Correia / Wilson Microtubules, in vitro

1. Auflage 2010
ISBN: 978-0-08-088858-3
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 115, 699 Seiten, Web PDF

Reihe: Methods in Cell Biology

There continues to be intense interest in the microtubule cytoskeleton; the assembly, structure and regulation of microtubules; and the numerous motors and accessory proteins that control cell cycle, dynamics, organization and transport. The field continues to grow and explore new aspects of these issues driven immensely by developments in optical imaging and tracking techniques. This volume (complemented by the forthcoming companion volume by Cassimeris and Tran) brings together current research and protocols in the field of microtoubules in vitro and will serve as a valuable tool for cell biologists, biophysicists and pharmacologists who study the microtubule cytoskeleton, as well as for researchers in the biomedical and biotechnology communities with interest in developing drugs that target microtubules, MAPS and motors. * Chapters reflect both?experimental procedures and new developments in the field of microtubule in vitro research.
* Combines classical approaches and modern technologies
* Presents easy-to-use protocols and thorough background information, compiled by leaders?in the field

Autoren/Hrsg.

Correia, John J.

Wilson, Leslie

Inhaltsverzeichnis

1;cover page;1
2;Series editors;3
3;Title page;4
4;copyright page;5
5;Contents;6
6;Contributors;14
7;Preface;20
8;SECTION I;24
8.1;CHAPTER 1;26
8.1.1;I. Introduction;26
8.1.2;II. Protocols;27
8.1.3;III. Buffer Compositions;36
8.1.4;IV. Concluding Comments;37
8.1.5;Acknowledgments;37
8.1.6;References;37
8.2;CHAPTER 2;40
8.2.1;Abstract;40
8.2.2;I. Why Tubulin from Nonneural Sources?;41
8.2.3;II. General Principles of Tubulin Purification;41
8.2.4;III. Specific Properties of Distinct Nonneural Sources;42
8.2.5;IV. Genetic Manipulation of Tubulin Genes;45
8.2.6;V. Isolation of Tubulin: Theme and Variation;47
8.2.7;VI. Protocol Notes;51
8.2.8;VII. Summary;52
8.2.9;Acknowledgments;52
8.2.10;References;52
8.3;CHAPTER 3;56
8.3.1;Abstract;56
8.3.2;I. Introduction;56
8.3.3;II. The Characterization of AA2, a Pan-specific Anti-beta-tubulinMonoclonal Antibody That Reacts with All Vertebrate beta-Tubulin Isotypes (Gene Products);57
8.3.4;III. The Characterization of Isotype-Specific beta-TubulinMonoclonal and Polyclonal Antibodies;59
8.3.5;IV. The Characterization of Antibodies That Recognizethe Glutamyl Side Chain of Glutamylated Proteins;60
8.3.6;V. Summary;67
8.3.7;References;67
8.4;CHAPTER 4;70
8.4.1;Abstract;70
8.4.2;I. Introduction and Rationale;71
8.4.3;II. Methods and Materials;71
8.4.4;III. Results and Discussion;77
8.4.5;IV. Summary;81
8.4.6;References;81
8.5;CHAPTER 5;82
8.5.1;I. Introduction;83
8.5.2;II. Rationale;83
8.5.3;III. Methods;84
8.5.4;IV. Materials;95
8.5.5;V. Discussion;96
8.5.6;VI. Summary;96
8.5.7;Acknowledgments;96
8.5.8;References;97
8.6;CHAPTER 6;99
8.6.1;Abstract;99
8.6.2;I. Introduction;100
8.6.3;II. Methods;104
8.6.4;III. Results and Discussion;109
8.6.5;IV. Conclusion;120
8.6.6;Acknowledgments;121
8.6.7;References;121
8.7;CHAPTER 7;127
8.7.1;Abstract;127
8.7.2;I. Introduction;128
8.7.3;II. Methods;129
8.7.4;III. Summary;145
8.7.5;Acknowledgments;146
8.7.6;References;146
9;SECTION II;149
9.1;CHAPTER 8;151
