The Nuclear Pore Complex Methods and Protocols

This volume covers all methods used to discover the composition, structure, and dynamics of the nuclear pore complex (NPC), as well as the soluble transport factors involved in the transport process. Chapters detail structural biology methods, biophysical, transport factors , and a wide range of model organisms that have been used over the years. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Affinity Isolation of Endogenous Saccharomyces cerevisiae Nuclear Pore Complexes.- Transformation of Chaetomium thermophilum and affinity-purification of native thermostable protein complexes.- Nuclear Pore Complex Assembly using Xenopus Egg Extract.- Analysis of Nuclear Pore Complex Permeability in Mammalian Cells and Isolated Nuclei Using Fluorescent Dextrans.- Hormone-inducible Transport Reporter Assay to Study Nuclear Import Defects in Neurodegenerative Diseases.- Subcellular Fractionation Suitable for Studies of RNA and Protein Trafficking.- Localising Total mRNA in Plant Cells.- Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.- Analysis of Nucleoporin Function using Inducible Degron Techniques.- Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC).- Analysis of Nuclear Pore Complexes in Caenorhabditis elegans by Live Imaging and Functional Genomic.- Protein Retargeting in Aspergillus nidulans to Study the Function of Nuclear Pore Complex Proteins.- Split-GFP Complementation to Study the Nuclear Membrane Proteome using Microscopy.- Bimolecular Fluorescence Complementation: Quantitative Analysis of In Cell Interaction of Nuclear Transporter Importin a with Cargo Protein.- Validation of Nuclear Pore Complex Protein-Protein Interactions by Transient Expression in Plants.- Binding affinity measurement of Nuclear Export Signal peptides to their exporter CRM1.- Analysis of Ubiquitylation and SUMOylation of Yeast Nuclear Pore Complex Proteins.- Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.- Crystallization of Nuclear Export Signals or Small Molecule Inhibitors Bound to Nuclear Exporter CRM1.- Atomic Force Microscopy for Structural and Biophysical Investigations on Nuclear Pore Complexes.- Multivalent Interactions with Intrinsically DisorderedProteins Measured by Surface Plasmon Resonance.- Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology during Confined Migration.- SPEED Microscopy: High-Speed Single-Molecule Tracking and Mapping of Nucleocytoplasmic Transport.- Imaging Fluorescent Nuclear Pore Complex Proteins in C. elegans.- Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts.- TEM Imaging of Membrane Choreography During Mitosis of Drosophila Tissue Culture Cells.- Scanning Electron Microscopy (SEM) and Immuno-SEM of Nuclear Pore Complexes from Amphibian Oocytes, Mammalian Cell Cultures, Yeast and Plants.- NPC Structure in Model Organisms: Transmission Electron Microscopy and Immuno-gold Labelling using High Pressure Freezing/Freeze Substitution of Yeast, Worms and Plants.- High Resolution Imaging and Analysis of Individual Nuclear Pore Complexes.- Live CLEM imaging of Tetrahymena to Analyze the Dynamic Behavior of the Nuclear Pore Complex.- Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy.