Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells

Oh Yeun Kwon; Ick Chan Kwon; Hyun Kyu Song; Hyesung Jeon

doi:10.1016/j.bbagen.2008.08.003

Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells

Oh Yeun Kwon, Ick Chan Kwon, Hyun Kyu Song, Hyesung Jeon

Research output: Contribution to journal › Article › peer-review

8 Citations (Scopus)

Abstract

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 + Ca²⁺) 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-3β, a calcineurin-opposing regulator. General Significance: This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.

Original language	English
Pages (from-to)	1403-1407
Number of pages	5
Journal	Biochimica et Biophysica Acta - General Subjects
Volume	1780
Issue number	12
DOIs	https://doi.org/10.1016/j.bbagen.2008.08.003
Publication status	Published - 2008 Dec

Bibliographical note

Funding Information:
We thank Dr. Hiroshi Takayanagi, Tokyo Medical and Dental University, Japan for generously providing NF-ATc1 plasmid. This work was supported by Molecular Imaging and GRL “Theragnosis” grants from the Korea Government (MOST), by the internal grant from the Korea Institute of Science and Technology to HJ, and partly by the Korea Science and Engineering Foundation Grant (KOSEF, R01-2007-000-20032-0) funded by MOST, Korea to HKS.

Keywords

Calcineurin
Dronpa
GSK-3β
NF-AT
Nucleocytoplasmic shuttling
Real-time imaging

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

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10.1016/j.bbagen.2008.08.003

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title = "Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells",

abstract = "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 + Ca2+) 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-3β, a calcineurin-opposing regulator. General Significance: This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.",

keywords = "Calcineurin, Dronpa, GSK-3β, NF-AT, Nucleocytoplasmic shuttling, Real-time imaging",

author = "Kwon, {Oh Yeun} and Kwon, {Ick Chan} and Song, {Hyun Kyu} and Hyesung Jeon",

note = "Funding Information: We thank Dr. Hiroshi Takayanagi, Tokyo Medical and Dental University, Japan for generously providing NF-ATc1 plasmid. This work was supported by Molecular Imaging and GRL “Theragnosis” grants from the Korea Government (MOST), by the internal grant from the Korea Institute of Science and Technology to HJ, and partly by the Korea Science and Engineering Foundation Grant (KOSEF, R01-2007-000-20032-0) funded by MOST, Korea to HKS.",

year = "2008",

month = dec,

doi = "10.1016/j.bbagen.2008.08.003",

language = "English",

volume = "1780",

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journal = "Biochimica et Biophysica Acta - General Subjects",

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AU - Kwon, Oh Yeun

AU - Kwon, Ick Chan

AU - Song, Hyun Kyu

AU - Jeon, Hyesung

N1 - Funding Information: We thank Dr. Hiroshi Takayanagi, Tokyo Medical and Dental University, Japan for generously providing NF-ATc1 plasmid. This work was supported by Molecular Imaging and GRL “Theragnosis” grants from the Korea Government (MOST), by the internal grant from the Korea Institute of Science and Technology to HJ, and partly by the Korea Science and Engineering Foundation Grant (KOSEF, R01-2007-000-20032-0) funded by MOST, Korea to HKS.

PY - 2008/12

Y1 - 2008/12

N2 - 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 + Ca2+) 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-3β, a calcineurin-opposing regulator. General Significance: This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.

AB - 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 + Ca2+) 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-3β, a calcineurin-opposing regulator. General Significance: This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.

KW - Calcineurin

KW - Dronpa

KW - GSK-3β

KW - NF-AT

KW - Nucleocytoplasmic shuttling

KW - Real-time imaging

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JF - Biochimica et Biophysica Acta - General Subjects

IS - 12

ER -

Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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