In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.

Although nuclear pore complexes (NPC) are considered to be key structures in gene expression, little is known about their regulatory control. In order to explore the regulatory mechanism of passive transport of small macromolecules we examined the influence of different factors on the diffusional pathway of NPCs in isolated Xenopus laevis oocyte nuclei. Diffusion of fluorescence-labeled 10-kD dextran was measured across the nuclear envelope with confocal fluorescence microscopy. Surprisingly, the filling state of the perinuclear Ca(2+) store had no influence on passive transport of 10-kD dextran. Furthermore, nuclear envelope permeability was independent of cytoplasmic pH (pH range 8.3-6.3). In contrast, nuclear swelling, induced by omission of the endogenous cytosolic macromolecules, clearly increased nuclear permeability. An antibody against the glycoprotein gp62, located at the central channel entrance, reduced macromolecule diffusion. In addition, nuclei from transcriptionally active, early developmental stages (stage II) were less permeable compared to transcriptionally inactive, late-developmental-stage (stage VI) nuclei. In stage II nuclei, atomic force microscopy disclosed NPC central channels with plugs that most likely were ribonucleoproteins exiting the nucleus. In conclusion, the difference between macromolecule permeability and previous measurements of electrical resistance strongly indicates separate routes for macromolecules and ions across the nuclear envelope.

Throughout oogenesis, huge amounts of RNA are produced that are needed for early development. Early stages of oocyte development are characterized by high transcriptional activity whereas translation of maternal RNA dominates late stages. Nuclear pore complexes (NPCs), located in the nuclear envelope (NE), mediate bidirectional macromolecule exchange between the nuclear and cytosolic compartments including RNA export. Here, we report on structural correlates of this transport pathway at single NPC level. Using atomic force microscopy (AFM), we imaged the nucleoplasmic ("inner") surface of the NE of Xenopus laevis oocytes in different stages of development. We found that NPC frequency per nucleus increases with maturation. However, individual NPCs are more active in immature stages. In early stages, known for high transcriptional activity, we found nearly 10% of NPC central channels plugged with a 400-800 kDa mass. In contrast, the incidence of plugged NPCs was below 1% in late oocyte stages. On-site RNA digestion led to a change in plug shape from prominent to flat while plug mass decreased by almost 20%. Quantitative AFM analysis revealed that RNase exposure reduced total nucleoplasmic NPC mass by about 58 and 25% in early and late stage oocytes, respectively. We conclude: (i) NPCs of immature oocytes are more active in RNA transport, (ii) Plugs identified at the nucleoplasmic entrance of NPC central channels represent ribonucleoproteins exiting the nucleus, (iii) RNA is a structural component of the NPC nanomachine.

The mineralocorticoid hormone aldosterone controls fluid and electrolyte transport in target cells of the kidney and the cardiovascular system. Classic genomic aldosterone action involves the activation of cytosolic mineralocorticoid receptors and translocation into the cell nucleus where specific transcription processes are initiated. A key barrier of the intracellular signalling pathway is the nuclear envelope, which physically separates the nucleoplasm from the cytoplasm. It was shown recently that aldosterone changes ion conductivity of the nuclear envelope mediated by nuclear pore complexes. The latter are supramolecular nanomachines responsible for import and export of inorganic ions and macromolecules. The aim of the present study was to test whether aldosterone changes the macromolecule permeability of the nuclear envelope. Aldosterone-responsive Xenopus laevis oocytes were used as a model system. We isolated the cell nuclei at defined times after hormone injection. By means of confocal fluorescence microscopy and fluorescence-labelled dextrans we evaluated passive macromolecule import and export in isolated nuclei. 10 minutes after aldosterone injection nuclear envelope permeability of 10 kD dextran was found sharply increased. At the same time cell nuclei were found swollen by about 28%. Changes in nuclear volume and nuclear envelope permeability lasted 5 to 15 minutes and could be inhibited by the mineralocorticoid receptor blocker spironolactone. We conclude that aldosterone transiently changes the barrier function of the nuclear envelope. This short-lasting permeability change signals the start of a sustained transcription process that follows in response to steroids.