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1 Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2 Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
3 CREST of JST and Communications Research Laboratory, Kansai Advanced Research Centre, Kobe, Hyogo 651-2492, Japan
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Abstract |
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/ß, is down-regulated, while the nuclear import of 70 kD heat-shock cognate protein is up-regulated in heat-shock cells. Among the factors involved in the mediation of the conventional nuclear import, significant levels of importin accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin becomes less mobile and its nucleocytoplasmic recycling is impaired in heat-shock cells. These data coincided well with biochemical and cytological studies. Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin , resulting in the suppression of conventional nuclear import. This suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock. |
Introduction |
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The nucleocytoplasmic trafficking of most proteins is mediated by a large family of nuclear transport receptors of the importin ß superfamily, referred to as importins or exportins (reviewed in Görlich & Kutay 1999; Imamoto 2000; Macara 2001). Importins recognize specific import signals present within the import cargoes, and carry the bound cargo from the cytoplasm into the nucleus. Exportins recognize specific export signals present in cargoes and carry the bound cargoes from the nucleus out to the cytoplasm. Small GTPase Ran, in its GTP form, which plays a role in the disassembly of import complexes, and the assembly of export complexes, is essential in these transport pathways (reviewed in Görlich & Kutay 1999). The Ran GTPase cycle, which generates RanGTP in the nucleus by the Ran guanine nucleotide exchange factor RCC1 (RanGEF), and RanGDP in the cytoplasm by the Ran GTPase-activating protein (RanGAP), creates a steep RanGTP gradient across the nuclear envelope, and assures the directionality of transport of importin/exportin cargoes. In metazoans, more than 20 members of the importin ß family have been identified, in which 11 have been shown to act as importins, and 6 as exportins (reviewed in Macara 2001).
The first import receptor characterized was the importin /ß heterodimer, which was initially identified as playing a role in mediating the nuclear import of cargo proteins containing conventional nuclear localization signals (cNLS). Importin proteins are adapters that mediate the interaction of cNLS-proteins with importin ß, while importin ß is the founding member of a family of transport receptors. The cNLS is recognized by importin (Adam & Gerace 1991; Görlich et al. 1994; Imamoto et al. 1995a), and forms a trimeric complex with importin ß (Imamoto et al. 1995b). Importin ß mediates the NPC-translocation of a cargo-importin complex owing to its ability to interact with NPC components (nucleoporins). After translocation, RanGTP binds to importin ß and displaces the cargo-importin complex in the nucleus, while the cNLS cargo is displaced from importin by an exportin called cellular apoptosis susceptibility gene (CAS) and RanGTP (Kutay et al. 1997). Importin ß-Ran GTP complex, and importin -Ran GTP-CAS complex, recycle back to the cytoplasm, disassembles, and then mediate the another round of nuclear import.
Human cells employ at least 6 members of importin , which can be largely subdivided into three classes (for reviews, see Jans et al. 2000; Yoneda 2000). All importin proteins have molecular masses of 50–60 kD and share common sequence features, such as an N-terminus importin ß binding (IBB) domain (Görlich et al. 1996; Weis et al. 1996), a central domain composed of 10 armadillo (arm) repeats (Conti et al. 1998), and a short acidic domain in the C-terminus, which binds to CAS (Herold et al. 1998). The expression patterns of multiple importin suggest that each may transport a discrete set of nuclear proteins or that some may have unique functions in different cells (Kamei et al. 1999; Kohler et al. 1999). In vitro binding studies have shown that there is both redundancy and specificity for NLS recognition (Miyamoto et al. 1997; Nadler et al. 1997; Kohler et al. 1999). A particular family member of importin not only recognizes the conventional basic NLS, but also binds to non-basic NLS containing cargo, such as signal activated Stat1 (signal transducer and activator of transcription 1) (Sekimoto et al. 1997). Specific conditional mutations in the Saccharomyces cerevisiae importin (Srp1p) results in a mitotic arrest at G2/M (Yano et al. 1994), and one of the two importin proteins (Cut15) expressed in Shizosaccharomyces pombe is required for chromosome condensation (Matsusaka et al. 1998). It has been suggested that in Caenorhabditis elegans, a member of the importin family (IMA-3) is involved in the developmental roles of germ-line (Geles & Adam 2001), and in Drosophila melanogaster, it has been shown that one member is involved in the developmental regulation of heat-shock response (Fang et al. 2001). The employment of importin as an adapter protein widens the scope of cargo recognition for importin ß, and may acquire an additional regulatory step for diverse cellular function.
