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Address correspondence to Y. Yoneda, Dept. of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-4605. Fax: 81-6-6879-4609. email: yyoneda{at}anat3.med.osaka-u.ac.jp; or T. Haraguchi, email: tokuko{at}nict.go.jp
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Abstract |
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Key Words: cellular stress; importin/karyopherin; nuclear transport; nuclear localization signal; Ran
Abbreviation used in this paper: hsc, heat shock cognate.
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Introduction |
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A variety of cellular stresses affect multiple aspects of cellular physiology, and an important part of the cellular stress response involves the translocation of stress-responsible factors from the cytoplasm to the nucleus. However, how these factors are translocated into the nucleus in response to stress or how the nuclear transport pathways are regulated in stressed cells is unclear.
In this paper, we report that importin accumulates in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock stress, resulting in the inhibition of classical nuclear import. We demonstrated that the decrease in nuclear RanGTP levels actually triggers the accumulation of importin
in the nucleus by suppressing nuclear export. Moreover, we show that both nuclear retention and the importin ß/Ran-independent nuclear import of importin
are also involved in its rapid nuclear accumulation.
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Results and discussion |
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To determine if the nuclear accumulation of importin in response to UV irradiation is directly involved in the repair of UV-damaged DNA, we examined the issue of whether importin
effectively accumulates only in the irradiated nucleus but not other nonirradiated nuclei of a polykaryon constructed by cell fusion. At 30 min after the fusion of HeLa cells transfected with pEGFP-importin
, only one nucleus (Fig. 1 B, arrow) was irradiated by UV. As shown in Fig. 1 B (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200312008/DC1), EGFP-importin
accumulated in all the nuclei of the fused cells at almost the same rate. In addition, the nuclear accumulation of EGFP-importin
was also reversible in the polykaryon (unpublished data). Therefore, it is likely that the nuclear accumulation of importin
induced by UV irradiation is the result of cellular responses to UV irradiation, probably one of the stress responses, rather than that of the repair of damaged DNA.
Next, to determine whether or not the nuclear accumulation of importin is a common response to various stress conditions, we treated HeLa cells with hydrogen peroxide (H2O2) or incubated them at 42°C. When the cells were incubated in a medium including 200 µM H2O2 at 37°C, endogenous importin
detected by antibodies specific for importin
was rapidly localized in the nucleus after approximately a 30-min incubation (Fig. 2 A). Furthermore, when the cells were placed in fresh medium without H2O2 after treatment with H2O2 for 1 h, the importin
began to redistribute to the cytoplasm within 4 h, and the distribution of endogenous importin
returned to the original state 24 h after the replacement (Fig. 2 B).
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Based on the data showing that importin accumulates in the nucleus in response to cellular stress, we hypothesized that the nuclear import efficiency of the classical NLS substrates is decreased due to the suppressed recycling of importin
to the cytoplasm. To analyze the import rate, we performed time-lapse experiments. After treating HeLa cells with H2O2 for 30 min, recombinant GST-SV40 T antigen NLS-GFP (GST-NLS-GFP) proteins were injected into the cytoplasm, and the rate of nuclear import of the substrate was measured under the stress conditions. In unstressed cells, the substrate rapidly migrated into the nuclei (Fig. 3 A, top). In contrast, in stress-exposed cells the nuclear import rate of the substrate was dramatically diminished, although eventually substrates gradually accumulated in the nucleus (Fig. 3 A, bottom).
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What induces the nuclear accumulation of importin ? It is possible that the stress-induced nuclear accumulation of importin
is caused by the inhibition of its export pathway. Because it is known that the cellular apoptosis susceptibility gene product CAS (also referred to as exportin 2) exports importin
from the nucleus in a nuclear RanGTP-dependent manner (Kutay et al., 1997), we analyzed the distribution of CAS under the stress conditions using indirect immunofluorescence. Endogenous CAS showed no change in distribution under any of the stress conditions tested (unpublished data). Next, we attempted to examine if the subcellular localization of Ran is altered in response to cellular stress. HeLa cells were incubated with H2O2 for 1 h, irradiated with UV, or incubated at 42°C for 1 h, and the subcellular localization of endogenous Ran was observed using indirect immunofluorescence. As shown in Fig. 4 A, a significant amount of Ran was distributed to the cytoplasm in response to all three types of stress. In addition, in each stress condition, the level of the cytoplasmic distribution of Ran was mutually correlated with that of the nuclear accumulation of importin
, suggesting that the stress-induced alteration in Ran distribution may suppress the efficiency of the CAS-mediated nuclear export of importin
.
