Regulation of nuclear import by light-induced activation of caged nuclear localization signal in living cells.
A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine-128 residue in SV40 T-antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78+/-0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm(2)). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally.
Watai
Y
Y
Laboratory of Molecular BioPhotonics, 5000 Hamakita, 434-8555, Shizuoka, Japan.
Sase
I
I
Shiono
H
H
Nakano
Y
Y
D021581:Active Transport, Cell Nucleus / Q000528:radiation effects; D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008027:Light; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D010782:Photolysis / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D013539:Simian virus 40
D000957:Antigens, Viral, Tumor; D005456:Fluorescent Dyes; D019913:Nuclear Localization Signals; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C421106:caged nuclear localization signal; D008239:Lysine