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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.
List of contents
Purification of Mammalian Tubulins and Tubulin-Associated Proteins using a P2A-based Expression System.- Expression and Purification of Microtubule Associated Proteins from HEK293T Cells for In Vitro Reconstitution.- In vitro Reconstitution Assays of Microtubule Amplification and Lattice Repair by the Microtubule Severing Enzymes.- In vitro Microtubule Dynamics Assays using Darkfield Microscopy.- In vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Laments in Emulsion Droplets.- Studying Tau-microtubule Interaction using Single Molecule TIRF Microscopy.- In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis.- Methods for Studying Membrane-associated Bacterial Cytoskeleton Proteins in vivo by TIRF Microscopy.- Processing TIRF Microscopy Images to Characterize the Dynamics and Morphology of Bacterial Actin-like Assemblies.- Studying Centriole Duplication and Elongation in Human Cells.- Assaying Microtubule Nucleation.- Detection of Microtubule Nucleation Hotspots at the Golgi.- Ultrastructural Analysis of Microtubule Ends.- Optogenetic Control of Microtubule Dynamics.- Measurement of Microtubule Half-Life and Poleward Flux in the Mitotic Spindle by Photoactivation of Fluorescent Tubulin.- Functional Dissection of Mitosis using Immortalized Fibroblasts from the Indian Muntjac, A Placental Mammal with only Three Chromosomes.- Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes.- Using the Four-cell C. elegans Embryo to Study Contractile Ring Dynamics during Cytokinesis.- Knocking out Multiple Genes in Cultured Primary Neurons to Study Tubulin Posttranslational Modifications.- Measuring the Role of Tubulin Posttranslational Modifications on Axonal Transport.- Methods to Measure Perinuclear Actin Dynamics during Nuclear Movement in Migrating Cells.
