Microtubules, in vitro (eBook)

cover page 1
Series editors 3
Title page 4
copyright page 5
Contents 6
Contributors 14
Preface 20
SECTION I 24
CHAPTER 1 26
I. Introduction 26
II. Protocols 27
III. Buffer Compositions 36
IV. Concluding Comments 37
Acknowledgments 37
References 37
CHAPTER 2 40
Abstract 40
I. Why Tubulin from Nonneural Sources? 41
II. General Principles of Tubulin Purification 41
III. Specific Properties of Distinct Nonneural Sources 42
IV. Genetic Manipulation of Tubulin Genes 45
V. Isolation of Tubulin: Theme and Variation 47
VI. Protocol Notes 51
VII. Summary 52
Acknowledgments 52
References 52
CHAPTER 3 56
Abstract 56
I. Introduction 56
II. The Characterization of AA2, a Pan-specific Anti-beta-tubulinMonoclonal Antibody That Reacts with All Vertebrate beta-Tubulin Isotypes (Gene Products) 57
III. The Characterization of Isotype-Specific beta-TubulinMonoclonal and Polyclonal Antibodies 59
IV. The Characterization of Antibodies That Recognizethe Glutamyl Side Chain of Glutamylated Proteins 60
V. Summary 67
References 67
CHAPTER 4 70
Abstract 70
I. Introduction and Rationale 71
II. Methods and Materials 71
III. Results and Discussion 77
IV. Summary 81
References 81
CHAPTER 5 82
I. Introduction 83
II. Rationale 83
III. Methods 84
IV. Materials 95
V. Discussion 96
VI. Summary 96
Acknowledgments 96
References 97
CHAPTER 6 99
Abstract 99
I. Introduction 100
II. Methods 104
III. Results and Discussion 109
IV. Conclusion 120
Acknowledgments 121
References 121
CHAPTER 7 127
Abstract 127
I. Introduction 128
II. Methods 129
III. Summary 145
Acknowledgments 146
References 146
SECTION II 149
CHAPTER 8 151
Abstract 152
I. Introduction 152
II. Rationale 156
III. Methods 160
IV. Discussion 171
V. Summary 174
Acknowledgments 174
References 175
CHAPTER 9 179
Abstract 180
I. Introduction to AFM Imaging of Biomolecules 180
II. Rationale 181
III. AFM Principle and Operation Mode 181
IV. Practical Aspects of Microtubules Adsorption on Surface and AFM Imaging 183
V. AFM Imaging in Air of Microtubule 189
VI. AFM Imaging in Air of Tubulin or Microtubule:Protein Interactions 191
VII. Conclusion 193
References 194
CHAPTER 10 197
Abstract 197
I. Introduction 198
II. Mathematical and Computational Modeling: A Primer 199
III. Using Modeling to Understand Microtubule Dynamics 203
IV. Conclusions 208
References 209
CHAPTER 11 211
Abstract 212
I. Introduction 212
II. Method 214
III. Summary and Conclusion 222
Acknowledgments 222
References 223
CHAPTER 12 229
Abstract 229
I. Introduction 230
II. Rationale 231
III. Methods 231
IV. Materials 234
V. Discussion 236
VI. Summary 239
Acknowledgments 239
References 239
CHAPTER 13 243
Abstract 244
I. Introduction 244
II. Single-Molecule TIRF Microscopy 246
III. List of Reagents 249
IV. Choice of Fluorophore/Protein Labeling 251
V. (Anti-)blinking/Photo-Toxicity/Photo-Bleaching Cocktails 252
VI. Preparation of GMPCPP-Stabilized Microtubules 255
VII. Glass Treatment and Sample Chamber Preparation 258
VIII. Binding of Microtubules and Passivation of Surfaces 261
IX. Dynamic Microtubule Assays 263
References 265
CHAPTER 14 269
Abstract 269
I. Introduction 270
II. Setup of Gliding Motility Assays 271
III. Analysis of Microtubule and Quantum Dot Movements 281
IV. Future Directions 288
Acknowledgments 289
Reagents 289
References 291
SECTION III 295
CHAPTER 15 297
Abstract 297
I. Introduction 298
II. Materials and Methods 298