Heat-shock induces a strong stress response and affects all aspects of cellular physiology, which involves dynamic changes in the nucleocytoplasmic distributions of a variety of proteins. However, the issue of how a particular nuclear transport pathway is regulated under heat-shock condition remains elusive. In this study, we report on a study of the nuclear import of a conventional NLS cargo under heat-shock conditions which indicates that conventional import is down-regulated in heat-shock cells. In contrast, the nuclear import of the 70 kD heat-shock cognate protein (hsc70) is up-regulated in response to heat-shock.
In order to better understand the factors that causes the nuclear import depression of conventional NLS cargoes in heat-shock cells, we examined the behaviour of transport factors that are involved in importin /ß mediated transport pathways under heat-shock and normal conditions. Among the factors examined, importin showed significant differences in its subcellular localization. Biochemical and cytological studies indicate that importin becomes tightly attached to the nuclear structure in response to heat-shock. Moreover, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) studies using live cells that were microinjected with green fluorescent protein (GFP) tagged importin showed that nuclear importin becomes less mobile and that its nucleocytoplasmic recycling is impaired in heat-shock cells.
Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin , resulting in the suppression of conventional nuclear import. The regulation of importin behaviour may be involved in the regulatory role for the adaptation of cells to environmental changes, such as heat-shock.
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Results |
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In order to determine whether a particular transport pathway is regulated under heat-shock condition, we examined the nuclear import of a cargo protein bearing the conventional basic nuclear localization signal (cNLS) of the SV40 large T-antigen, and a 70 kD heat-shock cognate protein (hsc70). The former substrate is known to possess strong nuclear import activity that is mediated by importin and importin ß in normal cells. The latter substrate is generally localized in the cytoplasm of normal cells, but accumulates in the nucleus under heat-shock conditions (Welch & Feramisco 1984; Figs 1A and 2A). These substrates were expressed in bacteria, tagged with glutathione-S-transferase and green fluorescent protein (GST-cNLS-GFP), or labelled with Cy3 (Cy3-hsc70), and used to examine transport efficiency in living cells.
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In contrast to the behaviour of Cy3-hsc70 examined above, the opposite effect of heat-shock on the nuclear import of cNLS cargo was found. As shown in Fig. 1B, in normal cells, the cytoplasmically injected cNLS cargo rapidly migrated into all the nuclei within 5 min in normal cells, while its nuclear migration was slower after heat-shock treatment. For example, at 10 min, a time when complete nuclear accumulation was observed in normal cells, some cNLS substrate still remained in the cytoplasm under heat-shock. In most of the cells, the complete nuclear accumulation of cNLS substrate was observed at 30 min after injection. Quantification of import showed that the extent of decline of nuclear import of the cNLS cargo under heat-shock varied widely from cell to cell (Fig. 1C).
Importin accumulates in the nucleus under heat-shock conditions
In order to determine the cause of the import depression of the cNLS cargo under heat-shock, we first examined whether any of the known factors involved in conventional nuclear import might cause change in the subcellular localization in response to heat-shock. Among the transport factors examined by indirect immunofluorescence studies, we found a significant change in the distribution of endogenous importin in response to heat-shock. As shown in Fig. 2A, importin showed cytoplasmic distribution in normal cells, but it accumulated strongly in the nucleus after heat-shock. Importin gradually recovered to the normal cytoplasmic distribution after release from heat-shock (Fig. 2B). While differences in the localization of importin ß or CAS were not detected, the distribution of small GTPase Ran shifted slightly to cytoplasmic localization after heat-shock treatment.