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In contrast, after pretreating the HeLa cells with H2O2 for 30 min, GST-Q69LRanGTP was injected into the nucleus and the cells were incubated for 30 min under the stress condition. Indirect immunofluorescence showed that endogenous importin was detected in the nucleus (Fig. 4 B, bottom), suggesting that importin
can be retained in the nucleus after migration induced by the collapse of the Ran gradient. Consistently, we observed that DNase I treatment, but not RNase, dramatically abolished nuclear importin
localization (unpublished data). It should be noted that a variety of NLSs overlap with DNA-binding regions (Cokol et al., 2000). Therefore, it is possible that importin
migrates into the nucleus to bind to the NLS regions of DNase Isensitive chromatin components exposed as the result of the stress conditions. It is reasonable to speculate that the nuclear retention of importin
after nuclear migration in stress-exposed cells may accelerate the rapid nuclear accumulation of importin
triggered by a collapse of the Ran gradient.
Consistent with our results, it has recently been reported that alterations in Ran distribution play a role in regulating nucleocytoplasmic transport under stress conditions (Czubryt et al., 2000; Stochaj et al., 2000). What induces the distribution change of Ran? Czubryt et al. (2000) reported that activation of the mitogen-activated protein kinase ERK2, an extracellular signal-regulated kinase, mediates the collapse of the Ran gradient by H2O2 treatment. In addition, it should be noted that nuclear import efficiency is affected by the activation of kinases (Kehlenbach and Gerace, 2000). Thus, it will be intriguing to determine whether kinase/phosphatase or other stress-inducible modifications directly alter the distribution of Ran under different stress conditions.
In contrast, one can speculate that nucleotide depletion may affect the Ran gradient. It has been shown that DNA strand breaks result in a drop in cellular ATP levels by the consumption of NAD for poly(ADP-ribose) synthesis (Carson et al., 1986). In addition, UV irradiation was shown to directly induce a decrease in mitochondrial respiratory activity, leading to ATP depletion (Djavaheri-Mergny et al., 2001). It has also been reported that hydrogen peroxide treatment results in lowered cellular ATP levels (Wu et al., 1996). In fact, Schwoebel et al. (2002) demonstrated that when cells were depleted of ATP by the addition of sodium azide and 2-deoxyglucose, Ran-dependent nuclear transport was rapidly inhibited. Thus, the drop in ATP level can lead to intracellular GTP depletion by the cellular interconversion of nucleotides. Therefore, a drop in GTP concentration should affect the production of RanGTP in the nucleus, resulting in the collapse of the Ran gradient. Further work will be required to address what actually triggers the change in distribution of Ran.
Next, we analyzed the nuclear import manner of importin in response to the cellular stress. It has already been shown that importin
is able to migrate into the nucleus via two independent pathways, importin ß/Ran-dependent and -independent (Miyamoto et al., 2002). It is known that, whereas WGA inhibits both pathways, Q69LRanGTP blocks only the importin ß/Ran-dependent pathway. The nuclear import of the injected GST-NLS-GFP was consistently blocked by the coinjection of either WGA or GST-Q69LRanGTP, whereas that of the injected GFP-importin
was not inhibited by the coinjection of GST-Q69LRanGTP (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200312008/DC1).
Under the same assay conditions, WGA or GST-Q69L RanGTP was injected into the cytoplasm of HeLa cells, and the cells were incubated in the presence of H2O2 for 30 min. As shown in Fig. 4 C, endogenous importin did not accumulate in the nucleus of the WGA-injected cells, whereas the importin
became localized in the nucleus of the Q69LRanGTP-injected cells, like the surrounding uninjected cells. Furthermore, after HeLa cells were exposed to H2O2 for 30 min, GFP-importin
was injected into the cytoplasm of the cells followed by incubation for 30 min. Under the condition, in which GST-NLS-GFP was not transported into the nucleus, a considerable amount of GFP-importin
accumulated in the nucleus (Fig. 4 D). These results indicate that, in stress-induced cells, importin
is able to migrate into the nucleus, at least in part, in an importin ß/Ran-independent manner. Along with the other aforementioned findings, we propose that the nuclear accumulation of importin
in response to cellular stress can occur through a combination of the following events: (a) the inhibition of the nuclear export of importin
by CAS, resulting from a collapse in Ran distribution, (b) the nuclear retention of importin
, and (c) the importin ß/Ran-independent nuclear import of importin
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We demonstrated that the nuclear accumulation of importin in response to the cellular stresses actually suppressed the classical NLS nuclear import pathway in vivo (Fig. 3). To address the issue of how the nuclear transport pathway for classical NLS-containing proteins and that of stress-responsible factors are mutually regulated in stressed cells, we focused on heat shock cognate (hsc) 70, a well known typical heat shock protein that appears to be able to enter the nucleus efficiently without the aid of the classical nuclear import pathway under conditions of stress (Lamian et al., 1996).
We first confirmed the subcellular localization of hsc70 under the stress conditions using a specific antibody. HeLa cells were either exposed to 43°C for 1 h or incubated with 200 µM H2O2 for 1 h. In this case, to observe the nuclear localization of hsc70 clearly, the cells were incubated at 43°C instead of 42°C. As expected, endogenous hsc70 accumulated in the nucleus under heat shock condition (Fig. 5 A). In contrast, under conditions of oxidative stress, hsc70 was mainly localized in the cytoplasm and was slightly detected in the nucleoli, although importin clearly accumulated in the nucleus (Fig. 5 A). In UV-exposed cells, hsc70 was also observed mainly in the cytoplasm, analogous to oxidative stress (unpublished data).
