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First published online June 29, 2006
Journal of Experimental Biology 209, 2660-2677 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02292

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The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis

Bradley A. Buckley^*, Andrew Y. Gracey and George N. Somero

Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950, USA

View larger version (12K): [in a new window]   Fig. 1. Experimental heating regime. Control fish were held at 18°C for 480 min; fish were sampled at three time points (triangles) from the control tank at 0, 240 and 480 min. Another group of fish was ramped to 32°C, and sampled at six time points (circles). A third group was ramped to 32°C then ramped back down to 18°C and allowed to recover. Samples were taken from this group at 360, 420, 480 min (squares). N=4 for all samples.

View larger version (47K): [in a new window]   FIG. 2. Gene expression profiles in gill tissue from control and heat shocked Gillichthysmirabilis. Each row represents a single cDNA clone and each column represents a timepoint in the heat shock exposure. Genes depicted are those that were up- or downregulated at least twofold compared to the averaged values of the three control timepoints. Genes are clustered by cellular process, according to their gene ontology (GO) classification. Clustered processes are: (1) protein rescue and folding, (2) protein degradation, (3) protein synthesis, (4) proteolysis, (5) cell signaling, (6) cell proliferationand growth, (7) transcriptional regulation, (8) cytoskeletal structure and reorganization,(9) cell-cell or cell-matrix adhesion, (10) carbohydrate metabolism, (11) fatty acidmetabolism, (12) transport, (13) apoptosis and (14) other functions. For clarity, only two rows are dedicated to genes represented by two spots on the array. C, control; HS, heat shock, R; recovery.

View larger version (57K): [in a new window]   Fig. 3. Gene expression profiles in white muscle tissue from control and heat shocked Gillichthys mirabilis. Each row represents a single cDNA clone and each columnrepresents a time point in the heat shock exposure. Genes depicted are those that were up- or downregulated at least twofold compared to the averaged values of the three control time points. Genes are clustered by cellular process, according to their gene ontology (GO) classification (classifications are numbered identically to those described in the legend to Fig. 2). For clarity, only two rows are dedicated to genes represented by two spots on the array. C, control; HS, heat shock; R, recovery.

View larger version (21K): [in a new window]   Fig. 4. Ubiquitin-conjugated proteins in gill and white muscle from Gillichthys mirabilis. For determination of the concentration of ubiquitin-conjugated proteins, thawed tissues were homogenized and dot-blotted onto nitrocellulose membranes. Following incubation in primary and secondary antibodies, blots were visualized with enhanced chemiluminescence reagent and exposed to X-ray film; exposed film was scanned and spot intensity quantified densitometrically. Average pixel intensity for each spot was normalized to the average for the four time 0 individuals. Values are means ± s.d. (N=4). *Heat shock time points, in gill tissue, that differ significantly (ANOVA, P 0.001) from time 0; recovery time points, in gill tissue, that differ significantly from time 0; heat shock time points, in muscle tissue, that differ significantly (P 0.001) from time 0. In some cases, error bars are contained within symbols.

View larger version (26K): [in a new window]   Fig. 5. Hsp40 mRNA (filled symbols) and protein (open symbols) in the (A) gill and (B) white muscle from Gillichthys mirabilis. Average protein concentrations and mRNA levels are shown for fish (N=4) from each time point, either during heat shock (solid lines; circles) or during recovery (broken lines; squares). At a given time point, fish were sacrificed and frozen immediately in liquid nitrogen. The mRNA values are taken from the microarray analyses (see Materials and methods and Figs 2, 3). For protein concentration determination, thawed tissues were homogenized and dot-blotted onto nitrocellulose membranes. Following incubation in primary and secondary antibodies, blots were visualized with enhanced chemiluminescence reagent and exposed to X-ray film; exposed film was scanned and spot intensity quantified densitometrically. Average pixel intensity for each spot was normalized to the average for the four time 0 individuals. For protein levels, values are means ± s.d. (N=4). *Heat shock time points that differ significantly (ANOVA, P 0.001) from time 0; recovery time points that differ significantly from time 0. In some cases, error bars are contained within symbols.

View larger version (28K): [in a new window]   Fig. 6. Hsp70 mRNA (closed symbols) and protein (open symbols) in (A) the gill and (B) white muscle from Gillichthys mirabilis. Average protein concentrations and mRNA levels are shown for fish (N=4) from each time point, either during heat shock (solid lines; circles) or during recovery (dashed lines; squares). Methods were identical to those described in the legend to Fig. 5, except that antibodies used were specific to Hsp70. Average pixel intensity for each spot was normalized to the average for the four time 0 individuals. For protein levels, values are means ± s.d. (N=4). *Heat shock time points that differ significantly (ANOVA, P 0.001) from time 0; recovery time points that differ significantly from time 0. In some cases, error bars are contained within symbols.

View larger version (26K): [in a new window]   Fig. 7. Hsp90 mRNA (filled symbols) and protein (open symbols) in (A) the gill and (B) white muscle from Gillichthys mirabilis. Average protein concentrations and mRNA levels are shown for fish (N=4) from each time point, either during heat shock (solid lines; circles) or during recovery (broken lines; squares). Methods were identical to those described in the legend to Fig. 5, except that antibodies used were specific to Hsp90. Average pixel intensity for each spot was normalized to the average for the four time 0 individuals. For protein levels, values are means ± s.d (N=4). *Heat shock time points that differ significantly (ANOVA, P 0.001) from time 0; recovery time points that differ significantly from time 0. In some cases, error bars are contained within symbols.

View larger version (23K): [in a new window]   Fig. 8. Protein disulfide isomerase (PDI) mRNA (filled symbols) and protein (open symbols) in the (A) gill and (B) white muscle from Gillichthys mirabilis. Average protein concentrations and mRNA levels are shown for fish (N=4) from each time point, either during heat shock (solid lines; circles) or during recovery (broken lines; squares). Methods were identical to those described in the legend to Fig. 5, except that the antibodies used were specific to PDI. Average pixel intensity for each spot was normalized to the average for the four time 0 individuals. For protein levels, values are means ± s.d (N=4). *Heat shock time points that differ significantly (ANOVA, (P 0.001) from time 0; recovery time points that differ significantly from time 0. In some cases, error bars are contained within symbols.

View larger version (23K): [in a new window]   Fig. 9. Actin mRNA (filled symbols) and protein (open symbols) in the (A) gill and (B) white muscle in from Gillichthys mirabilis. Average protein concentrations and mRNA levels are shown for fish (N=4) from each time point, either during heat shock (solid lines; circles) or during recovery (broken lines; squares). Methods were identical to those described in the legend to Fig. 5, except that the antibodies used were specific to actin. Average pixel intensity for each spot was normalized to the average for the four time 0 individuals. For protein levels, values are means ± s.d. (N=4). *Heat shock time points that differ significantly (ANOVA, P 0.001) from time 0; recovery time points that differ significantly from time 0. In some cases, error bars are contained within symbols.