Centrosomes and Spindle Pole Bodies

by Palazzo; Davis

• ISBN13: 9780125439855
• ISBN10: 0125439857
• Edition: 1st
• Format: Paperback
• Copyright: 2001-08-27
• Publisher: Elsevier Science

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