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associates tightly with nuclear structure resistant to non-ionic detergent, nuclease, and high-salt treatment in heat-shock cells
The GTPase cycle of Ran is essential in transport pathways mediated by the importin ß super family. Therefore, a collapse in Ran distribution could cause the nuclear accumulation of importin through the inhibition of its nuclear export, mediated by the importin ß family member CAS. However, as shown in Fig. 1A(e), nuclear import of Cy3-hsc70 was inhibited when it was co-injected with Ran mutant defective in GTP hydrolysis (Q69L Ran) after the heat-shock treatment. This indicates that heat-shock induced nuclear import of hsc70 is Ran-dependent. Localization of endogenous hsc70, which had accumulated into the nucleus in response to heat-shock, was not affected, when Q69L Ran was injected after the treatment of heat-shock (Fig. 1A(f)). Therefore, Ran-dependent nuclear transport pathways would be expected to function under heat-shock, at least to certain extent. This prompted us to examine whether heat-shock affects any property of importin itself.
We first examined the biochemical properties of importin . In whole cell extracts of heat-shock cells or normal cells, differences in the total amount or a mobility shift in importin was not detected by SDS-PAGE. Subsequent fractionation studies showed that importin shifted from the soluble fraction to the insoluble fraction in response to heat-shock. As shown in Fig. 3A, when cells were sequentially fractionated, the majority of importin in normal cells could be extracted with 0.1% Triton X-100, while importin in heat-shock cells was not extracted with such non-ionic detergent. Importin remaining in normal cells after the detergent treatment was completely extracted with DNase I and a high-salt treatment. In contrast, most of importin in heat-shock cells was not extracted by non-ionic detergent, nuclease, or high-salt.
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is localized in the nucleus, we examined the localization of endogenous importin after treating cells with detergent, nuclease, or high salt by immunostaining. Immunostaining signals of importin of normal cells were dramatically decreased after the detergent and DNase I treatment (Fig. 3B, left panels). On the other hand, importin of heat-shock cells was found to be associated with the nucleus throughout the treatment with detergent, nuclease, and high-salt (Fig. 3B, right panels). Together with fractionation data, these results indicate that importin becomes tightly attached to some nuclear structure that is resistant to detergent, nuclease, and high-salt treatments, in response to heat-shock.
Heat-shock induces nuclear retention of importin in living cells
We next focused on how heat-shock affects the behaviour of importin in living cells. For this, bacterially expressed GFP-importin was microinjected into the cytoplasm of cells. The majority of microinjected GFP-importin is localized in the cytoplasm and some in the nucleus in normal cells, but when cells are exposed to heat-shock, GFP-importin becomes concentrated in the nucleus (Fig. 4), like endogenous importin (Fig. 2). Using cells microinjected with GFP-importin , we carried out fluorescence recovery after photobleaching (FRAP) to examine the mobility of nuclear GFP-importin . A square area within the nucleus was photobleached, and images were collected at 5 min intervals for a further 
5 h (see Experimental procedures). The fluorescence intensity of the bleached area was monitored in parallel with the region surrounding the bleached area. The nuclear region of an adjacent cell was monitored for the same time of period to serve as a control.
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was photobleached, the fluorescence intensity was uniformly reduced throughout the cells, but no discernible bleached zone was observed (Fig. 4d). This shows that in normal cells, GFP-importin is mobile, that it repopulates the bleached area within the time of the pulse and during the subsequent image collection. In contrast, when nuclear GFP-importin in a heat-shock cell was photobleached at the same condition, the bleached area clearly persisted (Fig. 4b). This indicates that nuclear importin becomes significantly less mobile in response to heat-shock, and little was able to enter the square area during the same periods of time. GFP-CAS and GFP, either in normal cells or in heat-shocked cells, showed a uniform reduction in fluorescence intensity throughout the bleached cells without the persistence of a bleached area (Fig. 4f,h,j,l). The photobleaching of GFP in fixed cells resulted in a prominent bleached zone with no recovery (Fig. 4n). Therefore, fluorescence recovery would depend on the movement of unbleached GFP molecules. These data show that a significant reduction in the mobility of importin , which occurred in response to heat-shock, was specific.
