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Depletion of nucleoporins from HeLa nuclear pore complexes to facilitate the production of ghost pores for in vitro reconstitution

  • Method in Cell Science
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Abstract

During cell division, Nuclear Pore Complexes (NPCs) are broken down into protein subcomplexes that are the basis for reassembly in daughter cells. This is the driving force for the establishment of an in vitro reconstitution system to study aspects of NPC reassembly. In this study, nuclear envelope (NE) was isolated from HeLa cells. NE was treated with increasing concentrations of heparin to extract nucleoporins (Nups) for the production of “ghost pores” which are pores severely deficient in Nups, while still containing Pore Membrane proteins (POM) needed to anchor the NPC. Ghost pores have been subjected to incubation with previously stripped Nups and some re-binding has been shown to occur by western blot analysis. This in vitro assay provides a powerful tool to investigate the protein–protein interactions of NPC reassembly from a human cell line. Through a better understanding of the process of NPC reassembly, we can continue to piece together the puzzle of this macromolecular structure. It is most advantageous to establish a straightforward reconstitution procedure at the mammalian level.

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Abbreviations

NE:

Nuclear envelope

NPC:

Nuclear pore complex

Nups:

Nucleoporins

FG-Nup:

Phenylalanine–glycine nucleoporins

CE:

Cell extract

INM:

Inner nuclear membrane

ONM:

Outer nuclear membrane

POM:

Pore membrane

EM:

Electron microscopy

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PMSF:

Phenylmethylsulfonyl fluoride

PBS:

Phosphate buffered saline

DTT:

Dithiothreitol

TEA:

Triethanolamine

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Acknowledgments

We thank Thomas Cattabiani and Yifei Bao for critical reading, figure preparation and helpful discussions. We are grateful to Sarah Whitcomb and Samuel Dales for their contributions to the early portions of the project. A.L. DiGuilio, and J.S. Glavy were supported by Stevens Institute of Technology.

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  1. Department of Chemistry, Chemical Biology & Biomedical Engineering, Stevens Institute of Technology, Castle Point on the Hudson, Hoboken, NJ, 07030, USA

    Amanda L. DiGuilio & Joseph S. Glavy

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  1. Amanda L. DiGuilio
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  2. Joseph S. Glavy
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Correspondence to Joseph S. Glavy.

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DiGuilio, A.L., Glavy, J.S. Depletion of nucleoporins from HeLa nuclear pore complexes to facilitate the production of ghost pores for in vitro reconstitution. Cytotechnology 65, 469–479 (2013). https://doi.org/10.1007/s10616-012-9501-y

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  • DOI: https://doi.org/10.1007/s10616-012-9501-y

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