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Biophysical Journal 84:1317-1327 (2003)
© 2003 The Biophysical Society

Real-Time Imaging of Nuclear Permeation by EGFP in Single Intact Cells

Xunbin Wei, Vanessa G. Henke, Carsten Strübing, Edward B. Brown* and David E. Clapham

Howard Hughes Medical Institute, Children's Hospital, * E.L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115

Correspondence: Address reprint requests to Dr. David E. Clapham, Rm. 1309, Enders Bldg., P.O. Box EN-306, Children's Hospital, 320 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6163; Fax: 617-731-0787; E-mail: dclapham{at}enders.tch.harvard.edu.

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 [Ca2+] 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 ~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 Ca2+ stores, as assayed by a nuclear cisterna-targeted Ca2+ indicator. We also found that EGFP exchange was not altered significantly during the cell cycle.




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