Department of Physiology, McGill University, Montreal, Quebec, Canada
Submitted 1 December 2004 ; accepted in final form 25 May 2005
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ABSTRACT |
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nuclear transport; chaperone; nuclear retention; nucleoli
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MATERIALS AND METHODS |
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Treatment with leptomycin B, latrunculin B, and cytochalasin B.
To analyze the potential role of the importin- family member Crm1, we have tested the effect of leptomycin B (LMB), a compound that selectively inhibits this exporter (12). To this end, cells were incubated with 10 ng/ml LMB (gift of M. Yoshida, University of Tokyo, Tokyo, Japan) dissolved in ethanol or with the solvent ethanol for 15 h at 37°C after heat stress. Unstressed cells were incubated with LMB for up to 24 h at 37°C. Latrunculin B and cytochalasin B (Calbiochem, San Diego, CA) were dissolved in DMSO. Cells recovering from stress were incubated for 15 h at 37°C with 1 mM latrunculin B, 10 µM cytochalasin B, or the solvent DMSO. Unstressed cells were treated with latrunculin B, cytochalasin B, or DMSO for 3 h at 37°C, as indicated in Figs. 3 and 4. In control experiments, the effect of latrunculin B or cytochalasin B on actin polymerization was tested with FITC-labeled phalloidin, following the suppliers protocol (Sigma, Oakville, ON, Canada).
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Human-mouse heterokaryons.
Heterokaryons between HeLa and mouse NIH3T3 cells were generated by a modification of published procedures (5). In brief, HeLa cells transiently synthesizing EGFP-hsc70 were trypsinized and seeded on coverslips 24 h after transfection to reach 60% confluency on the next day. HeLa cells were then exposed to 1 h of heat stress at 45.5°C, and 3 x 105 NIH3T3 cells were added to each well of a six-well plate. After 1.5 h, mouse cells adhered to the coverslips, and cycloheximide was added to 75 µg/ml for 30 min. Cells were fused subsequently for 2 min with 50% polyethylene glycol (PEG) 3350. After removal of PEG, samples were washed three times with PBS and incubated at 37°C in growth medium containing 100 µg/ml cycloheximide. Cells were fixed 3, 5, and 15 h after heat stress, i.e., 1, 3, and 13 h after fusion. Nuclei were stained with DAPI, and heterokaryons or homokaryons were monitored for the distribution of EGFP-hsc70.
Analysis of nuclear retention.
Nontransfected HeLa cells were exposed for 1 h to 45.5°C and subsequently treated for 5 min with 40 µg/ml digitonin in buffer B on ice (19). Digitonin-extracted cells were incubated with buffer B [containing 5 mg/ml BSA and 0.05% Nonidet P-40 (NP-40)] for 15 min at room temperature. The buffer was supplemented with 2.5 mM ATP, 2.5 mM ADP, or 1 mM nonhydrolyzable ATP analog adenosine 5'-(,
-imido)triphosphate (AMP-PNP), as indicated in Figs. 5 and 6. Samples were washed extensively in buffer B-BSA-NP-40, in buffer B, and twice in PBS and fixed and processed for indirect immunofluorescence with anti-hsc70 antibodies as described above. To monitor the intactness of nuclear envelopes, cells were extracted with digitonin, treated with buffer B-BSA-NP-40, and washed as described above. Washed samples were fixed, blocked with PBS-2 mg/ml BSA, and incubated with anti-lamin B antibodies (0.5 µg/ml; Santa Cruz Biotechnology, sc-6217). Control cells were treated with digitonin only before blocking and incubation with antibodies.
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Western blot analysis. Western blotting and enhanced chemiluminescence detection was carried out essentially as described previously (4), using a Lumigen PS-3 detection kit (Amersham Biosciences).
