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Methods in cell biology. v. 95, Microtubules, in vitro / edited by Leslie Wilson, John J. Correia. — Amsterdam : Elsevier, c2010. – (58.1574/M592/v.95) |
Contents
CONTENTS
Contributors
Preface
SECTION I Isolation and Biochemistry of Tubulin and Characterization of Antibodies and Isotypes
1. Preparation of Microtubule Protein and Purified Tubulin from Bovine Brain by Cycles of Assembly and Disassembly and Phosphocellulose Chromatography
I. Introduction
II. Protocols
III. Buffer Compositions
IV. Concluding Comments
References
2. Isolating Tubulin from Nonneural Sources
I. Why Tubulin from Nonneural Sources?
II. General Principles of Tubulin Purification
III. Specific Properties of Distinct Nonneural Sources
IV. Genetic Manipulation of Tubulin Genes
V. Isolation of Tubulin: Theme and Variation
VI. Protocol Notes
VII. Summary
References
3. Characterization of Anti-β-tubulin Antibodies
I. Introduction
II. The Characterization ofAA2， a Pan-specific Anti-β-tubulin Monoclonal Antibody That Reacts with All Vertebrate β-Tubulin Isotypes (Gene Products)
III. The Characterization of Isotype-Specific β-Tubulin Monoclonal and Polyclonal Antibodies
IV. The Characterization of Antibodies That Recognize the Glutamyl Side Chain of Glutamylated Proteins
V. Summary
References
4. Expression Profiling of Tubulin Isotypes and Microtubule-Interacting Proteins Using Real-Time Polymerase Chain Reaction
I. Introduction and Rationale
II. Methods and Materials
III. Results and Discussion
IV. Summary
References
5. Nondenaturing Electrophoresis as a Tool to Investigate Tubulin Complexes
I. Introduction
II. Rationale
III. Methods
IV. Materials
V. Discussion
VI. Summary
References
6. Mass Spectrometry Analysis of C-Terminal Posttranslational Modifications of Tubulins
I. Introduction
II. Methods
III. Results and Discussion
IV. Conclusion
References
7. Methods in Tubulin Proteomics
I. Introduction
II. Methods
III. Summary
References
SECTION II Microtubule Structure and Dynamics
8. Cryo-EM Studies of Microtubule Structural Intermediates and Kinetochore-Microtubule Interactions
I. Introduction
II. Rationale
III. Methods
IV. Discussion
V. Summary
References
9. High-Resolution Imaging of Microtubules and Cytoskeleton Structures by Atomic Force Microscopy
I. Introduction to AFM Imaging of Biomolecules
II. Rationale
III. AFM Principle and Operation Mode
IV. Practical Aspects of Microtubules Adsorption on Surface and AFM Imaging
V. AFM Images in Air of Microtubule
VI. AFM Imaging in Air of Tubulin or Microtubule: Protein Interactions
VII. Conclusion
References
10. Using Computational Modeling to Understand Microtubule Dynamics: A Primer for Cell Biologists
I. Introduction
II. Mathematical and Computational Modeling: A Primer
III. Using Modeling to Understand Microtubule Dynamics
IV. Conclusions
References
11. Analysis of Dynamic Instability of Steady-State Microtubules In Vitro by Video-Enhanced Differential Interference Contrast Microscopy with an Appendix by Emin Oroudjev
I. Introduction
II. Method
III. Summary and Conclusion
References
Appendix
I. Introduction
II. MT-LHAP and Its Features
III. Instructions to Use MT-LHAP
IV. Conclusion
References
12. Nanometer-Resolution Microtubule Polymerization Assays Using Optical Tweezers and Microfabricated Barriers
I. Introduction
II. Rationale
III. Methods
IV. Materials
V. Discussion
VI. Sunnnary
References
13. Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy
I. Introduction
II. Single-Molecule TIRF Microscopy
III. List of Reagents
IV. Choice of Fluorophore/Protein Labeling
V. (Anti-)blinking/Photo-Toxicity/Photo-Bleaching Cocktails
VI. Preparation of GMPCPP-Stabilized Microtubules
VII. Glass Treatment and Sample Chamber Preparation
VIII. Binding of Microtubules and Passivation of Surfaces
IX. Dynamic Microtubule Assays
References
14. Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays
I. Introduction
II. Setup of Gliding Motility Assays
III. Analysis of Microtubule and Quantum Dot Movements
IV. Future Directions
Reagents