Figure 5 shows the recovery of the fluorescence of GFP-importin . The mobility of importin in heat-shock cells was significantly slow. The half-time (t1/2) of fluorescent recovery shown in the figure therefore may reflect the time of recovery of the mobility of importin as a result of release from heat-shock than the rate of mobility of nuclear GFP-importin in heat-shock cells. Images of an example of cell that was followed after photobleaching are shown in Fig. 5A. After the recovery of mobility, importin becomes dispersed throughout the cell nucleus and cytoplasm. The half-time of fluorescent recovery of GFP-importin in 20 individual cells was quantified and plotted and these data are shown in Fig. 5B. The half-time of recovery of mobility of GFP-importin was significantly heterogeneous. Such heterogeneity may have a close correlation with the heterogeneity of the extent of import decline observed with the cNLS cargo under heat-shock condition (Fig. 1C).
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is impaired in heat-shock cells
In order to determine whether nuclear or nuclear structure-associated importin could exchange with the very small population of importin in the cytoplasm, we next performed a fluorescence loss in photobleaching (FLIP) analysis. For this, the beam was focused on the cytoplasm of GFP-importin injected cells, bleached repeatedly, and the decline in nuclear fluorescence was monitored for a time period followed. In normal cells, when a small cytoplasmic region was bleached, the fluorescence signal of nuclear GFP-importin decreased to nearly the background levels (Fig. 6A(a)). In heat-shock cells, the cytoplasmic bleach caused a much slower decline in the nuclear fluorescence signal. In some cells, the fluorescence of nuclear GFP-importin decreased only slightly (Fig. 6A(b)), whereas in other cells, the nuclear fluorescence gradually decreased, but did not disappear completely for the time period followed here (Fig. 6A(c)). In these experiments, cytoplasmic GFP-importin showed a rapid loss in fluorescence regardless of heat shock, indicating that cytoplasmic importin is mobile regardless of heat shock or normal conditions. The cytoplasmic bleach had no effect on the rate of disappearance of fluorescence of nuclear GFP regardless of heat-shock, nor did it reduce the nuclear fluorescence in fixed cells (Fig. 6B). The delay in the loss of fluorescence of nuclear GFP-importin show that the nuclear export and recycling of importin is impaired in heat-shock cells.
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is localized in the interheterochromatin space in heat-shock cells
The above FRAP and FLIP analysis show that importin is tightly retained in the nucleus, and recycling is impaired in heat-shock cells, which is in agreement with fractionation results (Fig. 3). The nuclear retention and defect in the recycling of importin could be the major factor in the inhibition of cNLS-mediated nuclear import under heat-shock.
To obtain further information, we examined the localization of endogenous nuclear importin in heat-shock cells. Importin accumulated strongly into the nucleus under heat-shock conditions, and was located in the interheterochromatin space (Fig. 7d,f). On the other hand, importin in the nucleus of a normal cell was diffusely distributed, and no structural image was observed (Fig. 7a,c).
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Discussion |
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accumulated to a significant extent in the nucleus in response to heat-shock. The nuclear accumulated importin was tightly retained in the nucleus, and its nucleocytoplasmic recycling was impaired. No changes in the subcellular distribution of importin ß and CAS were detected, but small GTPase Ran was shifted slightly to cytoplasmic localization.
