Centrosomes and spindle pole bodies / edited by Robert E. Palazzo, Trisha N. Davis.

Format:

Book

Contributor:

Palazzo, Robert E.

Davis, Trisha, 1954-

Series:

Methods in cell biology 0091-679X ; v. 67.

Methods in cell biology, 0091-679X ; v. 67

Language:

English

Subjects (All):

Centrosome--physiology.

Spindle Apparatus--physiology.

Centrosomes.

Spindle (Cell division).

Medical Subjects:

Centrosome--physiology.

Spindle Apparatus--physiology.

Physical Description:

xv, 375 pages, 16 unnumbered pages of plates : illustrations (some color) ; 25 cm.

Place of Publication:

San Diego : Academic Press, [2001]

Summary:

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.

Contents:

1. Purification and Reconstitution of Drosophila [gamma] - Tubulin Complexes / Ruwanthi N. Gunawardane, Yixian Zheng, Karen Oegema, Christiane Wiese

II. Isolation of [gamma]-Tubulin-Containing Complexes from Drosophila Embryo Extracts 3

III. [gamma]TuSC Reconstitution in Sf9 Cells 12

IV. Functional Assays for Isolated [gamma]TuSC or [gumma]TuRC 18

2. 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

II. How Rapid Freezing and Freeze-Substitution Work 29

III. Rapid Freezing Techniques 31

IV. Freeze-Substitution Protocols 33

V. Embedding Protocols 34

VI. Sectioning and Imaging 34

VII. Immunolabeling for Electron Microscopy 35

VIII. Three-Dimensional Modeling of Spindle Pole Bodies and Other Organelles 37

3. Digital Fluorescence Microscopy of Cell Cytoplasts with and without the Centrosome / Vladimir Rodionov, Elena Nadezhdina, John Peloquin, Gary Borisy

II. Experimental Procedures 44

4. Methods for the Study of Pericentrin in Centrosome Assembly and Function / Aruna Purohit, German A. Pihan, Stephen J. Doxsey

II. Pericentrin Localization to Centrosomes 54

III. Pericentrin Recruitment to Centrosomes 58

IV. Obtaining Enriched Fractions of Pericentrin 63

V. Artificial Elevation of Pericentrin Levels 66

VI. Pericentrin Staining to Detect Centrosome Defects in Tumor Tissues and Cell Lines 67

5. Molecular Dissection of Yeast Spindle Pole Bodies by Two Hybrid, in Vitro Binding, and Co-purification / C. Schramm, C. Janke, E. Schiebel

II. The Two-Hybrid Approach 73

III. Immuno- and Affinity Precipitation of Spindle Pole Body Components 84

IV. In Vitro Binding of Spindle Pole Body Proteins 89

6. Genetic Analysis of Yeast Spindle Pole Bodies / Trisha N. Davis

II. Dosage-Dependent Suppressor Screen 97

III. Extragenic Suppressor Screen 100

IV. Synthetic Lethal Screen 103

V. Synthetic Dosage Lethality 108

7. Methods for the Study of Centrosomes in Drosophila during Embryogenesis / Uyen Tram, Blake Riggs, Carol Koyama, Alain Debec, William Sullivan

II. Materials 114

8. Methods for Identification of Centrosome-Associated Proteins / Ryoko Kuriyama, Toshiro Ohta, Jacalyn Vogel, Gang Peng

II. Preparation of Centrosomes and Anticentrosomal Antibodies 128

III. Molecular Cloning of cDNA Encoding Centrosome-Associated Proteins 129

IV. Evaluation of Clone Specificity 132

V. Identification of a Novel 135-kDa Centrosomal Protein, Cep 135 135

9. Reconstitution of Centrosome Microtubule Nucleation in Drosophila / Michelle Moritz, Michael B. Braunfeld, Bruce M. Alberts, David A. Agard

