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. 2004 Jun 7;165(5):617-23.
doi: 10.1083/jcb.200312008.

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

Yoichi Miyamoto  1 , Takuya Saiwaki, Junichi Yamashita, Yoshinari Yasuda, Ippei Kotera, Satoshi Shibata, Masaki Shigeta, Yasushi Hiraoka, Tokuko Haraguchi, Yoshihiro Yoneda
Affiliations

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

Yoichi Miyamoto et al. J Cell Biol. .

Abstract

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.

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Figures

Figure 1.
Figure 1.
Importin α accumulates in the nucleus in response to UV irradiation. The nuclei of cells expressing either EGFP-importin α or β were irradiated with UV (364 nm) using a laser in conjunction with the confocal microscopy, and the subcellular localization of EGFP fusion proteins was observed by time-lapse photography. (A, top and middle) EGFP-importin α-expressing cells. (bottom) EGFP-importin β-expressing cell. The nucleus (arrows) was irradiated with UV. (B) After EGFP-importin α-expressing cells were fused by hemagglutinating virus of Japan, one nucleus in the fused cell (arrow) was irradiated with UV and the cell was scanned.
Figure 2.
Figure 2.
Importin α accumulates in the nucleus in response to either the oxidative stress or the heat shock stress. After HeLa cells were treated with hydrogen peroxide or incubated at 42°C at the indicated time, the cells were stained by specific antibodies for importin α (mRch1) and Alexa488-conjugated secondary antibodies. (A) HeLa cells were cultured with a medium containing 200 μM H2O2. (B) After HeLa cells were treated with 200 μM H2O2 for 1 h, the cells were incubated with fresh medium in the absence of H2O2. (C) HeLa cells were incubated at 42°C. (D) After HeLa cells were incubated for 1 h at 42°C, they were incubated at 37°C for the indicated times.
Figure 3.
Figure 3.
The classical nuclear import pathway is suppressed under conditions of stress. After HeLa cells were treated without or with 200 μM H2O2 for 30 min, 1 mg/ml GST-NLS-GFP was injected into the cytoplasm of the cells without (A) or with (B) a molar equivalent of recombinant importin α or β, and the subcellular localization of GST-NLS-GFP was observed by time-lapse photography. Images were collected at 10-s intervals.
Figure 4.
Figure 4.
Distribution change of Ran in response to the cellular stresses induces the nuclear accumulation of importin α. (A–C) After HeLa cells were exposed to various stresses, indirect immunofluorescence was performed to detect either endogenous importin α (mRch1) or endogenous Ran using specific antibodies. (B and C) Green, importin α stained with Alexa488-conjugated second antibodies; red, an injection marker (Alexa568-conjugated antibodies). (A) HeLa cells were incubated with 200 μM H2O2 for 1 h, irradiated with UV (254 nm, 0.3 J/cm2), or incubated at 42°C for 1 h. (B, top) After 4 mg/ml GST-Q69LRanGTP was injected into the nucleus of HeLa cells, they were further incubated with 200 μM H2O2 for 30 min. (bottom) After HeLa cells were treated with 200 μM H2O2 for 30 min, 4 mg/ml GST-Q69LRanGTP was injected into the nucleus of HeLa cells, and they were further incubated for 30 min. (C) After either 2 mg/ml WGA or 8 mg/ml GST-Q69LRanGTP was injected into the cytoplasm of HeLa cells, they were further incubated with 200 μM H2O2 for 30 min. (D) After HeLa cells were incubated with 200 μM H2O2 for 30 min, either 1 mg/ml of GST-NLS-GFP or GFP-importin α was injected into the cytoplasm of the cells, and then they were further incubated for 30 min.
Figure 5.
Figure 5.
The nuclear accumulation of importin α, but not hsc70, is commonly observed in response to various stresses. (A) HeLa cells were either exposed to 43°C for 1 h or incubated with 200 μM H2O2 for 1 h. The cells were stained by either endogenous importin α (mRch1) or endogenous hsc70 using specific antibodies. Green, hsc70, Alexa488-conjugated second antibodies; red, importin α, Alexa568-conjugated second antibodies. (B) After HeLa cells were either exposed to 43°C for 30 min or incubated with 200 μM H2O2 for 30 min, Alexa488-labeled recombinant hsc70 proteins were injected into the cytoplasm of the cells, and they were incubated for 1 h at 43°C or in the presence of 200 μM H2O2.
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