9.1.1;Abstract;152
9.1.2;I. Introduction;152
9.1.3;II. Rationale;156
9.1.4;III. Methods;160
9.1.5;IV. Discussion;171
9.1.6;V. Summary;174
9.1.7;Acknowledgments;174
9.1.8;References;175
9.2;CHAPTER 9;179
9.2.1;Abstract;180
9.2.2;I. Introduction to AFM Imaging of Biomolecules;180
9.2.3;II. Rationale;181
9.2.4;III. AFM Principle and Operation Mode;181
9.2.5;IV. Practical Aspects of Microtubules Adsorption on Surface and AFM Imaging;183
9.2.6;V. AFM Imaging in Air of Microtubule;189
9.2.7;VI. AFM Imaging in Air of Tubulin or Microtubule:Protein Interactions;191
9.2.8;VII. Conclusion;193
9.2.9;References;194
9.3;CHAPTER 10;197
9.3.1;Abstract;197
9.3.2;I. Introduction;198
9.3.3;II. Mathematical and Computational Modeling: A Primer;199
9.3.4;III. Using Modeling to Understand Microtubule Dynamics;203
9.3.5;IV. Conclusions;208
9.3.6;References;209
9.4;CHAPTER 11;211
9.4.1;Abstract;212
9.4.2;I. Introduction;212
9.4.3;II. Method;214
9.4.4;III. Summary and Conclusion;222
9.4.5;Acknowledgments;222
9.4.6;References;223
9.5;CHAPTER 12;229
9.5.1;Abstract;229
9.5.2;I. Introduction;230
9.5.3;II. Rationale;231
9.5.4;III. Methods;231
9.5.5;IV. Materials;234
9.5.6;V. Discussion;236
9.5.7;VI. Summary;239
9.5.8;Acknowledgments;239
9.5.9;References;239
9.6;CHAPTER 13;243
9.6.1;Abstract;244
9.6.2;I. Introduction;244
9.6.3;II. Single-Molecule TIRF Microscopy;246
9.6.4;III. List of Reagents;249
9.6.5;IV. Choice of Fluorophore/Protein Labeling;251
9.6.6;V. (Anti-)blinking/Photo-Toxicity/Photo-Bleaching Cocktails;252
9.6.7;VI. Preparation of GMPCPP-Stabilized Microtubules;255
9.6.8;VII. Glass Treatment and Sample Chamber Preparation;258
9.6.9;VIII. Binding of Microtubules and Passivation of Surfaces;261
9.6.10;IX. Dynamic Microtubule Assays;263
9.6.11;References;265
9.7;CHAPTER 14;269
9.7.1;Abstract;269
9.7.2;I. Introduction;270
9.7.3;II. Setup of Gliding Motility Assays;271
9.7.4;III. Analysis of Microtubule and Quantum Dot Movements;281
9.7.5;IV. Future Directions;288
9.7.6;Acknowledgments;289
9.7.7;Reagents;289
9.7.8;References;291
10;SECTION III;295
10.1;CHAPTER 15;297
10.1.1;Abstract;297
10.1.2;I. Introduction;298
10.1.3;II. Materials and Methods;298
10.1.4;III. Results and Discussion;304
10.1.5;IV. Summary;308
10.1.6;References;308
10.2;CHAPTER 16;311
10.2.1;Abstract;311
10.2.2;I. Introduction;312
10.2.3;II. Methods;314
10.2.4;III. Materials;320
10.2.5;Acknowledgments;320
10.2.6;References;320
10.3;CHAPTER 17;323
10.3.1;Abstract;324
10.3.2;I. Introduction;324
10.3.3;II. Colchicine Binding to Tubulin;325
10.3.4;III. Vinblastine Binding to Tubulin;332
10.3.5;IV. Taxol Binding to Microtubules;338
10.3.6;V. Determination of Binding Constants Using Extrinsic Fluorescent Probes;346
10.3.7;VI. Conclusion;347
10.3.8;Acknowledgments;348
10.3.9;References;348
10.4;CHAPTER 18;353
10.4.1;Abstract;353
10.4.2;I. Introduction;354
10.4.3;II. Rationale;355
10.4.4;III. Methods;364
10.4.5;IV. Summary;371
10.4.6;References;372
10.5;CHAPTER 19;375
10.5.1;Abstract;376
10.5.2;I. Introduction;376
10.5.3;II. Materials;377
10.5.4;III. Methods;377
10.5.5;IV. Applications and Discussion;384
10.5.6;V. Summary;390
10.5.7;Acknowledgments;392
10.5.8;References;392
10.6;CHAPTER 20;395
10.6.1;Abstract;396
10.6.2;I. Introduction;396
10.6.3;II. Rationale;397
10.6.4;III. Structural Studies of the Tubulin–Vinca Domain Ligand Interactions;398
