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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 Second Edition brings together current research and protocols in the field of microtubules 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 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
1. Isolation of Antarctic fish tubulin and MAPs2. Making, isolating and characterizing tubulin alpha antibodies3. Techniques for labeling tubulin and drugs4. Single-particle 3D reconstruction methods for decorated and undecorated microtubules5. A new microtubule gliding assay and analysis of microtubule persistence length6. Characterization of gamma tubulin 7. Biochemical and Structural Studies of the doublecortin family of MAPs8. Detection and quantification of microtubule detachment from centrosomes and spindle poles9. Regulation of tubulin expression by micro RNAs: Implications for Drug Resistance10. A portable magnetic tweezers device to visualize three-dimensional deformation of reconstituted microtubule networks11. Mitochondrial/microtubule interactions – mmbp112. Going Solo: Measuring the motions of microtubules with an in vitro assay for TIRF microscopy13. Analysis of Microtubules and Motors in Isolated Axoplasm from the Squid Giant Axon14. Analysis of drug binding to microtubules and steady state microtubule growth/treadmilling by use of a sedimentation assay in vitro15. Specific recombinant antibody technology for the study of microtubule dynamics and regulatory proteins16. Tubulin-specific Chaperones: Components of a Molecular Machine that Assembles the aB Heterodimer17. G-protein tubulin interactions18. Katanin, a microtubule severing enzyme: expression, purification, and biophysical characterization19. Measuring tubulin polymerization in vitro – comparison of methods and possible artifacts20. Imaging microtubules in living plant cells21. How to use which antibody known in the field, what to expect from this antibody and how semi-quantitative data can be reliably obtained with antibody staining22. Preparation of bacterial tubulin BtubA/B and constructs bearing eukaryotic tubulin sequences23. Microtubule-associated proteins and tubulin interaction by isothermal titration calorimetry24. TBC25. Characterization of the mechanism of action of the covalent-binding microtubule-stabilizing agent zampanolide26. Studying kinetochore-fibre ultrastructure using correlative light-electron microscopy27. Fluorescence-based Assays for Microtubule Structure28. Yeast Tubulin-structure-function analysis29. Microtubule Binding SIm to simulate binding of proteins to polymers under different binding models