Cytoskeleton Dynamics Methods and Protocols

This volume details comprehensive state-of-the-art methods on actin microfilaments and microtubules and how they work to achieve different cellular functions in different cellular contexts. Chapters guide readers through protein purification, in vitro reconstitution of several cytoskeleton properties, analyses of microtubule- and actin-based structures, functional dissection of post-translational modifications, and roles in several biological processes. 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.

 Authoritative and cutting-edge, Cytoskeleton Dynamics: Methods and Protocols aims to provide a wide range of experimental approaches and be an invaluable resource for present and future generations of cytoskeleton researchers.

The chapter “Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes” is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

1. Purification of Mammalian Tubulins and Tubulin-Associated Proteins using a P2A-based Expression System

Nuo Yu and Niels Galjart

2. Expression and Purification of Microtubule Associated Proteins from HEK293T Cells for In Vitro Reconstitution

Shasha Hua and Kai Jiang

3. In vitro Reconstitution Assays of Microtubule Amplification and Lattice Repair by the Microtubule Severing Enzymes Katanin and Spastin

4. In vitro Microtubule Dynamics Assays using Darkfield Microscopy

Jeffrey O. Spector, Annapurna Vemu, and Antonina Roll-Mecak

 5. In vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Laments in Emulsion Droplets

Kim J.A. Vendel, Celine Alkemade, Nemo Andrea, Gijsje H. Koenderink, and Marileen Dogterom

6. Studying Tau-microtubule Interaction using Single Molecule TIRF Microscopy

7. In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis

Stephanie C. Ems-McClung and Claire E. Walczak

8. Methods for Studying Membrane-associated Bacterial Cytoskeleton Proteins in vivo by TIRF Microscopy
Charlène Cornilleau, Arnaud Chastanet, Cyrille Billaudeau, and Rut Carballido-López

9. Processing TIRF Microscopy Images to Characterize the Dynamics and Morphology of Bacterial Actin-like Assemblies

Cyrille Billaudeau, Arnaud Chastanet, and Rut Carballido-López

10. Studying Centriole Duplication and Elongation in Human Cells

Catarina Peneda, Carla A.M. Lopes, and Mónica Bettencourt-Dias

11. Assaying Microtubule Nucleation

Artur Ezquerra, Ricardo Viais, and Jens Lüders

12. Detection of Microtubule Nucleation Hotspots at the Golgi

Roslin J. Thoppil, Anna A.W.M Sanders, and Irina Kaverina

13. Ultrastructural Analysis of Microtubule Ends

J. Richard McIntosh, Eileen O’Toole, Cynthia Page, and Garry Morgan

14. Optogenetic Control of Microtubule Dynamics

Jeffrey van Haren, Lauren Adachi, and Torsten Wittmann

15. Measurement of Microtubule Half-Life and Poleward Flux in the Mitotic Spindle by Photoactivation of Fluorescent Tubulin

Hugo Girão and Helder Maiato

16. Functional Dissection of Mitosis using Immortalized Fibroblasts from the Indian Muntjac, A Placental Mammal with only Three Chromosomes

Ana C. Almeida, Danica Drpic, Naoyuki Okada, Joana Bravo, Marta Madureira, and Helder Maiato

17. Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes

Binyam Mogessie

18. Using the Four-cell C. elegans Embryo to Study Contractile Ring Dynamics during Cytokinesis

Fung Yi Chan, Ana Marta Silva, and Ana Xavier Carvalho

19. Knocking out Multiple Genes in Cultured Primary Neurons to Study Tubulin Posttranslational Modifications

Satish Bodakuntla, Carsten Janke, and Maria M. Magiera

20. Measuring the Role of Tubulin Posttranslational Modifications on Axonal Transport

Satish Bodakuntla, Maria M. Magiera, and Carsten Janke

21. Methods to Measure Perinuclear Actin Dynamics during Nuclear Movement in Migrating Cells

Cátia S. Janota, Francisco J. Calero-Cuenca, and Edgar R. Gomes