genetic Methods for Working with A. nidulans

365

(2)

References

367

(2)

19. Using Green Fluorescent Protein Fusion Proteins to Quantitate Microtubule and Spindle Dynamics in Budding Yeast

369

(17)

Kerry Bloom

Dale L. Beach

Paul Maddox

Sidney L. Shaw

Elaine Yeh

E. D. Salmon

I. Introduction

369

(1)

II. Construction of Protein-GFP Fusion and Promoter Selection

370

(3)

III. Quantifying Fluorescence in Cell Populations

373

(2)

IV. The Imaging System

375

(1)

V. Quantitative Solution to the Imaging Problem

376

(2)

VI. Image Acquisition and Processing

378

(1)

VII. Applications and Examples: Expression of Dynein-GFP in Vivo

379

(2)

References

381

(5)

20. The Use of Xenopus Egg Extracts to Study Mitotic Spindle Assembly and Function in Vitro

386

(27)

Arshad Desai

Andrew Murray

Timothy J. Mitchison

Claire E. Walczak

I. Introduction

386

(1)

II. Preparation of CSF Extracts for Spindle Assembly

387

(6)

III. Spindle Assembly Reaction

393

(4)

IV. Monitoring Spindle Assembly Reactions

397

(4)

V. Manipulation of Extracts

401

(3)

VI. Data Analysis and Interpretation

404

(1)

VII. Anaphase in Vitro

405

(5)

VIII. Conclusions

410

(1)

References

411

(2)

21. Methods for Studying Cell Division in Higher Plants

413

(27)

Jan W. Vos

Aline H. Valster

Peter K. Hepler

I. Introduction

413

(1)

II. Cell Types of the Study of Cell Division

414

(14)

III. Microinjection of Plant Cells

428

(5)

IV. Conclusions

433

(1)

References

434

(6)

22. Using Sea Urchin Gametes for the Study of Mitosis

440

(33)

Greenfield Sluder

Fredrick J. Miller

Edward H. Hinchcliffe

I. Introduction

440

(1)

II. The Experimental System

440

(1)

III. Maintenance of the Organisms

441

(2)

IV. Obtaining Gametes

443

(1)

V. Zygotes

444

(4)

VI. Mounting Cells for Observation

448

(14)

VII. Other Methods

462

(2)

VIII. Annotated List of References

464

(8)

References

472

(1)

Index

473

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Methods in Cell Biology: Mitosis and Meiosis

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