Elsevier

Methods

Volume 51, Issue 1, May 2010, Pages 170-176
Methods

Review Article
Oocytes as an experimental system to analyze the ultrastructure of endogenous and ectopically expressed nuclear envelope components by field-emission scanning electron microscopy

https://doi.org/10.1016/j.ymeth.2010.01.015Get rights and content

Abstract

Xenopus oocytes provide a powerful model system for studying the structure and function of the nuclear envelope and its components. Firstly, the nuclear envelope is easily isolated by hand under gentle conditions that have little effect on its structural organization. They can then be prepared for several types of electron microscopy (EM) including field-emission scanning EM (feSEM) (described here) and cryo-EM. They can be immuno-gold labeled to determine the localization of individual proteins. There is also enough material to analyze biochemically. Secondly, they possess an efficient transcription and translation system so that proteins of interest can be ectopically expressed by injection of either mRNA into the cytoplasm or plasmids into the nucleus. Such proteins can be tagged and mutated. They are post-translationally modified and usually incorporate into the correct compartment. We describe here methods developed to analyze the structural organization of the nuclear envelope by feSEM including the structural organization of ectopically expressed nuclear envelope proteins.

Introduction

In eukaryotic cells the nucleoplasmic and cytoplasmic compartments are separated by the nuclear envelope (NE). The NE is composed of two lipid bilayers, the outer and inner nuclear membranes. The outer nuclear membrane is continuous with the endoplasmic reticulum and is studded with ribosomes. The nucleoplasmic side of the inner nuclear membrane is associated with a filamentous network, the nuclear lamina, which itself is closely associated with the peripheral chromatin. Outer and inner nuclear membranes are fused at sites where nuclear pore complexes (NPCs) are embedded. NPCs are the sole gateways for trafficking between cytoplasm and nucleus. Vertebrate NPCs are large supramolecular assemblies with a total mass of 125 MDa [1]. They have an eightfold symmetry and consist of eight spokes located at the core of the NPC, a cytoplasmic ring with cytoplasmic filaments, and a nucleoplasmic ring to which the nuclear basket is attached [2]. Nuclei of Xenopus laevis oocytes have been the system of choice to study the molecular architecture of NPCs for a long time [3], [4], [5], [6]. Several features make Xenopus oocytes particular suitable for ultrastructural studies of the NE. Nuclei of full-grown oocytes, called germinal vesicles (GVs), are giant nuclei about 400–500 μm in diameter. They have a high density of NPCs and can be easily and gently isolated free of cytoplasm. Due to their high translational capacity proteins of choice can be expressed very efficiently in oocytes by injection of mRNA or DNA constructs into the cytoplasm or nucleus, respectively [7], [8], [9], [10], [11]. Moreover, Xenopus oocytes have a crucial advantage over all other cell types with respect to ultrastructural studies of the NE and particularly of the nuclear lamina. In somatic cells it is necessary to remove either the chromatin or the nuclear membranes by harsh treatments to allow an en face view of the lamina. The treatment compromises the structure of the lamina and the NPCs and prevents imaging at high-resolution. In contrast, chromosomes of Xenopus GVs are not in contact with the nuclear periphery [12]. It is therefore possible to isolate Xenopus NEs free of chromatin and other adhering material by manually opening the giant nuclei followed by removal of the nuclear content with a gentle stream of physiological buffer. Xenopus oocytes are therefore ideally suited for NE studies. The Xenopus oocyte is the only cell type so far for which the filamentous nature of the lamina has clearly been demonstrated [13], [14], [15].

Main components of the lamina are the lamin proteins that are members of the intermediate filament (IF) protein family. Lamins are located exclusively in the nucleus. A nuclear localization signal (NLS) and a C-terminal CaaX-motif, which is the site of post-translational isoprenylation, target lamins to the inner nuclear membrane where they assemble into filaments [16]. Two types of lamins, A and B, can be distinguished based on their domain structure and biochemical properties. B-type lamins are permanently isoprenylated and are more closely associated with the inner nuclear membrane. A-type lamins are only transiently or not isoprenylated at all. Prelamin A looses its isoprene moiety in the course of proteolytic processing to mature lamin A [17]. Association of lamin A with the NE is indirect, probably mediated by B-type lamins [18]. The use of Xenopus oocytes as an expression system in combination with high-resolution field-emission scanning electron microscopic (feSEM) analysis revealed that filaments made from somatic A- and B-type lamins differ in structure and organization [14], [15]. The methods applied for the analysis of lamin filaments are also suitable to analyze the molecular architecture of a wide variety of other NE components.

In this article we give a description of oocyte techniques, nuclear isolation, preparation of NE spreads and sample processing for the analysis of NEs by feSEM imaging.

Section snippets

Oocyte isolation and culturing

Basic techniques of oocyte isolation and culturing have been described in detail in several reviews [19], [20]. For the experiments described here only a few hundred oocytes are needed per experiment. To obtain oocytes it is therefore convenient and effective to surgically remove pieces of ovary from a Xenopus female. Surgery of the same animal can be done repeatedly, if regulations allow, otherwise it must be sacrificed. Clearly any work involving procedures with animals must be done in

Acknowledgment

We would like to thank Dr. Irm Huttenlauch for her contribution to the experimental part of this work.

References (27)

  • B. Fahrenkrog et al.

    Trends Biochem. Sci.

    (2004)
  • J.E. Hinshaw et al.

    Cell

    (1992)
  • D. Stoffler et al.

    J. Mol. Biol.

    (2003)
  • V.A. Moar et al.

    J. Mol. Biol.

    (1971)
  • J.D. Richter et al.

    Cell

    (1981)
  • J.G. Gall et al.

    Exp. Cell Res.

    (2004)
  • L.D. Smith et al.

    Methods Cell Biol.

    (1991)
  • T.G. Clark et al.

    Cell

    (1977)
  • B.K. Kay

    Methods Cell Biol.

    (1991)
  • R. Reichelt et al.

    J. Cell Biol.

    (1990)
  • P.N. Unwin et al.

    J. Cell Biol.

    (1982)
  • M.W. Goldberg et al.

    J. Cell Sci.

    (1993)
  • J.B. Gurdon et al.

    Annu. Rev. Genet.

    (1981)
  • Cited by (0)

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