Macromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC-free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules.

Nuclear pore complexes (NPCs) are multiprotein channels that span the nuclear envelope. They strongly limit the efficiency of gene transfection by restriction of nuclear delivery of exogenously applied therapeutic macromolecules. NPC dilation could significantly increase this efficiency. Recently, it was shown in oocytes of Xenopus laevis that NPCs dilate from about 82 to 110 nm within min after injection of the glucocorticoid analog dexamethasone (dex). In the present paper we analyzed by means of atomic force microscopy the structural details of NPC dilation and correlated them with functional changes in nuclear envelope permeability. 5-11 min after Dex injection NPC dilation was found at its maximum (approximately 140 nm). In addition, a yet unknown configuration, so-called giant pore, up to 300 nm in diameter, was visualized. Giant pore formation was paralleled by an increase in nuclear envelope permeability tested by electrophysiology and confocal fluorescence microscopy. Even large macromolecules lacking any nuclear localization signal (77 kDa FITC-dextran, molecule diameter up to 36 nm) could gain access to the nucleus. We conclude that dex transiently opens unspecific pathways for large macromolecules. Dex treatment could be potentially useful for improving the efficiency of nuclear gene transfection.