Microtubule Dynamics: Methods and Protocols

ISBN-13: 9781617792519 / Angielski / Twarda / 2011 / 319 str.

Anne Straube

Opis

Microtubules are at the heart of cellular self-organization, and their dynamic nature allows them to explore the intracellular space and mediate the transport of cargoes from the nucleus to the outer edges of the cell and back. In Microtubule Dynamics: Methods and Protocols, experts in the field provide an up-to-date collection of methods and approaches that are used to investigate microtubule dynamics in vitro and in cells. Beginning with the question of how to analyze microtubule dynamics, the volume continues with detailed descriptions of how to isolate tubulin from different sources and with different posttranslational modifications, methods used to study microtubule dynamics and microtubule interactions in vitro, techniques to investigate the ultrastructure of microtubules and associated proteins, assays to study microtubule nucleation, turnover, and force production in cells, as well as approaches to isolate novel microtubule-associated proteins and their interacting proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Definitive and practical, Microtubule Dynamics: Methods and Protocols provides the key protocols needed by novices and experts on how to perform a broad range of well-established and newly-emerging techniques in this vital field.

Spis treści

1. How to Measure Microtubule Dynamics?

            Anne Straube

 

2. Purification of Tubulin from Porcine Brain

            Christopher Gell, Claire T. Friel, Barbara Borgonovo, David N. Drechsel, Anthony A. Hyman, and Jonathon Howard

 

3. Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe

            Douglas R Drummond, Susan Kain, Anthony Newcombe, Christina Hoey, Miho Katsuki, and Robert A. Cross

 

4. Generation of Differentially Polyglutamylated Microtubules

            Benjamin Lacroix and Carsten Janke

 

5. Preparation of Pure Tyrosinated or Detyrosinated Tubulin Isoforms

            Laurence Lafanechère and Didier Job

 

6. ab-Tubulin and Microtubule Binding Assays

            Jaime N. Campbell and Kevin C. Slep

 

7. Analysis of Microtubule Assembly Kinetics Using Turbidimetry

            Felicia Gaskin

 

8. Isolation of Centrosomes from Cultured Cells

            Simone Reber

 

9. Preparation of Dual-Color Polarity-Marked Fluorescent Microtubule Seeds

            Miho Katsuki, Etsuko Muto, and Rob Cross

 

10. Reconstitution and Quantification of Dynamic Microtubule End Tracking In Vitro Using TIRF Microscopy

            Ivo A. Telley, Peter Bieling, and Thomas Surrey

 

11. Force Generation by Dynamic Microtubules In Vitro

            Svenja-Marei Kalisch, Liedewij Laan, and Marileen Dogterom

 

12. Variations on the Single Molecule Assay for Microtubule-Associated Proteins and Kinesins

            Susanne Bechstedt, Michal Wieczorek, Michael Noujaim, and Gary J. Brouhard

 

13. Analyzing the ATP Hydrolysis Cycle of Microtubule Motors

            Claire T. Friel, Clive R. Bagshaw, and Jonathon Howard

 

14. Cryo-Electron Tomography of Microtubules Assembled In Vitro from Purified Components

            Frédéric M. Coquelle, Sophie Blestel, Claire Heichette, Isabelle Arnal, Charles Kervrann, and Denis Chrétien

 

15. Kinetochore Microtubules, Analyzed by Correlated Light and Immuno-Electron Microscopy

            Andreas Merdes and Jan De Mey

 

16. Photoactivatable-GFP-a-Tubulin as a Tool to Study Microtubule Plus-End Turnover in Living Human Cells

            Catarina P. Samora and Andrew D. McAinsh

 

17. Quantification of Asymmetric Microtubule Nucleation at Sub-Cellular Structures

            Xiaodong Zhu and Irina Kaverina

 

18. Investigating Microtubule Dynamic Instability Using Microtubule Targeting Agents

            Stéphane Honore and Diane Braguer

 

19. Laser Ablation of the Microtubule Cytoskeleton: Setting Up and Working with an Ablation System

            Nicola Maghelli and Iva Tolić-Nørrelykke

 

20. Isolation, Identification, and Validation of Microtubule-Associated Proteins from Drosophila Embryos

            Robin Antrobus and James G. Wakefield

 

21. Isolation of Novel +TIPs and Their Binding Partners Using Affinity Purification Techniques

            Ka Lou Yu, Nanda Keijzer, Casper C. Hoogenraad, and Anna Akhmanova