The Cytoskeleton

This volume explores techniques for studying, in detail, cytoskeletal structural and dynamic aspects that regulate cell organization and affect cell-cell interactions. The book features methods involving numerous areas of research, including the production of different relevant cytoskeletal proteins in native conditions, either in cells or in vitro, coupled to CCT chaperonin isolation for correct folding, the isolation of relevant organelles, actin and tubulin polymerization and motor transport, cryoelectron microscopy (CryoEM), post-translational modifications and protein localization, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips for troubleshooting and avoiding known pitfalls.

Authoritative and practical, The Cytoskeleton: Methods and Protocols serves as an ideal guide for researchers looking to untangle this complex and dynamic network of filaments.

Purification of the Eukaryotic Chaperonin CCT from Native Tissues. - Single-Step Purification of Endogenous Human Chaperonin CCT. - Integrated Protocol for TuRC Expression and Purification for Microtubule Nucleation and Cryo-EM Analysis. - Isolation of Centrosomal Fractions for Proteomic Studies in Lymphocytes. - Method for Analyzing Nucleoskeletal Components from the Isolated Nucleus. - In Vitro Analysis of Profilin 1 Binding to Recombinant Actin. - In Vitro Production of Full-Length Myosin 2 and Visualization of Actin Filament Motility by Total Internal Reflection Microscopy. - Reconstituting the Motility of Intraflagellar Transport Trains Ex Vivo. - Planar Immunological Synapses Formed by Human CD4 T Cells as In Vitro System to Study Actin and Tubulin Dynamics Through TIRF Microscopy. - Experimental Tools and Techniques for Studying Microtubule Nucleation at the Centrosome and the Golgi Apparatus. - Study of Unconventional Cytoskeleton Components Regulating Actin Contraction. - Methods to Study the Role of Cytoskeletal Motor Proteins in the Organization of the Post-Synaptic Membrane. - STED Super-Resolution Microscopy for Studying Actin Cytoskeleton Organization. - Immunofluorescence-Based Study of Cargo Transport Mediated by Cytoskeleton-Associated Molecular Complexes. - Cryo-EM Strategies for Elucidating the Closed State Architecture of CCT. - The Open Conformation of CCT: How to Achieve High Resolution Using CryoEM. - Methods to Study CCT Expression and Impact on Cytoskeleton. - Methods to Study Arginylation of Tubulin. - Post-Translational Dynamics of the Subcellular Proteome by Mass Spectrometry-Based Proteomics. - Computational and Statistical Framework for Quantitative Proteomics Analysis.