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In contrast, importin clearly accumulated in the nucleus under all stress conditions tested, suggesting that importin
may accumulate and function in the nucleus as one of the common responses induced by a variety of stresses. It would be interesting to know if importin
is able to play a role in the intranuclear response specific for stresses as a "nuclear stress response."
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Materials and methods |
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Transfection and UV irradiation
Importin (mouse Rch1) and ß were constructed with pEGFP-C1 plasmid (CLONTECH Laboratories, Inc.). HeLa cells were plated onto a glass bottom dish (MatTek Corporation) cultured for 12 d before use. After the constructs were transfected using Effecten Transfection Reagent (QIAGEN) under the conditions recommended by the supplier for 24 h, the cells were treated with 1 µg/ml of Hoechst 33342 in culture medium without phenol red, followed by incubation for 15 min at 37°C. The cells were placed on the stage of a confocal laser-scanning microscope (model LSM510; Carl Zeiss MicroImaging, Inc.). A nucleus (
100 µm2) of the cells expressing the EGFP fused proteins was irradiated with a UV (364 nm) laser (iteration 50, a power of 100% transmission of 0.0550.057 mW was used). Images were collected at 10- or 20-s intervals after irradiation of the nucleus.
As described previously (Miyamoto et al., 2002), when the expression level was high, we observed the nuclear localization of transiently expressed EGFP-importin even in the absence of stress. However, when the expression level was not so high, transiently expressed EGFP-importin
was found to remain in the cytoplasm. In this work, we selected EGFP-importin
-expressing cells in which the expression level was not so high to assess the stress response more easily and convincingly.
Time-lapse imaging
Cells were plated on a glass bottom dish (MatTek Corporation) and maintained with MEM supplemented with 10% FBS, antibiotics, 8 mM of L-glutamine, and 30 mM Hepes, pH 7.1, without phenol red. Experiments were performed on an inverted microscope (model Axiovert 100M; Carl Zeiss MicroImaging, Inc.) equipped with a stage (model CZI-3; Carl Zeiss MicroImaging, Inc.) heated to 37°C in conjunction with an objective heater (Bioptechs) and a 63x NA 1.4 Plan-Apochromat (Fig. 1 and Fig. S1) or 40x NA 0.6 LD-Achroplan (Fig. 3). Time-lapse imaging was performed using a confocal laser-scanning microscope equipped with 488-nm lasers in conjunction with a BP 505550 for EGFP. The acquired images were processed by LSM510 software version 3.2 SP2.
Indirect immunofluorescence
Indirect immunofluorescence was performed as described previously (Miyamoto et al., 2002). The mAbs against importin (mRch1) and Ran (BD Biosciences) were used at 1:250 and 1:500, respectively. Anti-hsc70 antibodies (StressGen Biotechnologies) were used at 1:100.
Expression and purification of recombinant proteins
Recombinant untagged and GFP fused importin (mouse Rch1) and ß, GST-SV40 T antigen NLS-GFP (GST-NLS-GFP), and GST-Q69LRanGTP were prepared as described previously (Miyamoto et al., 2002). Full-length cDNA of hsc70 subcloned into pGEX6P-1 was provided by S. Kose (RIKEN, Wako, Japan). Purification of the recombinant hsc70 protein was performed in the same manner as for GFP-importin
. GST-free hsc70 was labeled with Alexa Fluor 488 dye (Molecular probes) according to the manufacturer's recommendation.
Cell fusion by Sendai virus
HeLa cells cultured on a glass bottom dish were transfected with pEGFP-importin and incubated for 24 h. The cells were fused as described previously (Tachibana et al., 1994). Hemagglutinating virus of Japan (Sendai virus) was provided by M. Nakanishi (National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan).
Online supplemental material
Video 1 shows an irreversible nuclear accumulation of EGFP-importin in a UV-irradiated cell. Video 2 shows a reversible nuclear accumulation of EGFP-importin
in a UV-irradiated cell. Video 3 shows a subcellular localization of EGFP-importin ß in a UV-irradiated cell. Video 4 shows an irreversible nuclear accumulation of EGFP-importin
in a UV-irradiated fusion cell. Fig. S1 shows that importin
accumulated in the nucleus repeatedly in response to the UV irradiation. Fig. S2 shows that importin
migrated into the nucleus in an importin ß/Ran-independent manner in an in vivo assay. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312008/DC1.
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Acknowledgments |
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This work was supported by the Japanese Ministry of Education, Science, Sports and Culture, the Human Frontier Science Program, and Nippon Boehringer Ingelheim (to Y. Yoneda) and by the Japan Science and Technology Corporation (Core Research for Evolutional Science and Technology to Y. Hiraoka and T. Haraguchi).
Submitted: 1 December 2003
Accepted: 22 April 2004
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