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The Nucleus pp 161-180 | Cite as

Investigation of Nuclear Envelope Structure and Passive Permeability

  • Victor Shahin
  • Yvonne Ludwig
  • Hans Oberleithner
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

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.

Keywords

Atomic force microscopy Confocal microscopy Nuclear envelope structure and permeability Nuclear hourglass technique Nuclear pore complex Xenopus laevis oocyte 

References

  1. 1.
    Fahrenkrog, B., Stoffler, D., and Aebi, U. (2001) Nuclear pore complex architecture and functional dynamics. Curr. Top. Microbiol. Immunol. 259, 95-117.PubMedCrossRefGoogle Scholar
  2. 2.
    Pante, N. and Aebi, U. (1993) The nuclear pore complex. J. Cell Biol. 122, 977-984.PubMedCrossRefGoogle Scholar
  3. 3.
    Shahin, V., Ludwig, Y., Schafer, C., Nikova, D., and Oberleithner, H. (2005) Glucocorticoids remodel nuclear envelope structure and permeability. J. Cell Sci. 118, 2881-2889.PubMedCrossRefGoogle Scholar
  4. 4.
    Shahin, V., Albermann, L., Schillers, H., Kastrup, L., Schafer, C., Ludwig, Y., Stock, C., and Oberleithner, H. (2005) Steroids dilate nuclear pores imaged with atomic force microscopy. J. Cell Physiol. 202, 591-601.PubMedCrossRefGoogle Scholar
  5. 5.
    Allen, T. D., Cronshaw, J. M., Bagley, S., Kiseleva, E., and Goldberg, M. W. (2000) The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. J. Cell Sci. 113, 1651-1659.PubMedGoogle Scholar
  6. 6.
    Bayliss, R., Corbett, A. H., and Stewart, M. (2000) The molecular mechanism of transport of macromolecules through nuclear pore complexes. Traffic. 1, 448-456.PubMedCrossRefGoogle Scholar
  7. 7.
    Corbett, A.H., and Silver, P.A. (1997) Nucleocytoplasmic transport of macromolecules. Microbiol. Mol. Biol. Rev. 61, 193-211.PubMedGoogle Scholar
  8. 8.
    Gorlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell. Dev. Biol. 15, 607-660.PubMedCrossRefGoogle Scholar
  9. 9.
    Mattaj, I. W. and Englmeier, L. (1998) Nucleocytoplasmic transport: the soluble phase. Annu. Rev. Biochem. 67, 265-306.PubMedCrossRefGoogle Scholar
  10. 10.
    Rout, M. P., Aitchison, J. D., Magnasco, M. O., and Chait, B. T. (2003) Virtual gating and nuclear transport: the hole picture. Trends Cell Biol. 13, 622-628.PubMedCrossRefGoogle Scholar
  11. 11.
    Wente, S. R. (2000) Gatekeepers of the nucleus. Science. 288, 1374-1377.PubMedCrossRefGoogle Scholar
  12. 12.
    Hinshaw, J. E., Carragher, B. O., and Milligan, R. A. (1992) Architecture and design of the nuclear pore complex. Cell. 69, 1133-1141.PubMedCrossRefGoogle Scholar
  13. 13.
    Mazzanti, M., Bustamante, J. O., and Oberleithner, H. (2001) Electrical dimension of the nuclear envelope. Physiol. Rev. 81, 1-19.PubMedGoogle Scholar
  14. 14.
    Oberleithner, H. (2004) Nuclear envelope: nanoarray responsive to aldosterone. J. Membr. Biol. 199, 127-134.PubMedCrossRefGoogle Scholar
  15. 15.
    Danker, T., Schillers, H., Storck, J., Shahin, V., Krämer, B., Wilhelmi, M., and Oberleithner, H. (1999) Nuclear hourglass technique: novel approach detects electrically open pores in xenopus laevis oocyte. Proc. Natl. Acad. Sci. USA. 96, 13531-13535.CrossRefGoogle Scholar
  16. 16.
    Schafer, C., Shahin, V., Albermann, L., Hug, M. J., Reinhardt, J., Schillers, H., Schneider, S. W., and Oberleithner, H. (2002) Aldosterone signaling pathway across the nuclear envelope. Proc. Natl. Acad. Sci. USA. 99, 7154-7159.PubMedCrossRefGoogle Scholar
  17. 17.
    Shahin, V., Danker, T., Enss, K., Ossig, R., and Oberleithner, H. (2001) Evidence for Ca2+and ATP-sensitive peripheral channels in nuclear pore complexes. FASEB. J. 15, 1895-1901.PubMedCrossRefGoogle Scholar
  18. 18.
    Kastrup, L., Oberleithner, H., Ludwig, Y., Schafer, C., and Shahin, V. (2006) Nuclear envelope barrier leak induced by dexamethasone. J. Cell Physiol. 206, 428-434.PubMedCrossRefGoogle Scholar
  19. 19.
    Kroeger, H. (1966) Elektrophysiologische und cytologische Untersuchungen an den Speicheldrüsen von Chironomus Thummi. Exp. Cell Res. 41, 64-80.PubMedCrossRefGoogle Scholar
  20. 20.
    Wünsch, S., Schneider, S., Schwab, A., and Oberleithner, H. (1993) 20-OH-ecdysone swells nulcear volume by alkalinization in salivary glands of Drosophila melanogaster. Cell Tissue Res. 274, 145-151.PubMedCrossRefGoogle Scholar
  21. 21.
    Oberleithner, H., Schafer, C., Shahin, V., and Albermann, L. (2003) Route of steroid-activated macromolecules through nuclear pores imaged with atomic force microscopy. Biochem. Soc. Trans. 31, 71-75.PubMedCrossRefGoogle Scholar
