Nucleus and Nuclear Envelope: Methods for Preparation

Abstract

The nucleus of eukaryotic cells contains the genome surrounded by a nuclear envelope (NE) consisting of two lipid membranes with embedded nuclear pores and an underlying nuclear lamina. The uniformity in size and density makes it possible to isolate pure intact nuclei at high yields from tissue homogenates by centrifugation through a sucrose cushion. NEs can be prepared from isolated nuclei by enzymatic degradation of their nucleic acid content. The resulting NE preparations contain structurally well‐conserved inner and outer nuclear membranes with attached ribosomes, nuclear pore complexes and nuclear lamina. Reliable methods for preparation of nuclei and NEs play an important role in the successful identification of components and their interacting partners that are located in nuclei and in nuclear subcompartments.

Key Concepts

  • Pure intact nuclei can be isolated at high yields from tissue homogenates, and nuclear envelopes can be prepared from the isolated nuclei.
  • The preparation of pure nuclei is important for the identification of nuclear and subnuclear components and their interacting partners.
  • Animal behaviourists must participate in conservation planning to protect the future of biodiversity.
  • Lipid bilayers provide the fundamental architecture of biological membranes.

Keywords: nucleus; nuclear envelope; nuclear membrane; nuclear pore; protein–protein interactions; MCLIP; proteomics

Figure 1. Flowchart showing the major steps from rat liver homogenate to isolated nuclei and isolated nuclear envelopes (NEs).
close

References

Andersen JS, Lyon CE, Fox AH, et al. (2002) Directed proteomic analyses of the human nucleolus. Current Biology 12: 1–11.

Aris JP and Blobel G (1988) Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. Journal of Cell Biology 107: 17–31.

Blobel G and Potter VR (1966) Nuclei from rat liver: isolation method that combines purity with high yield. Science 154: 1662–1665.

Collas P (1998) Modulation of plasmid DNA methylation and expression in zebrafish embryos. Nucleic Acids Research 26 (19): 4454–4461.

Cronshaw JM, Krutchinsky AN, Zhang W, et al. (2002) Proteomic analysis of the mammalian nuclear pore complex. Journal of Cell Biology 158: 915–927.

Dwyer N and Blobel G (1976) A modified procedure for the isolation of a pore complex‐lamina fraction from rat liver nuclei. Journal of Cell Biology 70: 581–591.

D'Angelo MA, Raices M, Panowski SH, et al. (2009) Age‐dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell 136: 284–295.

Fisher DZ, Chaudhary N and Blobel G (1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proceedings of the National Academy of Sciences of the USA 83 (17): 6450–6454.

Fisher PA and Blobel G (1983) Preparation of a nuclear matrix‐pore complex‐lamina fraction from embryos of Drosophila melanogaster. Methods in Enzymology 96: 589–596.

Hallberg E, Wozniak RW and Blobel G (1993) An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin‐like region. Journal of Cell Biology 122: 513–521.

Jafferali MH, Vijayaraghavan B, Figueroa RA, et al. (2014) MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells. Biochimica et Biophysica Acta 1838: 2399–2403.

Rout MP, Aitchison JD, Suprapto A, et al. (2000) The yeast nuclear pore complex: composition, architecture, and transport mechanism. Journal of Cell Biology 148: 635–651.

Roux KJ, Kim DI, Raida M and Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology 196: 801–810.

Schirmer EC, Florens L, Guan T, et al. (2003) Nuclear membrane proteins with potential disease links found by subtractive proteomics. Science 301: 1380–1382.

Starr CM, D'Onofrio M, Park MK and Hanover JA (1990) Primary sequence and heterologous expression of nuclear pore glycoprotein p62. Journal of Cell Biology 110 (6): 1861–1871.

Wozniak RW, Bartnik E and Blobel G (1989) Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. Journal of Cell Biology 108 (6): 2083–2092.

Further Reading

Dauer WT and Worman HJ (2009) The nuclear envelope as a signaling node in development and disease. Developmental Cell 17: 626–638.

Hetzer MW, Walther TC and Mattaj IW (2005) Pushing the envelope: structure, function, and dynamics of the nuclear periphery. Annual Review of Cell and Developmental Biology 21: 347–380.

Mèjat A and Misteli T (2010) LINC complexes in health and disease. Nucleus 1 (1): 40–52.

Stewart CL, Roux KJ and Burke B (2007) Blurring the boundary: the nuclear envelope extends its reach. Science 318: 1408–1412.

Worman HJ and Bonne G (2007) “Laminopathies”: a wide spectrum of human diseases. Experimental Cell Research 313: 2121–2133.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Jafferali, Mohammed H, Beckman, Marie, Kihlmark, Madeleine, and Hallberg, Einar(May 2015) Nucleus and Nuclear Envelope: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002602.pub3]