Nuclear Pore Complexes and Nucleocytoplasmic Transport - Methods

1st Edition, Volume 122 - May 20, 2014
Editor: Valérie Doye
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 4 1 7 1 6 0 - 2
eBook ISBN:
9 7 8 - 0 - 1 2 - 4 1 7 1 7 8 - 7

Volume 122 of Methods in Cell Biology describes modern tools and techniques used to study nuclear pore complexes and nucleocytoplasmic transport in diverse eukaryotic model sys… Read more

Nuclear Pore Complexes and Nucleocytoplasmic Transport - Methods

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Volume 122 of Methods in Cell Biology describes modern tools and techniques used to study nuclear pore complexes and nucleocytoplasmic transport in diverse eukaryotic model systems (including mammalian cells, Xenopus, C. elegans, yeast).

The volume enables investigators to analyze nuclear pore complex structure, assembly, and dynamics; to evaluate protein and RNA trafficking through the nuclear envelope; and to design in vivo or in vitro assays appropriate to their research needs.

Beyond the study of nuclear pores and transport as such, these protocols will also be helpful to scientists characterizing gene regulation, signal transduction, cell cycle, viral infections, or aging. The NPC being one of the largest multiprotein complexes in the cell, some protocols will also be of interest for people currently characterizing other macromolecular assemblies. This book is thus designed for laboratory use by graduate students, technicians, and researchers in many molecular and cellular disciplines.

Preface
Chapter 1: Fifty Years of Nuclear Pores and Nucleocytoplasmic Transport Studies: Multiple Tools Revealing Complex Rules
- Abstract
- Introduction
- Concluding Remarks
- Acknowledgments
Chapter 2: Imaging Metazoan Nuclear Pore Complexes by Field Emission Scanning Electron Microscopy
- Abstract
- Introduction
- Acknowledgments
Chapter 3: Imaging Yeast NPCs: From Classical Electron Microscopy to Immuno-SEM
- Abstract
- Introduction
- Conclusions and Perspectives
- Acknowledgments
Chapter 4: Exploring Nuclear Pore Complex Molecular Architecture by Immuno-Electron Microscopy Using Xenopus Oocytes
- Abstract
- Introduction
- 4.1 Materials
- 4.2 Experimental Strategies (Fig. 4.2)
- 4.3 Preparation of Antibodies Conjugated with Colloidal Gold Particles
- 4.4 Immunogold Labeling of Nucleoporins Using Anti-Nucleoporin Antibodies
- 4.5 Immunogold Labeling of Nucleoporins Using Epitope-Tagged Nucleoporins
- Concluding Remarks
- Acknowledgments
Chapter 5: Utilizing the Dyn2 Dimerization-Zipper as a Tool to Probe NPC Structure and Function
- Abstract
- Introduction
- Conclusion
- Acknowledgment
Chapter 6: The Use of Targeted Proteomics to Determine the Stoichiometry of Large Macromolecular Assemblies
- Abstract
- Introduction
- Conclusions
- Acknowledgments
Chapter 7: A Pulse–Chase Epitope Labeling to Study Cellular Dynamics of Newly Synthesized Proteins: A Novel Strategy to Characterize NPC Biogenesis and Ribosome Maturation/Export
- Abstract
- Introduction
- Conclusion and Perspectives
- Acknowledgments
Chapter 8: Analysis of Nuclear Reconstitution, Nuclear Envelope Assembly, and Nuclear Pore Assembly Using Xenopus In Vitro Assays
- Abstract
- Introduction
- 8.3 Demembranated Sperm Chromatin
- 8.4 In vitro Reconstitution of Nuclei
- 8.5 Assaying Assembly and Integrity of the Nuclear Envelope
- 8.6 A Nuclear Pore Complex Assembly Assay Using Pore-Free Nuclear Intermediates
- Conclusion
Chapter 9: Xenopus In Vitro Assays to Analyze the Function of Transmembrane Nucleoporins and Targeting of Inner Nuclear Membrane Proteins
- Abstract
- Introduction
- Conclusion
Chapter 10: Imaging the Assembly, Structure, and Function of the Nuclear Pore Inside Cells
- Abstract
- Introduction
- Acknowledgments
Chapter 11: Cell-Fusion Method to Visualize Interphase Nuclear Pore Formation
- Abstract
- Introduction
- Conclusions
- Acknowledgments
Chapter 12: An In Vitro System to Study Nuclear Envelope Breakdown
- Abstract
- Introduction
- Acknowledgments
Chapter 13: Modern Tools to Study Nuclear Pore Complexes and Nucleocytoplasmic Transport in Caenorhabditis elegans
- Abstract
- Introduction
- 13.1 Forward and Reverse Genetics
- 13.2 Transgenesis
- 13.3 Live Imaging of Embryos
- 13.4 In vivo methods to evaluate structural and functional integrity of the NE
- 13.5 Immunofluorescence and Electron Microscopy
- 13.6 Interaction of Nups with Chromatin
- Summary and Future Perspectives
- Acknowledgments
Chapter 14: Assessing Regulated Nuclear Transport in Saccharomyces cerevisiae
- Abstract
- Introduction
- Acknowledgments
Chapter 15: Analysis of Nucleocytoplasmic Transport in Digitonin-Permeabilized Cells Under Different Cellular Conditions
- Abstract
- Introduction
- Conclusions and Perspectives
- Acknowledgments
Chapter 16: Novel Approaches for the Identification of Nuclear Transport Receptor Substrates
- Abstract
- Introduction
- Conclusions
- Acknowledgments
Chapter 17: NPC Mimics: Probing the Mechanism of Nucleocytoplasmic Transport
- Abstract
- Introduction
- Conclusions
Chapter 18: Analysis of RNA Transport in Xenopus Oocytes and Mammalian Cells
- Abstract
- Introduction
- Concluding Remarks
- Acknowledgment
Chapter 19: Strategies for Investigating Nuclear–Cytoplasmic tRNA Dynamics in Yeast and Mammalian Cells
- Abstract
- Introduction
- Concluding remarks
Chapter 20: Dissecting Ribosome Assembly and Transport in Budding Yeast
- Abstract
- Introduction
- Conclusions
- Acknowledgments
Chapter 21: Approaches to Studying Subnuclear Organization and Gene–Nuclear Pore Interactions
- Abstract
- Introduction
- Concluding Remarks, Possible Caveats, and Troubleshooting
- Acknowledgments
Index
Volumes in Series

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Valérie Doye