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RESULTS AND DISCUSSION |
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In control experiments, LMB efficiently inhibited shuttling of NLS-NES-GFP2. This reporter protein carries a signal for nuclear localization (SV40-NLS) as well as nuclear export (PKI-NES); PKI-NES is recognized by Crm1. NLS-NES-GFP2 was both cytoplasmic and nuclear in the absence of LMB but accumulated in nuclei when LMB was added to the growth medium. Together, these results show that hsc70 nuclear export upon recovery from heat or under nonstress conditions does not rely on Crm1.
Actin filament-destabilizing drugs latrunculin B and cytochalasin B inhibit nuclear export of hsc70s in stressed and control cells. To further define hsc70 nuclear transport, we tested the effect of latrunculin B and cytochalasin B. These compounds are believed to affect actin located at the nuclear pore complex (NPC), thereby preventing nuclear export of various components (8). Incubation with latrunculin B or cytochalasin B drastically reduced the amount of actin filaments, which became obvious by the loss of phalloidin binding (not shown). Importantly, in cells recovering from heat shock, either drug prevented hsc70 export from the nucleus (Fig. 3A). Similarly, when unstressed cells were treated with latrunculin B or cytochalasin B, hsc70s concentrated in nuclei and nuclear accumulation were apparent after a 3-h treatment. These results support the idea that under normal physiological conditions, i.e., in the absence of stress, hsc70s shuttle between nucleus and cytoplasm in human culture cells. Furthermore, hsc70 export is abolished by the destabilization of filamentous actin, suggesting a role for actin in the translocation of nuclear hsc70s to the cytoplasm. In particular, actin located at the NPC could play a crucial role because it seems to be involved in nuclear export of multiple cargos (8).
Hsc70 shuttling is inhibited by heat shock and restored when cells recover from stress. Heterokaryons have been used to analyze the shuttling of proteins that are concentrated in nuclei at steady state under normal growth conditions. However, this approach has not been applied previously to monitor shuttling in heat-stressed cells. To achieve this, we used the fluorescent reporter protein EGFP-hsc70, which shares the biological properties of endogenous hsc70s when tested under a variety of stress conditions (Fig. 4A; M. Kodiha and U. Stochaj, unpublished observations). In unstressed cells, EGFP-hsc70 was distributed throughout nuclei and cytoplasm (Fig. 4A). Like endogenous hsc70s, EGFP-hsc70 accumulated in nuclei when cytochalasin B or latrunculin B was added to the growth medium (Fig. 4A).
Human-mouse heterokaryons were used to evaluate EGFP-hsc70 shuttling after stress exposure; these heterokaryons contain nuclei from both species, which share the same cytoplasm (5). EGFP-hsc70 was first concentrated in nuclei of HeLa cells by heat shock for 1 h at 45.5°C. HeLa cells were returned subsequently to the normal growth temperature and fused to mouse cells. In these heterokaryons, we localized EGFP-hsc70 at different time points during their recovery from heat exposure. (It should be noted that EGFP-hsc70 synthesized in HeLa cells is the only source of fluorescence seen in Fig. 4B, because cycloheximide prevents de novo synthesis of EGFP-hsc70 in heterokaryons.) Three hours after heat shock, EGFP-hsc70 remained restricted to human nuclei in human-mouse heterokaryons (Fig. 4B). By contrast, EGFP-hsc70 was absent from mouse nuclei and the common cytoplasm, demonstrating that the translocation from human nuclei to the cytoplasm was prevented at this point. To determine whether this export inhibition, and thereby the block in shuttling, was reversible, heterokaryons were allowed to recover for a longer period of time. At 5 h after heat shock, EGFP-hsc70 began to migrate out of the human nucleus and appeared in the common cytoplasm. After a 15-h recovery period, human and mouse nuclei displayed comparable signals for EGFP-hsc70, showing that shuttling of the chaperone had resumed.
