Physiol Genomics 25: 493–501, 2006. First published April 4, 2006; doi:10.1152/physiolgenomics.00195.2005. Thermoprotection of synaptic transmission in a Drosophila heat shock factor mutant is accompanied by increased expression of Hsp83 and DnaJ-1 Scott J. Neal,1,* Shanker Karunanithi,2,* Adrienne Best,2 Anthony Ken-Choy So,1 Robert M. Tanguay,3 Harold L. Atwood,2 and J. Timothy Westwood1 Departments of 1Biology and 2Physiology, University of Toronto, Mississauga and Toronto, Ontario; and 3 Laboratory of Cellular and Developmental Genetics, Department of Medicine and Centre de Recherche sur la Fonction, la Structure, et l’Ingénierie des Protéines, Université Laval, Ste-Foy, Quebec, Canada Submitted 4 August 2005; accepted in final form 27 March 2006 neuromuscular junction; heat stress; thermotolerance; microarray such as elevated temperatures pose a serious threat to all eukaryotic organisms. Developing Drosophila larvae, which are at risk of desiccation and other developmental defects when subjected to severe heat stress, have proven to be very useful for experimental studies of thermally induced responses (14). The Drosophila nervous system is particularly sensitive to thermal damage. Prior heat shock (brief exposure to sublethal temperatures) affords thermotolerance to Drosophila neuronal synapses, extending their performance to higher than normal temperatures (21, 22). We investigated the possible contributions of heat-induced proteins to thermoprotective mechanisms in this organism. In Drosophila, most protein synthesis is believed to be downregulated after heat shock except for a class of proteins ADVERSE ENVIRONMENTAL CONDITIONS * S. J. Neal and S. Karunanithi contributed equally to this work. Address for reprint requests and other correspondence: J. T. Westwood, Dept. of Biology, Univ. of Toronto, 3359 Mississauga Rd., Mississauga, ON, Canada L5L 1C6 (e-mail: t.westwood@utoronto.ca). Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org). called heat shock proteins (Hsp for individual proteins; HSP for families of proteins), whose levels are upregulated (35). Hsps serve to preserve cellular integrity by preventing protein damage, misfolding, and aggregation at high temperatures (24, 33, 35, 49). Members of the 70-kDa family of HSPs (HSP70) are the most abundantly expressed proteins in Drosophila after heat shock; however, their levels are below detection in unstressed animals (35, 56). Previously, the extent of synaptic thermotolerance was shown to correlate with the levels of HSP70 expressed in the organism (21–23). Our initial investigations were designed to confirm HSP70’s role in conferring synaptic thermotolerance by using a temperature-sensitive mutant (Drosophila mutant hsf 4) possessing a mutation in the heat shock transcription factor HSF. The hsf 4 mutation does not affect constitutive Hsp synthesis at the permissive temperature but blocks the heat-associated DNA binding activity of HSF at or above 36°C and compromises its transactivation ability at intermediate temperatures (20). It has previously been reported that heat shock at 36°C fails to induce Hsp70 expression and that no accumulation of other inducible Hsps could be detected (20). The lack of production of induced Hsps in the hsf 4 mutants was anticipated to significantly reduce synaptic thermotolerance. Third-instar hsf 4 mutant larvae that have progressed past the earlier temperature-sensitive developmental block display no developmental defects in the nervous system or musculature and survive heat shock at 37°C (20). Surprisingly, they also displayed substantial synaptic thermotolerance (S. Karunanithi, personal observations). The latter findings motivated our present investigation into elucidating the factors that afford thermotolerance in the absence of induced Hsp expression, especially those that confer thermoprotection at the level of the whole organism. Because thermotolerance is strongly associated with the upregulation in expression of stress-activated genes (17), we attempted to identify genes that are upregulated by heat shock in hsf 4 mutants. DNA microarrays were used to screen 6,600 genes from the Drosophila genome (34). Only a small number of genes showed similar levels of induction in response to heat in both the mutant and control strains. Unexpectedly, this list included dnaJ-1 and hsp83, both constitutively expressed chaperones. DnaJ-1 is a J domaincontaining HSP40 family protein. Hsp83, a member of the HSP90 family of HSPs, is one of the most abundant cellular proteins, thus making its strong upregulation particularly surprising (59). The significance of these unanticipated results in relation to thermotolerance at the level of the whole organism as well as at synapses is discussed. 