Different groups have previously reported that certain types of stress affect the cellular localization of Ran. Stochaj et al. (2000) showed that starvation, ethanol, heat and hydrogen peroxide altered the cellular distribution of Ran in Saccharomyces cerevisiae. Czubryt et al. (2000) reported that treatment of vascular smooth muscle cells with hydrogen peroxide increased the cytosolic level of Ran through the activation of mitogen-activated protein kinase ERK2. Such an alteration in the distribution and function of Ran can be implicated in the depression of the conventional NLS import pathway, as well as the inhibition of importin export, which is mediated by a member of the importin ß superfamily CAS. The slight shift of Ran to the cytoplasmic distribution was also observed in heat-shock HeLa cells in the present study (Fig. 2A). However, the nuclear import of hsc70, which is up-regulated in response to heat-shock, was found to be inhibited by a Ran mutant defective in GTP hydrolysis in heat-shock cells (Fig. 1A). Therefore, although the function of Ran would be affected to certain extent, it did not collapse to the level where it was unable to mediate the Ran-dependent transport pathway in heat-shock cells.
The depletion of importin from the cytoplasm could be a major cause for the suppressed conventional nuclear import pathway in heat-shock cells. Examination of the dynamic behaviour of importin in living cells provided us with the following information: The cytoplasmic bleaching of GFP-importin injected cells showed a clear delay in the loss of nuclear fluorescence, compared to normal cells (Fig. 6), therefore the recycling of importin appears to be depressed in heat-shock cells. The photobleaching analysis (Fig. 4), together with fractionation studies (Fig. 3), showed the strong nuclear retention of importin in heat-shock cells. Therefore, the nuclear accumulation of importin in response to heat-shock could be caused by at least two combination of following events, one is the inhibition of the nuclear export of importin , and the other is the nuclear retention of importin .
Fractionation studies demonstrated that most of the importin became resistant to detergent, DNase I, and high-salt extraction in response to heat-shock, while most of the importin in normal cells was readily extracted by the detergent (Fig. 3). The importin remaining in the detergent insoluble fraction in normal cells was completely extracted by DNase I and a high-salt treatment. Nuclear importin in heat-shock cells was found to be located in the interheterochromatin space (Fig. 7). Since the nuclear located importin was not released by DNase I or high-salt up to 2 M NaCl (Fig. 3A,B), it is unlikely that interheterochromatin space located importin is bound to euchromatin. The population of importin that became less mobile in heat-shock cells is most likely to be associated with nuclear scaffold, rather than chromatin structure or DNA.
It has been reported that importin is post-translationally modified. In Saccharomyces cerevisiae, importin is phosphorylated by casein kinase II (Azuma et al. 1997), and in mammalian cells, importin is acetylated by CREB (cAMP-response element binding protein)-binding protein (CBP/p300) (Bannister et al. 2000). Currently, we did not detect any mobility shift of importin either by 1-D SDS-PAGE or in 2-D PAGE. On the other hand, during the fractionation studies, we noticed that a significant population of the nuclear structural proteins, such as nuclear lamin-assoiated protein 2, and some nuclear pore complex components became resistant to extraction by high-salt in response to heat-shock (M. Furuta and N. Imamoto, unpublished observation). These observations indicate that a nuclear structure involving the nuclear scaffold could become more stabilized in response to heat-shock. Hsc70, which accumulates into the nucleus in response to heat-shock like importin , is localized in nucleolus and heat-shock granules, most of which was extracted with detergent, DNase I and high-salt treatment (data not shown), indicating that the nuclear localization of hsc70 is clearly different from those of importin . Our unpublished observations (M. Furuta and N. Imamoto) show that the nuclear accumulation of hsc70 in heat-shock cells is not mediated by importin /ß. In addition, as discussed below, hsc70 uniformly recover to cytoplasmic localization after 90 min release from heat-shock in most cells (data not shown), whereas the recovery of importin was sometimes slower than hsc70 (Fig. 2B) and the recovery rate was much more heterogeneous (Fig. 5). These observations indicate that importin and hsc70 neither co-migrate nor co-localize in the nucleus of heat-shock cells. Importin is tightly associated with some nuclear structural proteins involving the nuclear scaffold proteins which becomes stabilized in response to heat-shock. It would be of interest to examine whether this is caused by the inhibition of dissociation of NLS from importin , since the mechanism involved in NLS dissociation is presently unclear. The inclusion of importin in the nuclear scaffold structure as a physiological response of stress, such as heat-shock, also suggest that importin could be involved in the organization of the nuclear architecture.
