Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells.
BACKGROUND
The transcription factor NF-AT plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-AT translocates from the cytoplasm to the nucleus then returns in response to the intracellular calcium level.


METHODS
We have investigated NF-AT nucleocytoplasmic shuttling in real-time in living cells using NF-ATc1 tagged with the reversibly photoswitchable fluorescence protein, Dronpa. We monitored both nuclear import and export rate of Dronpa-tagged NF-AT in live cells upon stimulation with ionomycin plus calcium (I+Ca(2+)) or cyclosporin A (CsA).


RESULTS
The results show that NF-AT moved into the nucleus within 3-9 min after stimulation and moved back out into the cytoplasm within 15-50 min after CsA addition. In the absence of stimulation, NF-AT stayed in the cytoplasm as in the cells overexpressing GSK-3beta, a calcineurin-opposing regulator.


CONCLUSIONS
This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.
Kwon
Oh Yeun
OY
Biomedical Research Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Republic of Korea.
Kwon
Ick Chan
IC
Song
Hyun Kyu
HK
Jeon
Hyesung
H
D000818:Animals; D019556:COS Cells; D002122:Calcium Chloride / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D016572:Cyclosporine / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics*; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D013997:Time Factors
D007476:Ionophores; D008164:Luminescent Proteins; D050778:NFATC Transcription Factors; D015759:Ionomycin; D016572:Cyclosporine; D002122:Calcium Chloride