  22. 22.
    Schafer, C., Shahin, V., Albermann, L., Schillers, H., Hug, M. J., and Oberleithner, H. (2003) Intracellular calcium: a prerequisite for aldosterone action. J. Membr. Biol. 196, 157-162.PubMedCrossRefGoogle Scholar
  23. 23.
    Danker, T., Shahin, V., Schlune, A., Schafer, C., and Oberleithner, H. (2001) Electrophoretic plugging of nuclear pores by using the nuclear hourglass technique. J. Membr. Biol. 184, 91-99.PubMedCrossRefGoogle Scholar
  24. 24.
    Binnig, G., Quate, C. F., and Gerber, C. (1986) Atomic force microscope. Phys. Rev. Lett. 56, 930-933.PubMedCrossRefGoogle Scholar
  25. 25.
    Binnig, G., Rohrer, H., Gerber, C., and Weibel, E. (1982) Surface studies by scanning tunneling microscopy. Phys. Rev. Lett. 49, 57-61.CrossRefGoogle Scholar
  26. 26.
    Henderson, R. M. and Oberleithner, H. (2000) Pushing, pulling, dragging, and vibrating renal epithelia by using atomic force microscopy. Am. J. Physiol. Renal Physiol. 278, F689-F701.PubMedGoogle Scholar
  27. 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
  28. 28.
    Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K., and Schindler, H. (1996) Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc. Natl. Acad. Sci. USA. 93, 3477-3481.PubMedCrossRefGoogle Scholar
  29. 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
  30. 30.
    Oberleithner, H., Brinckmann, E., Schwab, A., and Krohne, G. (1994) Imaging nuclear pores of aldosterone sensitive kidney cells by atomic force microscopy. Proc. Natl. Acad. Sci. USA. 91, 9784-9788.PubMedCrossRefGoogle Scholar
  31. 31.
    Oberleithner, H., Schneider, S., Lärmer, J., and Henderson, R. M. (1996) Viewing the renal epithelium with the atomic force microscope. Kidney Blood Press. Res. 19, 142-147.PubMedCrossRefGoogle Scholar
  32. 32.
    Oberleithner, H., Schneider, S. W., and Henderson, R. M. (1997) Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: the “molecularsandwich” technique. Proc. Natl. Acad. Sci. USA. 94, 14144-14149.PubMedCrossRefGoogle Scholar
  33. 33.
    Oberleithner, H., Ludwig, T., Riethmuller, C., Hillebrand, U., Albermann, L., Schafer, C., Shahin, V., and Schillers, H. (2004) Human endothelium: target for aldosterone. Hypertension. 43, 952-956.PubMedCrossRefGoogle Scholar
  34. 34.
    Oberleithner, H. (2005) Aldosterone makes human endothelium stiff and vulnerable. Kidney Int. 67, 1680-1682.PubMedCrossRefGoogle Scholar
  35. 35.
    Oberleithner, H., Riethmuller, C., Ludwig, T., Shahin, V., Stock, C., Schwab, A., Hausberg, M., Kusche, K., and Schillers, H. (2006) Differential action of steroid hormones on human endothelium. J. Cell Sci. 119, 1926-1932.PubMedCrossRefGoogle Scholar
  36. 36.
    Perez-Terzic, C., Behfar, A., Mery, A., van Deursen, J. M., Terzic, A., and Puceat, M. (2003) Structural adaptation of the nuclear pore complex in stem cell-derived cardiomyocytes. Circ. Res. 92, 444-452.PubMedCrossRefGoogle Scholar
  37. 37.
    Shahin, V., Hafezi, W., Oberleithner, H., Ludwig, Y., Windoffer, B., Schillers, H., and Kuhn, J. E. (2006) The genome of HSV-1 translocates through the nuclear pore as a condensed rodlike structure. J. Cell Sci. 119, 23-30.PubMedCrossRefGoogle Scholar
  38. 38.
    Hansma, H. G., Kim, K. J., Laney, D. E., Garcia, R. A., Argaman, M., Allen, M. J., and Parsons, S. M. (1997) Properties of biomolecules measured from atomic force microscope images: a review. J. Struct. Biol. 119, 99-108.PubMedCrossRefGoogle Scholar
  39. 39.
    Rotsch, C. and Radmacher, M. (1997) Mapping local electrostatic forces with the atomic force microscope. Langmuir. 13, 2825-2832.CrossRefGoogle Scholar
  40. 40.
    Maul, G. G. (1977) The nuclear and the cytoplasmic pore complex: structure, dynamics, distribution, and evolution. Int. Rev. Cytol. Suppl. 6, 75-186.PubMedGoogle Scholar
  41. 41.
    Lenart, P., Rabut, G., Daigle, N., Hand, A. R., Terasaki, M., and Ellenberg, J. (2003) Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes. J. Cell Biol. 160, 1055-1068.PubMedCrossRefGoogle Scholar
  42. 42.
    Peters, R. (1986) Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim. Biophys. Acta. 864, 305-359.PubMedCrossRefGoogle Scholar
  43. 43.
    Enss, K., Danker, T., Schlune, A., Buchholz, I., and Oberleithner, H. (2003) Passive transport of macromolecules through Xenopus laevis nuclear envelope. J. Membr. Biol. 196, 147-155.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Victor Shahin
    • 1
  • Yvonne Ludwig
    • 1
  • Hans Oberleithner
    • 1
  1. 1.Institute of Physiology IIUniversity of MünsterMünsterGermany

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