The Nuclear Pore Complex: Methods and Protocols

ISBN-13: 9781071623367 / Angielski / Twarda / 2022 / 532 str.

Martin W. Goldberg

Spis treści

Part I: The Nuclear Pore Complex and Nucleoporins           

1.     Affinity Isolation of Endogenous Saccharomyces cerevisiae Nuclear Pore Complexes

Ilona Nudelman, Javier Fernandez-Martinez, and Michael P. Rout

 

2.     Transformation of Chaetomium thermophilum and affinity-purification of native thermostable protein complexes

Nikola Kellner and Ed Hurt

 

3.     Nuclear Pore Complex Assembly using Xenopus Egg Extract

Guillaume Holzer and Wolfram Antonin

 

 Part II:  Nucleo-cytoplasmic Passage           

4.     Analysis of Nuclear Pore Complex Permeability in Mammalian Cells and Isolated Nuclei Using Fluorescent Dextrans

Marcela Raices and Maximiliano A. D’Angelo

 

5.     Hormone-inducible Transport Reporter Assay to Study Nuclear Import Defects in Neurodegenerative Diseases

Saskia Hutten and Dorothee Dormann

 

6.     Subcellular Fractionation Suitable for Studies of RNA and Protein Trafficking

Biljana Culjkovic-Kraljacic and Katherine L.B. Borden

 

7.     Localising Total mRNA in Plant Cells

Geraint Parry

 

8.     Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues

 Jennifer R. Aleman, Shawn C. Little, and Maya Capelson

 

Part III: Functional Analysis of Nucleoporins

9.     Analysis of Nucleoporin Function using Inducible Degron Techniques

Vasilisa Aksenova, Alexei Arnaoutov, and Mary Dasso

 

10.  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)

Frida Niss, Cecilia Bergqvist, Anna-Lena Ström, and Einar Hallberg

 

11.  Analysis of Nuclear Pore Complexes in Caenorhabditis elegans by Live Imaging and Functional Genomic

 Patricia de la Cruz Ruiz, Raquel Romero-Bueno, and Peter Askjaer

 

12.  Protein Retargeting in Aspergillus nidulans to Study the Function of Nuclear Pore Complex Proteins

Subbulakshmi Suresh and Stephen A. Osmani

 

Part IV: Protein-Protein Interactions 

13.  Split-GFP Complementation to Study the Nuclear Membrane Proteome using Microscopy. Shary N. Shelton, Sarah E. Smith, and Sue L. Jaspersen

14.  Bimolecular Fluorescence Complementation: Quantitative Analysis of In Cell Interaction of Nuclear Transporter Importin a with Cargo Proteins

Alexander Lee, Marie A. Bogoyevitch and David A. Jans

 

15.  Validation of Nuclear Pore Complex Protein-Protein Interactions by Transient Expression in Plants

 Fumika Ikeda and Kentaro Tamura

 

16.  Binding affinity measurement of Nuclear Export Signal peptides to their exporter CRM1. Ho Yee Joyce Fung, and Yuh Min Chook

 

Part V: Post Translational Modifications      

17.  Analysis of Ubiquitylation and SUMOylation of Yeast Nuclear Pore Complex Proteins

Catherine Dargemont

 

18.  Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope

 Justin D. Blethrow, Amanda L. DiGuilio, and Joseph S. Glavy

 

Part VI: Biophysical Methods

19.  Crystallization of Nuclear Export Signals or Small Molecule Inhibitors Bound to Nuclear Exporter CRM1

Yee Joyce Fung and Yuh Min Chook

 

20.  Atomic Force Microscopy for Structural and Biophysical Investigations on Nuclear Pore Complexes

 Ivan Liashkovich, Gonzalo Rosso, and Victor Shahin

 

21.  Multivalent Interactions with Intrinsically Disordered Proteins Measured by Surface Plasmon Resonance

Larisa E. Kapinos and Roderick Y. H. Lim

 

22.  Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology during Confined Migration

Richa Agrawal, Aaron Windsor, and Jan Lammerding

 

Part VII:  Imaging NPCs and Transport

23.  SPEED Microscopy: High-Speed Single-Molecule Tracking and Mapping of Nucleocytoplasmic Transport

Steven J. Schnell, Mark Tingey, and Weidong Yang

 

24.  Imaging Fluorescent Nuclear Pore Complex Proteins in C. elegans

Courtney Lancaster, Giulia Zavagno, James Groombridge, Adelaide Raimundo, David Weinkove, Tim Hawkins, Joanne Robson and Martin W. Goldberg

 

25.  Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts

Sampada Mishra and Daniel L. Levy

 

26.  TEM Imaging of Membrane Choreography During Mitosis of Drosophila Tissue Culture Cells

Anton Strunov, Lidiya V. Boldyreva, Alexey V. Pindyurin, Maurizio Gatti, and Elena Kiseleva

 

27.  Scanning Electron Microscopy (SEM) and Immuno-SEM of Nuclear Pore Complexes from Amphibian Oocytes, Mammalian Cell Cultures, Yeast and Plants

Martin W. Goldberg and Jindřiška Fišerová

 

28.  NPC Structure in Model Organisms: Transmission Electron Microscopy and Immuno-gold Labelling using High Pressure Freezing/Freeze Substitution of Yeast, Worms and Plants

 A. Christine Richardson, Jindřiška Fišerová, and Martin W. Goldberg

 

29.  High Resolution Imaging and Analysis of Individual Nuclear Pore Complexes

 Boris Fichtman, Saroj G. Regmi, Mary Dasso, and Amnon Harel

 

30.  Live CLEM imaging of Tetrahymena to Analyze the Dynamic Behavior of the Nuclear Pore Complex

Tokuko Haraguchi, Hiroko Osakada, and Masaaki Iwamoto

 

31.  Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy

Helena Bragulat-Teixidor, M. Julius Hossain, and Shotaro Otsuka

Blurb

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.

 

Authoritative and cutting-edge, The Nuclear Pore Complex: Methods and Protocols: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.