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Biophys J. 2003 Feb;84(2 Pt 1):1317-27.

Real-time imaging of nuclear permeation by EGFP in single intact cells.

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  • 1Howard Hughes Medical Institute, Children's Hospital, Boston, Massachusetts 02115, USA.


The NPC is the portal for the exchange of proteins, mRNA, and ions between nucleus and cytoplasm. Many small molecules (<10 kDa) permeate the nucleus by simple diffusion through the pore, but molecules larger than 70 kDa require ATP and a nuclear localization sequence for their transport. In isolated Xenopus oocyte nuclei, diffusion of intermediate-sized molecules appears to be regulated by the NPC, dependent upon [Ca(2+)] in the nuclear envelope. We have applied real-time imaging and fluorescence recovery after photobleaching to examine the nuclear pore permeability of 27-kDa EGFP in single intact cells. We found that EGFP diffused bidirectionally via the NPC across the nuclear envelope. Although diffusion is slowed approximately 100-fold at the nuclear envelope boundary compared to diffusion within the nucleus or cytoplasm, this delay is expected for the reduced cross-sectional area of the NPCs. We found no evidence for significant nuclear pore gating or block of EGFP diffusion by depletion of perinuclear Ca(2+) stores, as assayed by a nuclear cisterna-targeted Ca(2+) indicator. We also found that EGFP exchange was not altered significantly during the cell cycle.

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