III. Results and Discussion 304
IV. Summary 308
References 308
CHAPTER 16 311
Abstract 311
I. Introduction 312
II. Methods 314
III. Materials 320
Acknowledgments 320
References 320
CHAPTER 17 323
Abstract 324
I. Introduction 324
II. Colchicine Binding to Tubulin 325
III. Vinblastine Binding to Tubulin 332
IV. Taxol Binding to Microtubules 338
V. Determination of Binding Constants Using Extrinsic Fluorescent Probes 346
VI. Conclusion 347
Acknowledgments 348
References 348
CHAPTER 18 353
Abstract 353
I. Introduction 354
II. Rationale 355
III. Methods 364
IV. Summary 371
References 372
CHAPTER 19 375
Abstract 376
I. Introduction 376
II. Materials 377
III. Methods 377
IV. Applications and Discussion 384
V. Summary 390
Acknowledgments 392
References 392
CHAPTER 20 395
Abstract 396
I. Introduction 396
II. Rationale 397
III. Structural Studies of the Tubulin–Vinca Domain Ligand Interactions 398
IV. Fluorescence Is Useful to Monitor Binding of Vinca Domain Ligands to Tubulin in Solution 404
V. Effects of Vinca Domain Ligands on Tubulin Biochemical Properties 405
VI. Discussion 409
Acknowledgments 410
References 410
CHAPTER 21 413
Abstract 413
I. Introduction and Rationale 414
II. Methods 415
III. Discussion 423
Acknowledgments 424
References 424
SECTION IV 427
CHAPTER 22 429
Abstract 429
I. Introduction 430
II. Rationale 431
III. Methods 436
IV. Application to Tubulin/Microtubules Interactions 444
V. Conclusion 463
VI. Appendix I. Materials 463
References 466
CHAPTER 23 471
Abstract 471
I. Introduction 472
II. Rationale 474
III. Materials and Methods 478
IV. Discussion 485
V. Concluding Remarks 496
Acknowledgments 497
References 497
CHAPTER 24 503
Abstract 504
I. Introduction and Rationale 504
II. Methods 512
III. Materials 517
IV. Summary 519
Acknowledgments 520
References 520
CHAPTER 25 527
Abstract 527
I. Introduction 528
II. Rationale 529
III. Methods 530
IV. Fluorescent Labeling for FPM 538
V. Discussion and Future Directions 539
Acknowledgments 540
References 540
CHAPTER 26 543
Abstract 543
I. Introduction 544
II. TIRF Optics 545
III. Labeling Molecules 550
IV. Examples and Protocols 550
V. Conclusions and Outlook 562
Acknowledgments 563
References 563
CHAPTER 27 565
Abstract 565
I. Introduction 566
II. Rationale 567
III. Methods 568
IV. Discussion 575
Acknowledgments 575
References 575
CHAPTER 28 577
Abstract 578
I. Introduction 578
II. Rationale 579
III. Materials 580
IV. Methods 581
V. Surface Chemistry on Glass 583
VI. Fluorescence Microscopy Assays 587
VII. Discussion 598
VIII. Conclusion 599
Acknowledgments 600
References 600
SECTION V 603
CHAPTER 29 605
Abstract 605
I. Introduction to Cytoskeletal Filament Dynamics 606
II. Single-Molecule Lifetime Measurements 607
III. Theoretical Foundations 609
IV. Results and Conclusion 619
References 621
CHAPTER 30 623
Abstract 623
I. Introduction 624
II. Rationale 626
III. Materials 628
IV. Methods 629
V. Discussion 635
VI. Summary 636
Acknowledgments 636
References 636
CHAPTER 31 639
Abstract 639
I. Introduction 640
II. Materials 642
III. Methods 643
IV. Results 655
V. Conclusion 656
Acknowledgments 658
References 658
CHAPTER 32 663
Abstract 663
I. Introduction 664
II. Methods 664
III. Conclusion 678
References 678
CHAPTER 33 679
Abstract 679
I. Introduction 680
II. Rationale 681
III. Materials and Methods 682
IV. Summary and Discussion 695
Acknowledgments 696
References 696
INDEX 699
VOLUMES IN SERIES 715