Passive Transport of Macromolecules through Xenopus laevis Nuclear Envelope

Abstract

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.

Glucocorticoids remodel nuclear envelope structure and permeability

“…Comparison of diffusion rates under different experimental conditions allows conclusions about passive NPC permeability. The diffusion of FITC (Fluorescein isothiocyanate; Sigma, St Louis, MO) labelled 20 kDa dextran was measured across the nuclear envelope with confocal fluorescence microscopy, as previously described (Enss et al, 2003). The light source of the confocal laser-scanning microscope (CLSM Fluoview, Olympus) was an argon/krypton ion laser (Omnichrome ® ), which generates three excitation wavelengths, 488, 568 and 647 nm.…”

Hot Spot Formation in the Nuclear Envelope of Oocytes in Response to Steroids

“…The light source of the confocal microscope was an argon/krypton ion laser (Omnichrome ® ) that generated an excitation wavelength of 488 nm. The underlying principle of exploring passive transport of macromolecules is that a change in NPC conformation is followed by a change in the passive diffusion rate of fluorescence-labeled 20 kDa dextran [18]. Comparison of diffusion rates under different experimental conditions allows conclusions about passive NPC permeability.…”

“…To prevent the cell nucleus from moving, it was kept in place by using Cell-tak (Biosciences, Bedford, USA). Diffusion of 20 kDa FITC-dextran across the nuclear envelope was measured and analyzed with confocal fluorescence microscopy as previously described [18]. Ninety seconds, 3, 5, 10, 20 minutes and 24 hours after injection of 50 nl of either TA or solvent into GR expressing oocytes, cell nuclei were isolated and collected in NIM.…”

Ethanol alters access to the cell nucleus

“…Comparison of diffusion rates under different experimental conditions allows conclusions to be drawn on NPC macromolecule permeability [9]. The diffusion of dextrans from the cytosol to the nucleus and vice versa inversely correlates with the molecular masses of the respective dextrans (nuclear exclusion limit between 17 and 41 kDa), indicating that the NE can act as a molecular sieve [22].…”

“…Indeed, nuclear volume did not remain constant but increased significantly with time of ethanol incubation (>0.5‰). The volume changes were used to calculate the respective surface/volume ratios of individual nuclei and taken for the permeability measurements [9]. Similar to the NEER experiments, we tested NE dextran permeability at the same ethanol concentrations and over 7 and 20 h of incubation (Fig.…”