The absence of EGFP-hsc70 from mouse nuclei at early time points after cell fusion is not simply a failure of the nonstressed mouse nuclei to import the chaperone. While generating heterokaryons, we also obtained fusions originating from a mixture of transfected and nontransfected HeLa cells, the latter were not synthesizing EGFP-hsc70. In these multinucleated cells, or homokaryons, 3 h after heat shock we detected nuclei that did not contain EGFP-hsc70 (Fig. 4B). As observed for heterokaryons, EGFP-hsc70 appeared in the common cytoplasm of homokaryons at 5 h upon heat exposure and began to migrate into all of the nuclei present. At 15 h after heat shock, EGFP-hsc70 was present in all of the nuclei of multinucleated cells, and nuclei from transfected and nontransfected cells could no longer be distinguished.
For comparison, heterokaryons were generated with the protocol described above, but with unstressed instead of heat-treated HeLa cells. When inspected 1 and 3 h after fusion, the times equivalent to 3 and 5 h after heat shock, EGFP-hsc70 was detected at the earlier time point in all mouse nuclei present in heterokaryons (Fig. 4C). At 1 h after fusion, the amount of EGFP-hsc70 in mouse nuclei was somewhat variable between different heterokaryons. Three hours after fusion, the time equivalent to 5 h after heat exposure for the stressed cells shown in Fig. 4B, the signal for EGFP-hsc70 was comparable to that for mouse and HeLa nuclei of heterokaryons (Fig. 4C). Together, the data obtained for heterokaryons support the idea that EGFP-hsc70 appears faster in mouse nuclei when unstressed HeLa cells are the source of the fusion protein.
Hsc70s are retained in nuclei of heat-shocked cells. Shuttling between nucleus and cytoplasm can be regulated on different levels; this includes import, export, and retention of the shuttling protein. As such, the movement of nuclear hsc70s to the cytoplasm could be controlled by retention in the nucleus. The liberation from nuclear anchors would be a rate-limiting step for shuttling because this release is a prerequisite for subsequent export to the cytoplasm.
Heat shock is likely to trigger hsc70 binding to a large number of nuclear proteins that require chaperone activity, a process that may contribute to nuclear retention of hsc70s. To test this hypothesis, we have developed an assay for hsc70 release from nuclear anchors, which is not complicated by transport across the nuclear envelope (see MATERIALS AND METHODSfor details). To this end, control and heat-shocked HeLa cells were first extracted with digitonin, which permeabilizes the plasma membrane and removes most of the cytoplasmic proteins but leaves the nuclear membranes intact. After digitonin extraction, the nonionic detergent NP-40 was used to solubilize the nuclear envelope, which would no longer restrict the movement of proteins. The proper permeabilization of membranes in our assays was verified in control experiments (Fig. 5A). As expected, anti-laminin B antibodies do not have access to the nuclear lamina in digitonin-treated cells, but subsequent incubation with NP-40 led to antibody binding.
We next used this assay to determine whether hsc70s are retained in nuclei of control and heat-shocked cells (Fig. 5, B and C). Samples were treated with digitonin followed by incubation in the absence or presence of NP-40. Heat-shocked samples retained most of the hsc70s even in the presence of NP-40, suggesting that binding to nuclear anchors contributes to hsc70 accumulation in nuclei. By contrast, little hsc70 was found in nuclei of unstressed cells under any of the conditions tested (Fig. 5B and Table 1).
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Nuclear retention of hsc70s changes during recovery from heat shock. We next monitored hsc70 nuclear retention in cells recovering from stress. To this end, HeLa cells exposed to heat were analyzed after 3-, 5-, and 15-h incubation at 37°C. The amount of hsc70s present in the nucleoplasm decreased during recovery, and nucleoplasmic chaperone could be liberated with ATP or AMP-PNP (Fig. 6). In addition, hsc70s transiently concentrated in nucleoli, albeit with kinetics different from their accumulation in the nucleoplasm. The hsc70 levels increased in nucleoli of most cells after a 3-h recovery period, but only in few nucleoli after 5 h (Fig. 6, Table 1). As observed after heat exposure, hsc70 associated with nucleoli was not fully liberated by incubation with ATP or AMP-PNP. At 15 h after heat shock, hsc70 distribution was similar to that in unstressed controls and no accumulation was seen in nuclei or nucleoli. Results of these in vitro experiments (summarized in Table 1) suggest that hsc70 binding to chaperone substrates contributes to its nuclear retention immediately after heat treatment and at early stages of recovery.
