Preparation of the Nuclear Envelope of Xenopus Laevis Nuclei for AFM Imaging

George Krohne and hans Oberleithner, Departments of Zoology and Physiology, University of Würzburg, Germany

Reprinted from TopoMetrix Applications Note #2-1293-01 (Dec. '93)

Transport of ions and macromolecules across the nuclear envelope occurs through so-called nuclear pore complexes (NPC). Each NPC is composed of 8 subunits that are arranged in a circle enclosing a central channel through which transport occurs. This channel is the only potential pathway for directed nucleocytoplasmic transport of ribonucleoproteins (from the nucleus into the cytoplasm) and of large macromolecules as polymerases (from the cytoplasm into the nucleus). It is known that transport through this pore is selective and regulated, however the underlying mechanisms are as yet unclear. Therefore, imaging the NPC and its central channel represents a first step in unraveling the molecular mechanisms of nucleocytoplasmic transport.

Click image for full 3D view (120k), click ICON for a large hi-res JPG file (495kB)

(Fig 1) Nuclear pore complexes on the cytoplasmic face of Xenopus Laevis

Oocyte nuclei of Xenopus laevis were manually isolated (82mM KCl, 17mM NaCl, 10mM Tris-HCl, pH 7.2) and transferred to a glass coverslip which was submersed in the same buffer. The nuclear envelopes were locally ruptured with needles and placed on the coverslip so that either its cytoplasmic or nucleoplasmic side was attached to the glass surface. Cover slips with attached nuclear envelopes were then washed twice each for two minutes with a half strength isolation buffer (41.5 mM KCl, 8.5 mM NaCl, 5mM Tris-HCl, pH 7.2) to remove soluble nuclear components and subsequently fixed for 10 minutes at room temperature with 1.25% glutaraldehyde (25mM KCl, 1.25 mM MgCl2, 25 mM cacodylate buffer, pH 7.2). Cover slips were then washed three times each for three minutes with distilled water and rapidly air dried.

The AFM images were acquired by Dr. Matthias Beckmann, Department of Physiology, University of Tübingen. The cytoplasmic face of the nuclear envelope was scanned with the TopoMetrix TMX 2010 Discoverer™ AFM in constant force mode using a high aspect ratio SuperTip™. The NPCs can be clearly imaged (Fig 1). Each NPC exhibits its central channel and the subunit structure of a single NPC can be disclosed. Further technical improvements, specifically in tip technology, should allow us to resolve the NPC at the molecular level.

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