Conventional nuclear import declined in response to heat-shock. However, the nuclear import of a cNLS cargo was not completely inhibited, as it accumulated in the nucleus in all cells within 30 min. The rate of nuclear accumulation varied considerably from cell to cell (Fig. 1C). Upon heat-shock treatment, endogenous hsc70 uniformly accumulated in the nucleus of all the cells observed in the field examined, and the relocalization of hsc70 to the cytoplasm required 90 min in most cells (data not shown). It is unlikely that the nuclear accumulation of a cNLS cargo is the result of release from heat-shock. The nuclear import of a cNLS cargo in heat-shock cells was inhibited by a Ran mutant that was defective in GTP hydrolysis. Therefore, a small portion of importin recycled back to the cytoplasm and contributed to importin ß-mediated nuclear import even under heat-shock conditions. The heterogeneity of the degree of import suppression of cNLS cargo may have a close correlation with the heterogeneity in the degree of recycling repression of importin (Fig. 6), as well as heterogeneity in the recovery time for the mobility of nuclear accumulated importin (Fig. 5). Regulation of the dynamic behaviour of importin could be caused by a combination of several up-stream and down-stream reactions.
What is the biological significance of the heat-shock induced nuclear accumulation and retention of importin leading to decline of importin /ß transport pathway? Heat-shock response is believed to be a universal and fundamental mechanism for cell protection against stress, and affect many process of cell function. For example, heat-shock induces transient cell cycle arrest, in which the mechanism have been extensively examined and explained in terms of stress-specific activation (or inhibition) of kinases or protein degradation that regulates activity or amount of cell cycle mediators, such as cyclin/Cdks (for review see Kuhl & Rensing 2000). However, it is well known that cell cycle regulated nuclear import and export of cyclin/Cdks also plays an important regulatory role in the cell cycle progression in normal cells. Cyclins/Cdks, whose nuclear import is mediated by importin /ß, for example cyclin E (Moore et al. 1999), may not accumulate into the nucleus in a timely manner in heat-shock cells because of decline importin /ß transport pathway. This would cause a cell cycle arrest in heat-shock cells. Alternation of activity of particular nucleocytoplasmic transport pathway like importin /ß, which is known to mediate nuclear import of variety of proteins (Imamoto 2000) should also cause a significant effect on gene expression and RNA processing, which is generally shut down in heat-shock cells. Therefore, depression of importin /ß pathway in response to heat shock would be an additional regulatory mechanism for cell protection against stress.
Many distinct nucleocytoplasmic transport pathways operate in eukaryotic cells. We have shown here that the conventional nuclear import pathway is specifically regulated through importin under heat-shock conditions. It is possible that different transport pathways or different transport factors function under different cellular conditions, leading to regulatory changes in gene expression in vivo.
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Experimental procedures |
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HeLa cells were incubated in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. For the heat-shock treatment, HeLa cells were incubated at 43 °C for 1 h with prewarmed DMEM supplemented with 10% FBS and 20 mM HEPES (pH 7.3). To assure heat-shock, the subcellular localization of endogenous hsc70, which accumulates in the nucleus in response to heat-shock, was detected by indirect immunofluorescence. The viability of cells exposed to heat-shock was examined through growth rate of cells, which confirmed that the heat-shock treatment did not affect cell viability.
