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Thermal acclimation changes DNA-binding activity of heat shock factor 1 (HSF1) in the goby Gillichthys mirabilis: implications for plasticity in the heat-shock response in natural populations

Bradley A. Buckley and Gretchen E. Hofmann^*

Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA

View larger version (26K): [in a new window]   Fig. 1. Map of Estero Morua, Mexico and water temperature data. (A) Experimental animals were collected on June 19, 2001 by baited trap in an estuary (Estero Morua) near Puerto Peñasco, Sonora, Mexico. (B) Water temperatures at the collection site for 53 days prior to sampling. Temperature data were gathered via a submerged data recorder.

View larger version (60K): [in a new window]   Fig. 2. Representative protein synthesis patterns in response to heat shock in the liver tissue of Gillichthys mirabilis (N=3) acclimated to (A) 21°C and (B) 28°C. Proteins were radiolabelled with 35S-methionine/cysteine after heat shock for 1 h at the temperatures listed below each lane. After heat shock, proteins were separated on 7.5% polyacrylamide gels, which were dried and exposed to X-ray films. The positions of molecular mass markers are denoted on right (arrows). N=3 for both acclimation groups.

View larger version (50K): [in a new window]   Fig. 3. HSF1—HSE complexes in the liver tissue of G. mirabilis visualized via electrophoretic mobility-shift assay (EMSA). A competition assay was run to determine HSE probe specificity. HSF-1 activation was visualized via EMSA, using a LightShiftTM chemiluminescent EMSA kit (PierceTM). After a 1 h heat shock at the indicated temperature, 3 µg of homogenized liver tissue were incubated with 15 pmol of biotinylated-DNA protein for 20 min at 18°C and then separated on a 5% non-denaturing polyacrylamide gel. Protein was blotted to nylon membrane, and HSF1—HSE complexes were visualized through a chemiluminescence reaction. Lane 1, liver tissue and biotinylated HSE probe only; lane 2, identical to lane 1 except for the addition of a 200 moll-1 excess of an unlabelled non-competitor DNA probe (AP2 from Promega); lane 3, identical to lane 1 except for addition of a 200 moll-1 excess of unlabelled HSE probe. Intensities of HSF1—HSE bands were determined by scanning densitometry (BioRadTM Fluor S Multimager).

View larger version (41K): [in a new window]   Fig. 4. Electrophoretic mobility-shift assay (EMSA) of HSF1 activation in liver tissue from G. mirabilis. Representative activation profiles of HSF1 in the liver tissues of differently acclimated G. mirabilis in response to heat shock (mean HSF1 activation levels for N=5 samples from each of the groups shown in Fig. 5). EMSAs were run as described in the legend to Fig. 3 and Materials and methods. Heat shock temperatures are labeled below each lane and acclimation groups are shown to the right of the figure.

View larger version (22K): [in a new window]   Fig. 5. The effect of thermal acclimation on the temperature sensitivity of HSF1 activation in liver tissue from G. mirabilis. Fish were collected in June, 2001 and either killed immediately (time 0), or acclimated for 5 weeks at 13°C, 21°C or 28°C. HSF1 activation levels were determined via electrophoretic mobility-shift assay. HSF1 activation levels are means ± S.E.M. (N=5). There was a significant effect of both heat shock temperature (P=0.011) and acclimation temperature (P<0.001) on HSF1 activation (ANOVA). Analyses of heat shock temperature effects within the acclimation groups are shown in Table 2. Analyses of the effect of acclimation group at a given heat shock temperature are listed in Table 3. *Within acclimation groups, heat shock temperature at which the mean HSF1 activity differed significantly (P<0.05; pairwise t-test) from that at the lowest heat shock temperature.

View larger version (18K): [in a new window]   Fig. 6. Concentrations of (A) HSF1, (B) Hsp70 and (C) actin in liver tissue from G. mirabilis. Levels of HSF1 and actin were determined by western blotting. Briefly, liver tissue was homogenized in 50mmol1-1 Tris-Cl, pH 6.8, 4% sodium dodecyl sulfate (SDS) and 1 mmol1-1 phenylmethylsulfonylfluoride (PMSF). Homogenates were boiled for 5 min and centrifuged at 12 000g for 10 min before loading on 7.5% polyacrylamide gels. After electrophoresis, proteins were blotted onto nitrocellulose membranes. Blots were incubated in primary antibody (for antibodies used, see Materials and methods) for 1.5 h and secondary HRP-conjugated antibody for 1 h. Bands were visualized by a chemiluminescence reaction and their densities determined by scanning densitometry. Values are means ± S.E.M. (N=5). Within a graph, values labeled with different letters differ significantly from one another (P<0.05: pairwise t-test).