title: Real-time imaging of nuclear permeation by EGFP in single intact cells.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, Children's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: Xunbin
authors.initials: X
authors.lastname: Wei
authors.forename: Vanessa G
authors.initials: VG
authors.lastname: Henke
authors.forename: Carsten
authors.initials: C
authors.lastname: Strübing
authors.forename: Edward B
authors.initials: EB
authors.lastname: Brown
authors.forename: David E
authors.initials: DE
authors.lastname: Clapham
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000493:pharmacokinetics* / Q000648:ultrastructure; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D009685:Nuclear Envelope / Q000378:metabolism*; D022022:Nuclear Pore / Q000378:metabolism* / Q000473:pathology*; D010539:Permeability; D011994:Recombinant Proteins / Q000493:pharmacokinetics / Q000648:ultrastructure; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium