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
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 TubulinVinca 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;6
36 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