For microinjection experiments, HeLa cells were plated on glass bottom dishes (MatTek Corporation) 36–48 h prior to each experiment. Fluorescently labelled recombinant proteins were injected through a glass capillary into the cytoplasm as previously described (Fig. 1, Yokoya et al. 1999; Figs 4–6, Haraguchi et al. 1997). To examine the behaviour of proteins in heat-shock cells, proteins were injected within 15 min after the heat-shock treatment (Fig. 1), or before heat-shock treatment (Figs 4–6).
Expression and purification of recombinant proteins
Recombinant hsc70 (Imamoto et al. 1992), GST-NLS-GFP (Yokoya et al. 1999), Q69L Ran (Tachibana et al. 2000), GFP-importin (Tachibana et al. 2000), and GFP-CAS (Hieda et al. 1999) proteins were prepared as previously described. Recombinant hsc70 was labelled with the FluoroLinkTM Cy3 Monofunctional Dye (Amersham) according to the manufacture's recommendation.
Indirect immunofluorescence studies
HeLa cells were fixed in 3.7% formaldehyde in PBS for 20 min at 37 °C and permeabilized with 0.5% TritonX-100 in PBS for 10 min at room temperature. After incubation in blocking solution (3% skim milk in PBS) for 20 min at room temperature, the cells were incubated with primary antibodies for 1 h at room temperature, washed five times with blocking solution, and the primary antibodies were detected with fluorescent-labelled secondary antibodies. The 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; 0.1 µg/mL; SIGMA) was included in the penultimate wash step to visualize DNA. Images were captured using Olympus IX70 microscope with Meta Morph (Figs 1–3; Nippon Roper) or a Delta Vision (Fig. 7; Plan Apo60x oil, NA = 1.4; Applied Precision Inc.) system (Haraguchi et al. 1997). For deconvolution microscopic image, serial optical section data (20–30 focal planes at 0.5 µm intervals) were collected and computationally processed by a 3D deconvolution method (WoRx 3.00 software) to remove out-of-field fluorescence (Agard et al. 1989).
Antibodies: anti-hsc70 (mAb1H5-1; preparation of mAb1H5-1 is the same as mAb2A11; Takagi et al. 1999), anti-importin (Rch1) (Santa Cruz Biotechnology; sc-6917), anti-importin (NPI1) (Santa Cruz Biotechnology; sc-6918), anti-CAS (Santa Cruz Biotechnology; sc-1708), anti-importin ß (Sekimoto et al. et al. 1997), anti-Ran (Transduction Laboratories; 610340), Cy3-labelled donkey anti-rabbit IgG (Jackson Immunoresearch; 286753), Cy3-labelled donkey anti-mouse IgG (Jackson Immunoresearch; 286343), Cy3-labelled donkey anti-goat IgG (Jackson Immunoresearch; 284733), and Alexa Fluor488-labelled goat anti-mouse IgG (Molecular Probes; A-11001).
Live cell imaging
HeLa cells were grown on glass bottom dishes (MatTek Corporation) 36–48 h prior to use. Recombinant proteins were injected through a glass capillary into the cytoplasm. Prior to imaging, the medium was removed and replaced with DMEM without phenol red supplemented with 10% FBS and 20 mM HEPES (pH 7.3), and the plates were sealed with mineral oil to prevent evaporation of the medium. The cells were maintained at 37 °C through all steps as previously described (Figs 4–6, Haraguti et al. (1997)). Imaging was performed using Olympus IX70 microscope (Figs 1–3) or a Zeiss LSM510 META confocal microscope (Figs 4–6).