Binding of hsc70s to nuclei of stressed cells. Members of the hsp/hsc70 family are involved in multiple interactions in the nucleus, and in response to heat stress hsc70s can be expected to interact with a large variety of nuclear components. For instance, the importance of hsc70s for the organization of nucleoli is well established, and chaperones are implicated in restoring nucleolar function upon stress (13, 16). On the basis of these earlier observations, nucleolar proteins were candidates for the interaction with hsc70s in heat-treated cells. To test this idea, we examined fibrillarin, a bona fide component of nucleoli, and the ribosomal protein rpS6, which is assembled into the small ribosomal subunits in nucleoli. When analyzed by indirect immunofluorescence (Fig. 7A), fibrillarin was concentrated in nucleoli of control cells but redistributed throughout nucleus and cytoplasm in response to heat exposure. During recovery, fibrillarin relocated to nucleoli, and after 15 h at 37°C, its distribution was similar to that in unstressed controls. In parallel, nuclear proteins were immunoprecipitated with antibodies against hsc70s, and immunoprecipitates that contained comparable amounts of hsc70 were probed with antibodies against fibrillarin (Fig. 7B). Although the nucleolar protein copurified with hsc70s for control, stressed, and recovering cells, clearly the highest amount of fibrillarin associated with hsc70s in heat-shocked cells.
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In conclusion, our study demonstrates that the nucleocytoplasmic shuttling of chaperones of the hsp/hsc70 family is inhibited by heat shock but restored when cells recover from stress-induced damage. Importantly, stress alters not only the steady-state distribution but also the movement of hsc70s between nucleus and cytoplasm. Herein we have shown that hsc70 retention in the nucleus is drastically increased in response to heat exposure, a process that prevents export of the chaperone to the cytoplasm and thereby shuttling. We have identified two different forms of hsc70 interaction with nuclear anchors, both of which can be expected to contribute to the sequestration of chaperone in nuclei. First, hsc70s bind to nuclear proteins in an ATP-sensitive fashion, which most likely represents binding of the chaperone to folding substrates. Second, hsc70s associate with nucleoli, and at least a portion of the nucleolar chaperone cannot be liberated by the addition of ATP. This could indicate an association of hsc70s with nucleolar components in a fashion that is distinct from a chaperone-folding protein interaction. Independent of the type of association that underlies hsc70s retention in nuclei, we have shown that this retention is low in control cells, high after heat shock, and gradually reduced during recovery from stress. These changes in nuclear retention of hsc70s upon stress and during recovery can be expected to affect a variety of biological processes that require chaperone activity. For instance, immediately after stress, the proper folding of chaperone substrates in the cytoplasm may be impaired until de novo synthesis or shuttling of hsp/hsc70s resumes. Moreover, stress may interfere with the chaperone-dependent targeting of cytoplasmic proteins to various organelles, including mitochondria and peroxisomes, both of which require cytoplasmic hsp/hsc70s for protein import.
On the basis of the results described herein, we have developed a simplified model for hsc70 shuttling (Fig. 8). Hsc70s accumulate in nuclei of heat-stressed cells, where they are initially retained in the nucleoplasm by binding to chaperone substrates in an ATP-sensitive fashion. During recovery from heat, hsc70s relocate within the nucleus and transiently concentrate in nucleoli; this interaction cannot be prevented by the addition of ATP. As recovery progresses, hsc70s are liberated from nuclear and nucleolar anchors, which precedes their relocation to the cytoplasm. We propose that the release from nuclear anchors is a limiting factor that regulates hsc70 nuclear export and thereby shuttling of the chaperone in cells exposed to heat.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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