Investigation of Nuclear Envelope Structure and Passive Permeability
We present an experimental approach by the help of which structure and passive permeability of the nuclear envelope (NE) can be investigated thoroughly, by combining imaging, fluorescent, and electrophysiological techniques. A mature Xenopus laevis oocyte features a large nucleus offering an excellent system for these investigations. Using the emerging technique of atomic force microscopy, NE structure and the conformational state of nuclear pore complexes (NPCs), known to rule NPC and inevitably NE permeability, can be visualised at high resolution and under near physiological conditions. Passive NE permeability to macromolecules can be determined by the long-established confocal laser scanning microscopy, applying fluorescent macromolecules (dextran). Passive NE permeability to small molecules, which has long remained confounded by the lack of an appropriate technique, can finally be investigated following development of a proper technique designated the “nuclear hourglass” technique. The experimental approach presented here thus opens unique perspectives towards understanding the correlation between NE structure and passive permeability. This chapter describes in detail the protocols for performing such investigations.
KeywordsAtomic force microscopy Confocal microscopy Nuclear envelope structure and permeability Nuclear hourglass technique Nuclear pore complex Xenopus laevis oocyte
- 27.Hillebrand, U., Hausberg, M., Stock, C., Shahin, V., Nikova, D., Riethmuller, C., Kliche, K., Ludwig, T., Schillers, H., Schneider, S. W., and Oberleithner, H. (2006) 17beta-estradiol increases volume, apical surface and elasticity of human endothelium mediated by Na+/H+ exchange. Cardiovasc. Res. 69, 916-924.PubMedCrossRefGoogle Scholar
- 29.Hinterdorfer, P., Schilcher, K., Baumgartner, W., Gruber, H. J., and Schindler, H. A. (1998) Mechanistic study of the dissociation of individual antibody-antigen pairs by atomic force microscopy. Nanobiology. 4, 177-188.Google Scholar