1094-8341/06 $8.00 Copyright © 2006 the American Physiological Society 493 Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 Neal, Scott J., Shanker Karunanithi, Adrienne Best, Anthony Ken-Choy So, Robert M. Tanguay, Harold L. Atwood, and J. Timothy Westwood. Thermoprotection of synaptic transmission in a Drosophila heat shock factor mutant is accompanied by increased expression of Hsp83 and DnaJ-1. Physiol Genomics 25: 493–501, 2006. First published April 4, 2006; doi:10.1152/physiolgenomics. 00195.2005.—In Drosophila larvae, acquired synaptic thermotolerance after heat shock has previously been shown to correlate with the induction of heat shock proteins (Hsps) including HSP70. We tested the hypothesis that synaptic thermotolerance would be significantly diminished in a temperature-sensitive strain (Drosophila heat shock factor mutant hsf 4), which has been reported not to be able to produce inducible Hsps in response to heat shock. Contrary to our hypothesis, considerable thermoprotection was still observed at hsf 4 larval synapses after heat shock. To investigate the cause of this thermoprotection, we conducted DNA microarray experiments to identify heatinduced transcript changes in these organisms. Transcripts of the hsp83, dnaJ-1 (hsp40), and glutathione-S-transferase gstE1 genes were significantly upregulated in hsf 4 larvae after heat shock. In addition, increases in the levels of Hsp83 and DnaJ-1 proteins but not in the inducible form of Hsp70 were detected by Western blot analysis. The mode of heat shock administration differentially affected the relative transcript and translational changes for these chaperones. These results indicate that the compensatory upregulation of constitutively expressed Hsps, in the absence of the synthesis of the major inducible Hsp, Hsp70, could still provide substantial thermoprotection to both synapses and the whole organism. 494 THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT MATERIALS AND METHODS Physiol Genomics • VOL 25 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 Drosophila strains and treatments. D. melanogaster strains were grown on standard yeast-agar medium supplemented with 0.05% (wt/vol) bromophenol blue at 25°C with a 12:12-h light-dark cycle. The strains dp cn bw cl and cn bw hsf4 (20), referred to simply as dp and hsf 4 strains herein, were kindly provided by Drs. C. Wu and M. Mortin (National Cancer Institute; Bethesda, MD). Adult flies were allowed to lay eggs for 3 days on fresh media, and wandering third-instar larvae were collected upon their emergence from the food during the illuminated period. Staging was verified by the blue gut method (29). Approximately 30 larvae were collected in 2-ml screwcap plastic vials for each treatment. Control larvae were returned to the 25°C incubator, whereas the remaining larvae were heat shocked in a 36°C incubator for 1 h and subsequently allowed to recover for 30 min at 25°C. An alternate method was also used to heat shock larvae, whereby the larvae-containing tubes were immersed in a Neslab GP-200 circulating water bath at 36°C for 1 h followed by a 30-min recovery in the 25°C incubator. Larvae were snap frozen in liquid nitrogen immediately after the respective treatments. Electrophysiology. Methods using focal macropatch electrodes to record and analyze synaptic currents from individual Ib boutons of motor neuron RP3 innervating muscle 6, segment 3, have been previously described (21, 22). Synaptic thermotolerance was assessed by monitoring the percentage of transmitting boutons (both evoked and spontaneous events) with increasing test temperatures (22, 27, 31, 35, and 39°C) for the different genotypes in nonshocked and heatshocked preparations. One synaptic bouton was recorded and analyzed from each larval preparation. Experiments were conducted in HL3 solution (50). Evoked responses were elicited at 1 Hz, and 300 events were recorded at each test temperature. RNA isolation. Treated larvae were briefly thawed on ice before the addition of TRIzol reagent (Invitrogen Canada; Burlington, ON, Canada). Larvae were homogenized with a handheld PRO200 homogenizer fitted with a Multi-Gen7 generator (Pro Scientific; Oxford, CT) for 10 s at settings 4 and 5. The RNA extraction was performed according to the manufacturer’s guidelines. This and other protocols used in this study are available at the Canadian Drosophila Microarray Centre (CDMC) website (http://www.flyarrays.com). Total RNA was resuspended in 18 M⍀ water (Sigma-Aldrich; Oakville, ON, Canada), and sample quality was evaluated using spectrophotometry. Gel electrophoresis of glyoxal-denatured samples was used to confirm sample integrity (41). Microarray hybridizations and data analysis. Microarray hybridizations were performed according to the methods previously described (34). Briefly, SuperScript II reverse transcriptase (Invitrogen) was used to generate fluorescently labeled cDNA from the total RNA template. cDNAs from one cyanine-3 (Perkin-Elmer; Boston, MA) reaction were combined with those from a cyanine-5 (Perkin-Elmer) reaction and were cohybridized to a cDNA microarray containing spots representing nearly 6,600 Drosophila genes [7k2 array, CDMC, Gene Expression Omnibus (GEO) Accession No. GPL311]. Images of the hybridized arrays were acquired using a ScanArray 4000 XL laser scanner (Perkin-Elmer) and were quantified using QuantArray 3.0 software (Perkin-Elmer). Microarray images and quantification data were imported into GeneTraffic Duo (Stratagene; La Jolla, CA), a Minimum Information About a Microarray Experiment (MIAME)-compliant software program (6), for analysis. Data were normalized using the Lowess algorithm at the subgrid level while ignoring flagged values. Normalized data were exported and analyzed using Statistical Analysis of Microarrays (SAM) software from Stanford University (55). The “delta” threshold was adjusted such that less than one result was expected to arise by chance. Gene lists generated in SAM were filtered in GeneTraffic to include only those genes that displayed at least a 1.5-fold difference and whose coefficient of variance was ⬍100%. Quantified microarray data and original TIFF images are available from GEO at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/). The 7k2 array platform has been updated with the present annotations. Each microarray hybridization is described as a sample (GSM65521– 65523 and GSM65567– 65569) within the series GSE2998. PCR primer design. PCR primers were designed using Whitehead Institute’s Primer3 software (39), and sequence data were acquired from GenBank (http://www.ncbi.nlm.nih.gov). The user-defined parameters were 1) amplicon length ⫽ 150 –250 bp, 2) oligo length ⫽ 18 –22 (20 optimal), 3) melting temperature ⫽ 57– 63°C (60°C optimal), 4) GC content ⫽ 35– 65% (50% optimal), and 5) maximum polynucleotide tract ⫽ 4. Other parameters were not changed from their default values. All oligonucleotide sequences and primer pairs were checked with OligoAnalyzer 3.0 (http://scitools.idtdna.com/Analyzer/) for secondary structure and dimer formation. The primers for hsp70 were designed to amplify a sequence that is shared between all of the hsp70 genes in D. melanogaster. In all other cases, each primer and amplicon sequence was tested using the nucleotide-nucleotide BLAST alignment tool (http://www.ncbi.nlm.nih.gov/blast/) to ensure minimal similarity with any other D. melanogaster sequence. The primer sequences used were as follows: HSP70 (CG31366, CG18743, CG31449, CG31359, and CG6489), 5⬘-CTCAGAACAGCAGCTGAACG-3⬘ and 5⬘GATGTCGTGGATCTGACCCT-3⬘; hsp83 (CG1242), 5⬘-CGATTAAGCGACCAGTCGAA-3⬘ and 5⬘-AAACGACAACTGCTCTTGAATG-3⬘; dnaJ-1 (CG10578), 5⬘-CATAAAGCAGCCCGTGTAGC-3⬘ and 5⬘-AGATGTTGAGGCACCGATTC-3⬘; gstE1 (CG5164), 5⬘-CTGAAGCTGCTGGAGACGTT-3⬘ and 5⬘-AGCTTATTGAGGCGATCCAA-3⬘; and actin 5C (CG4027), 5⬘-TACCCCATTGAGCACGGTAT-3⬘ and 5⬘-GGTCATCTTCTCACGGTTGG-3⬘. Real-time RT-PCR. A two-step approach was taken in which the initial RT was followed by the quantitative PCR amplification. Ten micrograms of total RNA were treated with 10 units of DNase I (Fermentas Life Sciences; Burlington, ON, Canada) in a 100-␮l reaction as recommended by the manufacturer. DNA-free RNA (500 ng) was reverse transcribed in a 20-␮l reaction using a dT20VN primer (Sigma Genosys; Oakville, ON, Canada) with SuperScript II for 1 h at 42°C. The reaction was stopped by the addition of EDTA to a final concentration of 5 mM and was diluted 1:8 for future use. Quantification of RNA-DNA hybrids was accomplished by spectrophotometry. One microliter of the diluted reaction was used as the template for each 25-␮l real-time PCR amplification. Reactions were assembled using components of the Brilliant SYBR Green QPCR Core Reagent Kit (Stratagene): 1⫻ core PCR buffer, 200 ␮M each dNTP, 2 mM MgCl2, 0.75 ␮l of 1:500 ROX (passive fluorescent dye), 1.25 ␮l of 1:1,000 SYBR green I, 8% glycerol, 1.25 units SureStart Taq polymerase, and 100 nM each gene-specific forward and reverse primer. Reactions were performed in 96-well