Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history
1 Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA and
2 Department of Biology, Bishops University, Lennoxville, Quebec, Canada J1M 1Z7
*Author for correspondence (e-mail: ghofmann{at}asu.edu)
Accepted July 20, 2001
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Summary |
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Key words: mussel, Mytilus trossulus, Mytilus californianus, heat shock, heat shock response, heat shock protein, Hsp70, temperature, thermal acclimation, gene expression.
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Introduction |
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One significant development in the pursuit of the ecological importance of Hsps has been the demonstration that the temperature at which these genes are activated, the threshold induction temperature, varies within the lifetime of a single organism and is subject to thermal acclimation and acclimatization. In particular, studies on marine fish and invertebrates have shown that there is a great deal of plasticity in the threshold induction temperature for Hsps as a function of season (Dietz and Somero, 1992; Roberts et al., 1997) and laboratory acclimation (Dietz, 1994; Hofmann and Somero, 1996; Tomanek and Somero, 1999). These observations have particular significance because intertidal organisms often face extreme variations in environmental temperature (see Helmuth, 1999) that may call for commensurately variable protein chaperoning.
However, despite the growing database regarding expression patterns of Hsps in nature, we know very little about how Hsp genes are regulated in response to variations in environmental temperature. What processes account for the season- or temperature-dependent manner in which the gene activation temperatures change? Furthermore, how is environmental temperature sensed and then transduced to the nucleus? The key to answering these questions is undoubtedly related to how the Hsp genes are transcriptionally activated. During gene expression, transactivation of heat-shock genes is mediated by the interaction between heat-shock transcription factor 1 (HSF1) and the heat-shock element (HSE), a series of pentameric units of the sequence 5'-nGAAn-3' found in the promoter of all Hsp genes (Xiao and Lis, 1988; Wu, 1995). Although several HSFs have been characterized in eukaryotic cells, HSF1 is the factor responsive to conditions and treatments that induce the heat-shock response (Sarge et al., 1993; Wu, 1995). It has been shown that the temperature of HSF1 activation, specifically the acquisition of promoter-binding ability, is influenced by evolutionary temperature in Drosophila melanogaster reared at different temperatures for more than 20 years in the laboratory (Lerman and Feder, 2001) and in phylogenetically distant lizards adapted to different habitat temperatures (Zatsepina et al., 2000). However, in D. melanogaster, the HSF1 activation temperature was found not to differ between strains with different thermotolerances and Hsp synthesis profiles (Zatsepina et al., 2001). At present, it is unclear what role HSF1 plays in the plasticity of the heat-shock response as a whole, especially within a single species during acclimatization in nature or during short-term acclimation in the laboratory. Particularly lacking are studies in non-model organisms that integrate simultaneous measurements of protein denaturation, molecular chaperone expression and the behavior of HSF1 in the face of realistic temperature stresses. With regard to HSF1, specific unanswered questions are (i) does the amount of activated HSF1 change during thermal acclimation/acclimatization and (ii) what is the relationship between the temperature of HSF1 activation and the threshold induction temperature of Hsp genes in organisms experiencing temperature stress in nature?
To begin to address these questions, the present study extends our examination of the heat-shock response in two species of intertidal mussel from the genus Mytilus. First, using established metabolic labeling techniques, we tested the plasticity of the threshold induction temperature in gill tissue both by comparing winter- and summer-acclimatized M. trossulus and through comparisons of laboratory-acclimated mussels with mussels simultaneously acclimatized in the field. Second, in the latter comparisons, western blotting was used to monitor levels of the constitutive and inducible isoforms of the molecular chaperones from the 70 kDa Hsp gene family and levels of ubiquitin-conjugated proteins (a direct measure of cellular protein denaturation) post-acclimation/acclimatization. Finally, concentrations of HSF1 were quantified in gill from the same mussels. In addition, a separate experiment was conducted in which we used electrophoretic mobility shift assays (EMSAs) to examine HSF1 DNA-binding activity in relation to the threshold induction temperature for the Hsp gene products in M. californianus.
The results presented here show that the threshold induction temperature for Hsp70 (i.e. the temperature at which the heat-inducible hsp70 gene was expressed) in gill tissue was subject to thermal acclimation/acclimatization and varied significantly with the thermal history of the organism. In addition, the shift in the set-point for the induction of Hsp70 genes was correlated with a concomitant sevenfold increase in levels of the inducible isoform of Hsp70 but no change in the levels of the constitutive cognate isoform of Hsp70 or HSF1. Finally, the temperature of HSF1 activation (i.e. acquisition of DNA-binding ability) in M. californianus was approximately 9°C lower than the threshold temperature of Hsp gene induction, which has important implications for our understanding of how these genes are regulated during adaptation to a given thermal regime.
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Materials and methods |
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Preparation of gill tissue
Gill lamellae were dissected from individual mussels, and approximately 100 mg of tissue was homogenized in 5 volumes of homogenization buffer (HB) consisting of 50 mmol l1 Tris-HCl, pH 6.8, 4 % sodium dodecyl sulfate (SDS) and 1 mmol l1 phenylmethylsulfonylfluoride (PMSF). The homogenate was heated at 100°C for 5 min and then centrifuged at 12 000 g for 15 min at room temperature (approximately 23°C). The resulting supernatant was analyzed for protein content using a modified Bradford assay (Pierce Coomassie Plus) and stored at 20°C prior to electrophoresis.
Heat-shock protein induction experiments: metabolic labeling with 35S-labelled amino acids
Metabolic labeling was conducted according to the protocol described by Hofmann and Somero (Hofmann and Somero, 1996). Dissected pieces of gill tissue (approximately 100 mg) were placed in 0.5 ml of Hepes-buffered artificial sea water [20 mmol l1 Hepes, 7.57 mmol l1 (NH4)2SO4, 375 mmol l1 NaCl, 9.35 mmol l1 KCl, 2.7 mmol l1 NaHCO3, 17.95 mmol l1 Na2SO4, 37.7 mmol l1 MgCl2.6H2O, 8 mmol l1 CaCl2.2H2O, 10 mmol l1 glucose] containing 3.7x106 Bq of 35S-labelled methionine/cysteine amino acid mixture (NEN). Each 1.5 ml microcentrifuge tube was pre-equilibrated to the desired incubation temperature prior to the addition of the tissue; the incubation temperatures used were 14, 17, 20, 23 26, 29, 32 and 35°C. After the 2 h incubation period, tissues were washed twice with 1 ml of Hepes-buffered sea water at 4°C, homogenized in 200 µl of HB and processed using the protocol described in the sample preparation section above. Samples from the induction experiments were not assayed for protein concentration; instead, a sample was analyzed in a liquid scintillation counter to determine the amount of radioactivity per milliliter for each gill extract.
Electrophoresis and fluorography
Patterns of protein synthesis during the temperature exposures were examined using SDSpolyacrylamide gel electrophoresis in combination with fluorography, as described by Hofmann and Somero (Hofmann and Somero, 1996). Proteins were separated on 10 % polyacrylamide gels, and each lane was loaded to give equivalent amounts of radioactivity (250 000 cts min1). Following electrophoresis, gels were treated with an autoradiographic enhancer (EN3HANCE; DuPont NEN), dried and exposed to X-ray film (Kodak X-OMAT AR5) at 70°C for an empirically determined period.
Solid-phase immunochemical measurement of Hsp70, HSF1 and ubiquitin conjugates
Immunochemical assays and scanning densitometry were employed to determine the levels of both the constitutive and inducible isoforms of Hsp70, HSF1 and ubiquitin-conjugated protein in gill tissue from laboratory-acclimated and field-acclimatized specimens of Mytilus trossulus. Hsp70 western blots were performed as described by Hofmann and Somero (Hofmann and Somero, 1995) except that wet electrophoretic transfer at 30 V for 15 h was used during the western protocol (transfer buffer: 20 mmol l1 Tris, 192 mmol l1 glycine, 20 % methanol). Equal amounts of protein (10 µg total protein) were separated on 7.5 % polyacrylamide gels. The immunodetection was performed using an anti-Hsp70 rat monoclonal antibody that crossreacts with the constitutive and inducible forms of Hsp70 (Affinity Bioreagents; MA3-001). For HSF1 western blots, the blot was sequentially incubated with an anti-HSF1 antibody (mouse anti-Drosophila; courtesy of Dr Carl Wu) diluted 1:10 000 in 0.5 % bovine serum albumin in phosphate-buffered saline (PBS)/0.1 % Tween 20 followed by a horseradish-peroxidase-conjugated secondary antibody (goat anti-mouse) diluted 1:20 000 in 5 % non-fat dry milk in PBS. Western blots were ultimately developed using an enhanced chemiluminescence (ECL) protocol according to the manufacturers instructions (Amersham). Ubiquitin conjugate analysis was performed as described previously (Hofmann and Somero, 1995). In all cases, the linear relationship between the chemiluminescent signal and the quantity of antigen was tested with known quantities of purified, commercially purchased protein and was further optimized for the gill extracts such that the amount of protein loaded was within the linear range of the ECL signal.
Electrophoretic mobility shift assay
For electrophoretic mobility shift assays (EMSAs), gill tissue (approximately 100 mg) was incubated in 1.5 ml microcentrifuge tubes containing 0.5 ml of Hepes-buffered artificial sea water. From each individual mussel, a piece of gill tissue was exposed to the desired temperature for 2 h; the temperature range was 1332°C. Following the temperature exposures, individual pieces of tissue were flash-frozen in liquid nitrogen prior to being processed for EMSA. To prepare tissue extracts, the gill samples were thawed on ice and lysed in a stabilization buffer containing 25 % glycerol, 20 mmol l1 Hepes (pH 7.9), 420 mmol l1 NaCl, 1.5 mmol l1 MgCl2, 0.2 mmol l1 EDTA, 0.5 mmol l1 PMSF and 0.5 mmol l1 dithiothreitol. EMSAs were conducted using LightShift chemiluminescent EMSA kits (Pierce). Briefly, gill extracts (7 µg total protein) were incubated with approximately 15 pmol of biotinylated HSE oligonucleotide probe (5'-GCCTCGAATGTTCGCGAATTT-3') (Airaksinen et al., 1998) in 25 mmol l1 Hepes (pH 7.6), 100 mmol l1 NaCl, 15 % glycerol, 0.1 % NP-40 and 0.5 mmol l1 PMSF in a final volume of 20 µl. Incubations were conducted for 30 min at 23°C. After incubation, assay mixtures were applied to 5 % acrylamide non-denaturing gels and electrophoresed for approximately 2 h at 100 V. Gels were then transferred to nylon membranes via electroblotting at 380 mA for 30 min. HSF1HSE complexes on each blot were visualized by a chemiluminescent reaction with streptavidin/horseradish peroxidase, according to the manufacturers protocol, and exposure of the blot to X-ray film (Kodak). The specificity of the HSE probe was confirmed in assays in which unlabelled HSE probe was added in excess as a competitor; in these assays, the intensity of the HSEHSF1 complex band was reduced on the resulting X-ray film.
Air and body temperature data
Mussel body temperatures were measured in the field during a low-tide aerial exposure. A digital thermometer was inserted into the shell of five mussels (of a similar size to those used in experiments), and their temperature was recorded every 20 min for 7 h. Ambient air temperature was also recorded over the same period.
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Results |
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There was an appreciable increase in the levels of both the 70 kDa class of Hsps and the low-molecular-mass class at 2023°C in laboratory-acclimated mussels, but in the field-acclimatized mussels these peaks occurred at 2629°C. Field-acclimatized mussels saw daily body temperatures fluctuations of 1534°C during tidal emersion, temperatures considerably higher than air temperature as a result of direct solar exposure (Fig. 3). These data agree with measurements of daily body temperature ranges of more than 20°C that have been recorded elsewhere in Mytilus californianus from the Pacific Northwest coast (Helmuth, 1999).
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Discussion |
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Four salient findings in this study address the effects of laboratory acclimation and field acclimatization on the heat-shock response and its regulation in the intertidal mussel Mytilus. First, in all cases, the threshold temperature for Hsp induction displayed a great deal of plasticity and varied with the thermal history of M. trossulus. Second, endogenous levels of the inducible isoform of Hsp70 were approximately seven times higher in the field-collected group than in the laboratory-acclimated group, and this corresponded with a fourfold increase in the level of ubiquitin conjugates (i.e. irreversibly damaged proteins) in the field-acclimatized group. Third, endogenous levels of HSF1 and of the constitutive isoform of Hsp70 did not vary between the two groups. Finally, the activation temperature of HSF1 was not identical to that of the threshold temperature for Hsp synthesis in the congeneric M. californianus.
The temperature at which the first noticeable expression of Hsps occurred shifted from 2023°C to 2629°C in 6 weeks of acclimatization to warm and varying environmental temperatures (Fig. 2A,B). Furthermore, there was a strong induction of low-molecular-mass Hsps (sHsps) in the laboratory-acclimated individuals at 23°C, whereas sHsps were constitutively expressed in the field-acclimatized mussels (Fig. 2A). sHsps (of which Hsp26 is the primary constituent) are thought to be involved in binding non-native, refoldable protein for subsequent refolding by other chaperones (Haslbeck et al., 1999). The regulation of sHsp activity involves a post-translational oligimerization and stress-responsive dissociation, so their expression patterns will not be discussed in the context of the HSF1-regulated mechanism of interest in the present study. Their constitutive expression in warm-acclimatized mussels in the field may reflect the need to bind and hold the large endogenous amount of denatured protein measured in these animals (see discussion of ubiquitin-conjugated protein below).
The finding that the induction set-point for Hsp synthesis changes with thermal history agrees with other studies in diverse organisms (for a review, see in Feder and Hofmann, 1999) including our own previous work on Mytilus (Hofmann and Somero, 1995; Hofmann and Somero, 1996). In general, organisms from warmer environments induce Hsps at a higher temperature than do closely related organisms from colder environments, whether that involves differences between congeners with different habitat distributions [e.g. in marine invertebrates (Tomanek and Somero, 1999); in insects (Huey and Bennett, 1990; Gehring and Werner, 1995)] or seasonal differences within a species (Dietz and Somero, 1992; Fader et al., 1994). In Mytilus, higher induction thresholds have been observed in M. galloprovincialis, a warm-water species, than in M. trossulus, which has a colder distribution in nature (Hofmann and Somero, 1995), and M. californianus has a demonstrably higher Hsp induction set-point in the summer than in the winter (Roberts et al., 1997). But what processes are responsible for this upward shift in threshold induction temperature?
Measurements of ubiquitin conjugation make it unlikely that the answer is that warm-acclimatized organisms simply sustain less total thermal denaturation of their protein pool (Fig. 4B). Ubiquitin is an abundant, low-molecular-mass protein that is crucial to the selective targeting of denatured proteins for proteolytic degradation (for a review, see Ciechanover, 1998). A thermally denatured protein that is not rescued through chaperone-mediated stabilization and refolding is targeted for degradation by conjugation with a ubiquitin tag [see (Wickner et al., 1999)]. Therefore, the content of ubiquitin-conjugated proteins is a direct measure of the amount of thermal damage incurred by the cellular protein pool. In the present study, levels of ubiquitin conjugates were four times higher in gill tissue from field-acclimatized mussels than in gill tissue from the laboratory-acclimated individuals. A similar result was found in seasonal comparisons of M. trossulus experiencing temperature variation in the field: levels of ubiquitin conjugates were higher in summer than in winter (Hofmann and Somero, 1995). It is apparent, therefore, that the process of warm-acclimatization in these organisms does not involve an increase in the inherent thermostability of the cellular protein pool (which would necessitate upregulation of a large number of more thermostable protein isoforms) and that significant temperature-related protein damage is occurring at elevated, but routinely experienced, environmental temperatures.
It is possible, however, that the gradual accumulation of Hsp70 during warm-acclimatization, such as that observed here and in Mytilus elsewhere (Hofmann and Somero, 1995; Roberts et al., 1997), might act as a buffer against subsequent heat stress, conferring increased thermotolerance. It is important to note that it was levels of the stress-inducible isoform of Hsp70 that increased in intracellular concentration and not of the constitutive isoform (Fig. 4A), which is involved in non-stress-related chaperoning functions [see (Fink, 1999)]. This means that the build-up in standing stocks of intracellular Hsp70 is probably due to repeated induction events during acclimatization to gradually warming environmental temperatures rather than to a thermally triggered increase in overall protein synthesis rates (which could result in a similar increase in levels of the constitutive isoform of Hsp70).
The increased levels of endogenous Hsp70 could affect the threshold induction temperature in at least two ways. First, increased levels of free Hsp70 in the cell might be sufficient to handle mild temperature stress, and therefore the induction of Hsp genes and de novo synthesis of Hsps would not be necessary until a higher threshold temperature had been experienced. The second possibility is that rising endogenous Hsp70 levels might result in an upward shift in threshold induction temperature through a classic negative feedback loop, a hypothesized model for the regulation of Hsp genes (Craig and Gross, 1991; Morimoto, 1993; Wu, 1995) (Fig. 7). In this model, the activity of the heat-shock transcription factor (HSF1) responsible for stress-induced upregulation of all families of Hsp genes is controlled post-translationally through the complexing of inactive HSF1 monomers with molecular chaperones, particularly Hsp70 and Hsp90 (Shi et al., 1998; Zou et al., 1998). During heat stress, Hsp70 and Hsp90 would be recruited to chaperone the denaturing protein pool, releasing HSF1 and enabling this molecule to trimerize, localize in the nucleus and upregulate Hsp genes. In warm-acclimatized M. trossulus, it is possible that increased levels of Hsp70 (and potentially Hsp90, which was not measured here) maintain HSF1 in an inactive state for longer, and only when temperatures reach the new higher threshold temperature is HSF1 released in order to transactivate Hsp genes.
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Acknowledgments |
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