10.6.5;IV. Fluorescence Is Useful to Monitor Binding of Vinca Domain Ligands to Tubulin in Solution;404
10.6.6;V. Effects of Vinca Domain Ligands on Tubulin Biochemical Properties;405
10.6.7;VI. Discussion;409
10.6.8;Acknowledgments;410
10.6.9;References;410
10.7;CHAPTER 21;413
10.7.1;Abstract;413
10.7.2;I. Introduction and Rationale;414
10.7.3;II. Methods;415
10.7.4;III. Discussion;423
10.7.5;Acknowledgments;424
10.7.6;References;424
11;SECTION IV;427
11.1;CHAPTER 22;429
11.1.1;Abstract;429
11.1.2;I. Introduction;430
11.1.3;II. Rationale;431
11.1.4;III. Methods;436
11.1.5;IV. Application to Tubulin/Microtubules Interactions;444
11.1.6;V. Conclusion;463
11.1.7;VI. Appendix I. Materials;463
11.1.8;References;466
11.2;CHAPTER 23;471
11.2.1;Abstract;471
11.2.2;I. Introduction;472
11.2.3;II. Rationale;474
11.2.4;III. Materials and Methods;478
11.2.5;IV. Discussion;485
11.2.6;V. Concluding Remarks;496
11.2.7;Acknowledgments;497
11.2.8;References;497
11.3;CHAPTER 24;503
11.3.1;Abstract;504
11.3.2;I. Introduction and Rationale;504
11.3.3;II. Methods;512
11.3.4;III. Materials;517
11.3.5;IV. Summary;519
11.3.6;Acknowledgments;520
11.3.7;References;520
11.4;CHAPTER 25;527
11.4.1;Abstract;527
11.4.2;I. Introduction;528
11.4.3;II. Rationale;529
11.4.4;III. Methods;530
11.4.5;IV. Fluorescent Labeling for FPM;538
11.4.6;V. Discussion and Future Directions;539
11.4.7;Acknowledgments;540
11.4.8;References;540
11.5;CHAPTER 26;543
11.5.1;Abstract;543
11.5.2;I. Introduction;544
11.5.3;II. TIRF Optics;545
11.5.4;III. Labeling Molecules;550
11.5.5;IV. Examples and Protocols;550
11.5.6;V. Conclusions and Outlook;562
11.5.7;Acknowledgments;563
11.5.8;References;563
11.6;CHAPTER 27;565
11.6.1;Abstract;565
11.6.2;I. Introduction;566
11.6.3;II. Rationale;567
11.6.4;III. Methods;568
11.6.5;IV. Discussion;575
11.6.6;Acknowledgments;575
11.6.7;References;575
11.7;CHAPTER 28;577
11.7.1;Abstract;578
11.7.2;I. Introduction;578
11.7.3;II. Rationale;579
11.7.4;III. Materials;580
11.7.5;IV. Methods;581
11.7.6;V. Surface Chemistry on Glass;583
11.7.7;VI. Fluorescence Microscopy Assays;587
11.7.8;VII. Discussion;598
11.7.9;VIII. Conclusion;599
11.7.10;Acknowledgments;600
11.7.11;References;600
12;SECTION V;603
12.1;CHAPTER 29;605
12.1.1;Abstract;605
12.1.2;I. Introduction to Cytoskeletal Filament Dynamics;606
12.1.3;II. Single-Molecule Lifetime Measurements;607
12.1.4;III. Theoretical Foundations;609
12.1.5;IV. Results and Conclusion;619
12.1.6;References;621
12.2;CHAPTER 30;623
12.2.1;Abstract;623
12.2.2;I. Introduction;624
12.2.3;II. Rationale;626
12.2.4;III. Materials;628
12.2.5;IV. Methods;629
12.2.6;V. Discussion;635
12.2.7;VI. Summary;636
12.2.8;Acknowledgments;636
12.2.9;References;636
12.3;CHAPTER 31;639
12.3.1;Abstract;639
12.3.2;I. Introduction;640
12.3.3;II. Materials;642
12.3.4;III. Methods;643
12.3.5;IV. Results;655
12.3.6;V. Conclusion;656
12.3.7;Acknowledgments;658
12.3.8;References;658
12.4;CHAPTER 32;663
12.4.1;Abstract;663
12.4.2;I. Introduction;664
12.4.3;II. Methods;664
12.4.4;III. Conclusion;678
12.4.5;References;678
12.5;CHAPTER 33;679
12.5.1;Abstract;679
12.5.2;I. Introduction;680
12.5.3;II. Rationale;681
12.5.4;III. Materials and Methods;682
12.5.5;IV. Summary and Discussion;695
12.5.6;Acknowledgments;696
12.5.7;References;696
13;INDEX;699
14;VOLUMES IN SERIES;715