Fractionations of cellular proteins
Aliquots of a 1 x 107 suspension of cultured HeLa cells were washed with ice-cold PBS and lysed with 100 µl of 0.1% TX-100 buffer (0.1% Triton X-100, 10 mM HEPES (pH 7.3), 100 mM NaCl, 0.1 mM MgCl2, 0.1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM DTT, 1 µg/mL aprotinin, leupeptin and pepstatinA) for 10 min on ice. After centrifugation (1000 g for 5 min at 4 °C), the debris was washed once more with the same volume of 0.1% TX-100 buffer. For DNase I extraction, the debris from the 0.1% TX-100 extraction was resuspended in 100 µl of ice-cold 0.1% TX-100 buffer supplemented with 1000 unit/mL DNase I (SIGMA) and incubated at 25 °C for 1 h. After incubation, insoluble and soluble materials were separated by centrifugation (1000 g for 5 min at 4 °C). For NaCl extraction, the debris from the DNase I extraction was resuspended in 100 µl of ice-cold 0.1% TX-100 buffer supplemented with 2 M NaCl and incubated for 10 min on ice. After incubation, insoluble and soluble materials were separated by centrifugation (1000 g for 5 min at 4 °C). For 1% SDS extraction, the debris from the NaCl extraction was resuspended in 100 µl 1% SDS buffer (1% SDS, 10 mM HEPES (pH 7.3), 100 mM NaCl, 0.1 mM MgCl2, 0.1 mM PMSF 1 mM DTT, 1 µg/mL aprotinin, leupeptin and pepstatinA) and incubated for 10 min at room temperature. After incubation, the insoluble and soluble materials were separated by centrifugation (15 000 g for 5 min at 25 °C). Samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes using a semidry transfer apparatus. After incubation in blocking solution (Tris-buffered saline containing 3% skim milk) at room temperature for 30 min, the membranes were first incubated with antibodies for 1 h, followed by incubation with peroxidase-labelled second antibodies. Antibody reactive protein bands were visualized using the ECL solution. For the experiments indicated in Fig. 3B, HeLa cells on eight-well multitest slides (ICN Biomedicals) were treated with the same buffer as was used for the fractionation study described above except that each buffer was supplemented with 3 mM MgCl2. Extracted cells were fixed, and subjected to indirect immunofluorescence.
Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses
FRAP was performed using a Zeiss LSM510 META confocal microscope (Zeiss; using the 488 nm line of an Ar laser; 40 x C-Apochromat/1.2 W corr). The cells injected with GFP-importin were used. One equatorial image was collected (100% laser power; 3% transmission; pinhole 200 µm; scan speed 5; 6 x zoom), and 10 x 10 µm2 square area was bleached. After photobleaching, scans were taken at the imaging intensity every 5 min until the fluorescence intensity reached a plateau.
All measurements were corrected for background fluorescence, as determined by measuring the average non-cellular fluorescence in the sample. The intensity (after background subtraction) of the bleached area relative to that of the whole nucleus in each frame was measured, and the intensity relative to the prebleached image was determined (Phair & Misteli 2000).
For FLIP analyses, one equatorial image was collected (80% laser power; 12% transmission; pinhole 249 µm; scan speed 5; 35 x zoom). A region of the cytoplasm was selected and 3 x 3 µm2 square area was bleached, followed by an imaging scan. This pattern was repeated 160 times at 30 s intervals, and the nuclear fluorescence was measured over time.
All measurements were corrected for background fluorescence, as determined by measuring the average non-cellular fluorescence in the sample. To correct for the loss of fluorescence during imaging, the nuclear fluorescence of control cells (not bleached) was measured in each scan. Fluorescence in the region of interest was normalized to the change in cellular fluorescence using the following equation: F(%) = C0It/CtI0 x 100 where F is the normalized intensity of the region of interest, C0 the intensity at the region of the control cell before photobleaching, Ct the intensity at the region of control cell at time t, I0 the intensity at the region of interest before photobleaching, and It the intensity in the region of interest at time t.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nimamoto{at}riken.jp
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References |
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Received: 26 December 2003
Accepted: 5 February 2004
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) followed by an imaging scan. This pattern was repeated 160 times, and nuclear fluorescence was measured (
) over time. To correct for the loss of fluorescence during imaging, the nuclear fluorescence of a control cell (X) was measured in each scan. Fluorescence in the region of interest was normalized as described in Experimental procedures. Top panel; quantification of fluorescence loss of GFP-importin 