II. Preparation of Extracts and Centrosomes from Drosophila Embryos 143

III. In Vitro Reconstitution of Microtubule Nucleation by Salt-Inactivated Centrosomes 144

10. Reconstitution of Centrosome Microtubule Nucleation in Spisula / Bradley J. Schnackenberg, Robert E. Palazzo

II. Preparations 151

III. Disassembly and Reassembly of Microtubule Nucleation Potential 154

IV. Fractionation of Extracts 159

11. 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

II. Protocol for Permeabilization 169

III. Protocol for Microtubule Nucleation 171

IV. Results 173

12. Gamma Tubulin and Microtubule Nucleation in Mammalian Cells / Harish C. Joshi, Jun Zhou

II. Interference with [gamma]-Tubulin Function in Mammalian Cells Abrogates Microtubule Nucleation 181

III. Overexpression of [gamma]-Tubulin in Mammalian Cells Causes Ectopic Microtubule Nucleation 187

13. Gamma Tubulin in Plant Cells / Yulia Ovechkina, Berl R. Oakley

II. Microtubule Arrays in Plant Cells 196

III. Methods for Cloning Plant [gamma]-Tubulin cDNAs and Genes 198

IV. Methods for Localizing [gamma]-Tubulin in Plant Cells 202

V. Functional Studies of [gamma]-Tubulin in Plants 209

VI. Future Prospects 210

14. Centrosomes and Parthenogenesis / Frederic Tournier, Michel Bornens

II. Parthenogenetic Test in Xenopus 214

III. Coupling between Egg Cell Cycle and Centrosome Duplication Pathway 216

IV. Species Barrier Specificity 219

V. Active or Inactive Centrosomes from the Same Species 220

VI. Prospects 221

15. In Vitro Approaches for the Study of Molecular Motors in Aster Formation / Duane A. Compton

II. Microtubule Aster Formation 228

III. Examination of Microtubule Motor Function in Aster Assembly 231

16. Methods for the Study of Centrosome-Independent Spindle Assembly in Xenopus Extracts / Sarah M. Wignall, Rebecca Heald

II. Preparation of Xenopus Egg Extract and Necessary Reagents 243

III. Microtubule Polymerization Assays in Extract 243

IV. Spindle Assembly Assays 245

V. Studying Proteins Involved in Spindle Assembly 249

VI. Methods to Observe Spindle Assembly 253

17. Methods for the Study of Centrosome Reproduction in Mammalian Cells / Ron Balczon

II. Induction of Centrosome Overproduction in Cultured Mammalian Cells 259

III. Preparation of Extracts from Hydroxyurea-Arrested Chinese Hamster Ovary Cells 262

IV. Analysis of Centrosome Protein Function Using Mammalian Cell Extracts and Salt-Stripped Centrosome Remnants 263

V. Conclusions and Prospects 266

18. Centrosome Reproduction in Xenopus Lysates / Edward H. Hinchcliffe, Greenfield Sluder

II. Reagents and Equipment 272

III. Methods 278

19. Centrosome Reproduction in Vitro: Mammalian Centrosomes in Xenopus Lysates / Matthieu Piel, Michel Bornens

II. Logic of the Assay 291

III. Duplication Assay 292

IV. Major Pitfalls 297

VI. Prospects 302

20. Identification of Centrosome Kinases / Andrew M. Fry, Alison J. Faragher

II. Subcellular Localization of Previously Characterized Protein Kinases 307

III. Isolation of Novel Centrosome Kinases 313

IV. Functional Analysis of Centrosome Kinases 315

V. Identifying Substrates for Centrosome Kinases 320

VI. Perspectives 321

21. Methods for the Analysis of Centrosome Reproduction in Cancer Cells / Wilma L. Lingle, Jeffrey L. Salisbury

II. Tissue Procurement and Storage 326

III. Preparation and Storage of Xenopus Egg Extract 327

IV. Reaction Mixture Composition and Use 328

V. Experimental Design and Data Analysis 330

22. Isolation of Centrosomes from Dictyostelium / Ralph Graf

II. Isolation of Dictyostelium Centrosomes 340

III. Analysis of Isolated Dictyostelium Centrosomes 344

IV. Generation of Monoclonal Antibodies against Dictyostelium Centrosomes 349.

Notes:

Includes bibliographical references and index.

ISBN:

0125441703

0125439857

OCLC:

47906398