Real-time imaging of nuclear permeation by EGFP in single intact cells.
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.
Wei
Xunbin
X
Howard Hughes Medical Institute, Children's Hospital, Boston, Massachusetts 02115, USA.
Henke
Vanessa G
VG
Strübing
Carsten
C
Brown
Edward B
EB
Clapham
David E
DE
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
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium