Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history
Bradley A. Buckley1,
Marie-Eve Owen2 and
Gretchen E. Hofmann1,*
1 Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA and
2 Department of Biology, Bishops University, Lennoxville, Quebec, Canada J1M 1Z7
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Fig. 1. Seasonal plasticity in the induction threshold temperature in the intertidal mussel Mytilus trossulus. Pieces of dissected gill tissue from mussels collected in the field in February and August were incubated at the indicated temperatures for 2 h and then metabolically labeled at (13°C) for 2 h in the presence of 35S-labelled methione/cysteine. Following metabolic labeling, proteins were separated on 10 % SDS–polyacrylamide gels and processed using standard fluorographic techniques. Individual lanes were loaded with equal counts of radioactivity (3.7x107 Bq), and the locations of molecular mass markers (not shown) are indicated on the right. The threshold temperature of heat-shock protein induction for each gel is given above the right-hand pair of columns.
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Fig. 2. Effects of thermal history on the induction of heat-shock protein (Hsp) synthesis in gill tissue from Mytilus trossulus. Gill sections from mussels acclimated to 10°C in the laboratory or acclimatized in the field for 6 weeks were treated identically to those in Fig. 1 (see Fig. 1 legend for methods) with the exception of the specific heat-shock temperatures (listed above each lane). Protein synthesis patterns in the gill are depicted for an individual from each acclimation/acclimatization group (A) and for four additional mussels from each group (B). In A, protein synthesis patterns across the entire experimental thermal gradient are shown. The threshold induction temperature for laboratory-acclimated mussels was 20°C and that for field-acclimatized mussels was 26°C. In B, the first lane depicts the temperature immediately prior to induction and the second lane demonstrates Hsp induction threshold temperature for that particular individual. The locations of molecular mass markers (not shown) are given on the right.
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Fig. 3. Mussel body temperature (means ± S.D., N=5) and ambient air temperatures on 29 July 1996. Measurements were taken using a hand-held digital thermometer equipped with a thermistor during tidal emersion, when the mussels were exposed to direct solar irradiation. All body temperatures were taken sequentially on the same five mussels.
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Fig. 4. Relative levels of Hsp70, its constitutive form Hsc70 and HSF1 (A) and relative levels of ubiquitin-conjugated protein (B) in gill tissue from laboratory-acclimated and field-acclimatized Mytilus trossulus (means ± S.D., N=5). For western blotting, equivalent amounts of protein (10 µg) were electrophoresed in each lane. Scanning densitometry was performed on western blots developed with a standard enhanced chemiluminescent technique (see Materials and methods). *Values that are significantly different (P<0.005).
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Fig. 5. Heat-shock protein induction and HSF1 activation profiles from Mytilus californianus (N=5). (A) HSF1 activation was visualized via an electrophoretic mobility shift assay, using a LightShift chemiluminescent EMSA kit (Pierce). Following a 1 h heat shock at the indicated temperature, homogenized gill tissue was incubated with approximately 15 pmol of biotinylated-DNA probe for 20 min at room temperature (approximately 20°C) and then separated on 5 % non-denaturing polyacrylamide gels. The arrowhead marks the induction of Hsp synthesis, which occurred at 26°C in all individuals. (B) A competitor/non-competitor assay in which the addition of excess non-biotinylated heat-shock element probe was used to outcompete the biotinylated probe for HSF1 binding activity. Unlabelled non-competitor sequence did not have the same effect, demonstrating the specificity of our probe.
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Fig. 6. Relative levels of activated HSF1 in Mytilus californianus. Densities of HSF1–HSE complex bands were calculated with Quantity One software and standardized to an internal standard (a single gill extract sample run on every gel). Values are means + S.D., N=5. *Values that are significantly different (P<0.005).
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Fig. 7. Model of the regulation of HSF1 (after Morimoto, 1993). At non-stressful temperatures, HSF1 is present in the cytosol as an inactive, monomeric protein. Following heat shock, HSF1 localizes in the nucleus, trimerizes and acquires DNA-binding ability (step 1). Following inducible phosphorylation of serine residues, HSF1 transactivates Hsp genes (step 2). In Mytilus, it appears that the temperatures at which steps 1 and 2 occur are uncoupled, and that the temperature at which HSF1 binds the Hsp promoter is lower than that at which gene product appears in the cell.
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© The Company of Biologists Ltd 2001
