9780125439855

Centrosomes and Spindle Pole Bodies

by ;
  • ISBN13:

    9780125439855

  • ISBN10:

    0125439857

  • Edition: 1st
  • Format: Paperback
  • Copyright: 2001-08-27
  • Publisher: Elsevier Science
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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.

Table of Contents

Contributors xi
Preface xv
Purification and Reconstitution of Drosophila γ-Tubulin Complexes
Ruwanthi N. Gunawardane
Yixian Zheng
Karen Oegema
Christiane Wiese
Introduction
2(1)
Isolation of γ-Tubulin-Containing Complexes from Drosophila Embryo Extracts
3(9)
γTuSC Reconstitution in Sf9 Cells
12(6)
Functional Assays for Isolated γTuSC or γTuRC
18(5)
Concluding Remarks
23(5)
References
24(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
28(1)
How Rapid Freezing and Freeze-Substitution Work
29(2)
Rapid Freezing Techniques
31(2)
Freeze-Substitution Protocols
33(1)
Embedding Protocols
34(1)
Sectioning and Imaging
34(1)
Immunolabeling for Electron Microscopy
35(2)
Three-Dimensional Modeling of Spindle Pole Bodies and Other Organelles
37(3)
Conclusion
40(3)
References
41(2)
Digital Fluorescence Microscopy of Cell Cytoplasts with and without the Centrosome
Vladimir Rodionov
Elena Nadezhdina
John Peloquin
Gary Borisy
Introduction
43(1)
Experimental Procedures
44(10)
References
51(3)
Methods for the Study of Pericentrin in Centrosome Assembly and Function
Aruna Purohit
German A. Pihan
Stephen J. Doxsey
Introduction
54(1)
Pericentrin Localization to Centrosomes
54(4)
Pericentrin Recruitment to Centrosomes
58(5)
Obtaining Enriched Fractions of Pericentrin
63(3)
Artificial Elevation of Pericentrin Levels
66(1)
Pericentrin Staining to Detect Centrosome Defects in Tumor Tissues and Cell Lines
67(4)
References
68(3)
Molecular Dissection of Yeast Spindle Pole Bodies by Two Hybrid, in Vitro Binding, and Co-purification
C. Schramm
C. Janke
E. Schiebel
Introduction
71(2)
The Two-Hybrid Approach
73(11)
Immuno- and Affinity Precipitation of Spindle Pole Body Components
84(5)
In Vitro Binding of Spindle Pole Body Proteins
89(6)
References
92(3)
Genetic Analysis of Yeast Spindle Pole Bodies
Trisha N. Davis
Introduction
95(2)
Dosage-Dependent Suppressor Screen
97(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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