References
SECTION III Drugs
15. Analysis of Tubulin Oligomers by Analytical Ultracentrifugation
I. Introduction
II. Materials and Methods
III. Results and Discussion
IV. Summary
References
16. Determination of Drag Binding to Microtubules In Vitro
I. Introduction
II. Methods
III. Materials
References
17. Fluorescence Spectroscopic Methods to Analyze Drug-Tubulin Interactions
I. Introduction
II. Colchicine Binding to Tubulin
III. Vinblastine Binding to Tubulin
IV. Taxol Binding to Microtubules
V. Determination of Binding Constants Using Extrinsic Fluorescent Probes
VI. Conclusion
References
18. A Tubulin Polymerization Microassay Used to Compare Ligand Efficacy
I. Introduction
II. Rationale
III. Methods
IV. Summary
References
19. Fluorescent Taxoid Probes for Microtubule Research
I. Introduction
II. Materials
III. Methods
IV. Applications and Discussion
V. Summary
References
20. The Binding of Vinca Domain Agents to Tubulin: Structural and Biochemical Studies
I. Introduction
II. Rationale
III. Structural Studies of the Tubulin-Vinca Domain Ligand Interactions
IV. Fluorescence Is Useful to Monitor Binding of Vinca Domain Ligands to Tubulin in Solution
V. Effects of Vinca Domain Ligands on Tubulin Biochemical Properties
VI. Discussion
References
21. Measurement of Ligand Binding to Tubulin by Sulfhydryl Reactivity
I. Introduction and Rationale
II. Methods
III. Discussion
References
SECTION IV Interactions with Motors and MAPs
22. Probing Interactions of Tubulin with Small Molecules， Peptides， and Protein Fragments by Solution Nuclear Magnetic Resonance
I. Introduction
II. Rationale
III. Methods
IV. Application to Tubulin/Microtubules Interactions
V. Conclusion
VI. Appendix I. Materials
References
23. Microtubule and MAPs: Themlodynamics of Complex Formation by AUC， ITC， Fluorescence， and NMR
I. Introduction
II. Rationale
III. Materials and Methods
IV. Discussion
V. Concluding Remarks
References
24. Quantitative Analysis of MAP-Mediated Regulation of Microtubule Dynamic Instability In Vitro-Focus on Tau
I. Introduction and Rationale
II. Methods
III. Materials
IV. Summary
References
25. Structure and Dynamics of the Kinesin-Microtubule Interaction Revealed by Fluorescence Polarization Microscopy
I. Introduction
II. Rationale
III. Methods
IV. Fluorescent Labeling for FPM
V. Discussion and Future Directions
References
26. Multiple Color Single Molecule TIRF Imaging and Tracking of MAPs and Motors
I. Introduction
II. TIRF Optics
III. Labeling Molecules
IV. Examples and Protocols
V. Conclusions and Outlook
References
27. Studying Plus-End Tracking at Single Molecule Resolution Using TIRF Microscopy
I. Introduction
II. Rationale
III. Methods
IV. Discussion
References
28. Fluorescence Microscopy Assays on Chemically Functionalized Surfaces for Quantitative Imaging of Microtubule， Motor， and +TIP Dynamics
I. Introduction
II. Rationale
III. Materials
IV. Methods
V. Surface Chemistry on Glass
VI. Fluorescence Microscopy Assays
VII. Discussion
VIII. Conclusion
References
SECTION V Functional Extracts and Force Measurements
29. Quantitative Characterization of Filament Dynamics by Single-Molecule Lifetime Measurements
I. Introduction to Cytoskeletal Filament Dynamics
II. Single-Molecule Lifetime Measurements
III. Theoretical Foundations
IV. Results and Conclusion
References
30. Extracting the Mechanical Properties of Microtubules from Thermal Fluctuation Measurements on an Attached Tracer Particle
I. Introduction
II. Rationale
III. Materials
IV. Methods
V. Discussion
VI. Summary
References
31. In Vitro Assays to Study Force Generation at Dynamic Microtubule Ends
I. Introduction
II. Materials
III. Methods
IV. Results
V. Conclusion/Discussion
References
32. Reconstitution and Functional Analysis of Kinetochore Subcomplexes
I. Introduction
II. Methods
Ill. Conclusion
References
33. In Vitro Assays to Study the Tracking of Shortening Microtubule Ends and to Measure Associated Forces
I. Introduction
II. Rationale
III. Materials and Methods
IV. Summary and Discussion
References
Index
Volume in Series