polypropylene PCR plates (Stratagene) fitted with 8-strip optical caps (Stratagene) and processed using the Stratagene Mx4000 Multiplex Quantitative PCR System. Samples were incubated at 95°C for 10 min before thermal cycling (40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s). Triplicate end-point observations were made at each annealing and extension step. The completed reactions were heated to 95°C for 1 min and cooled to 55°C. Reactions were reheated in 1°C increments back to 95°C with triplicate end-point observations made at each stage to plot a dissociation curve. The ROX-normalized fluorescence measurements were exported to Microsoft Excel, and the program LinRegPCR (37) was used to determine the efficiency of each reaction. These efficiencies were used in the final calculation of fold induction from the change in cycle threshold values. Protein isolation. Tubes containing 10 frozen larvae were homogenized in 300 ␮l of 2⫻ sample buffer [120 mM Tris 䡠 HCl (pH 6.8), 10% (vol/vol) glycerol, 3.4% (wt/vol) SDS, 2% (vol/vol) ␤-mercaptoethanol, and 100 mM DTT] for 5–10 s. Samples were boiled for 10 min, and the protein yield was assessed using the Bradford assay (5). THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT RESULTS Synaptic thermotolerance in hsf4 larvae. A prior heat shock treatment has been shown to prevent the decline in synaptic performance at elevated temperatures in Drosophila larvae (21, 22). One measure of synaptic performance is to assess the overall success rate of synaptic transmission by measuring the percentage of boutons responding at increasing test temperatures (22). At elevated temperatures, the percentage of responding boutons increased after heat shock (22), and the extent of increase corresponded to the levels of HSP70 expressed in the organism (21). The hsf 4 mutant used in this study should be defective in heat shock-inducible gene transcription. The hsf 4 strain produces a mutant HSF polypeptide containing a V57M substitution in the DNA-binding domain, leading to a temperaturesensitive phenotype (20). This system is somewhat paradoxical in that the major stress-sensing molecule, HSF, becomes dysfunctional at the heat shock temperature at which it is normally induced to act, yet the larvae remain viable. With the use of the hsf 4 strain, it was anticipated that the bulk of synaptic thermotolerance would be compromised due to the expected absence of induced Hsps, specifically, HSP70. Surprisingly, substantial synaptic thermoprotection was still present in the hsf 4 mutant after preheat shock at 36°C (Fig. 1). At 31°C, 71% of boutons generated a postsynaptic response in preheat-shocked hsf 4 larvae, whereas only 37% of the boutons responded in nonshocked hsf 4 larvae; 100% of the boutons respond at this temperature in the wild-type line Canton S (22). At 35°C, 47% of boutons responded in preheat-shocked hsf 4 larvae, 14% of boutons responded in nonshocked hsf 4 larvae, and 80% of boutons responded in the wild-type line. Gene expression after heat shock. Considering the substantial synaptic thermotolerance exhibited by heat-shocked hsf 4 mutant larvae, we sought to identify contributing transcript changes through DNA microarray experiments (Fig. 2). Lists of genes that were significantly affected by the heat shock treatment (60 min at 36°C plus 30 min at 25°C in air) were derived from raw expression data using the programs GeneTraffic DUO and SAM for both hsf 4 larvae and the parental fly line (dp) from which the hsf 4 mutant larvae were Physiol Genomics • VOL 25 • Fig. 1. Synaptic thermotolerance occurs in larvae containing a temperaturesensitive mutation in heat shock factor (HSF). Synaptic thermotolerance is not eliminated in hsf 4 mutant larvae. The percentage of boutons responding as a function of temperature is shown for nonshocked (hsf 4 unshocked; n ⫽ 8) and heat-shocked (hsf 4 heat pretreated; n ⫽ 13) hsf 4 mutants and wild-type [Canton-S heat pretreated; data from Karunanithi et al. (22)] wandering third-instar larvae. derived. Data normalization, a prerequisite for statistical analysis, was achieved by the Lowess algorithm implemented in GeneTraffic. The SAM program applies a modified t-test to the normalized experimental data and provides a false discovery rate correction in the form of the parameter “delta.” Because the data from duplicate spots were not considered independently, adjusting “delta” such that less than one result was expected to arise by chance enforced stringency. To obtain more manageable gene lists, we applied a minimum fold change threshold of 1.5 to the SAM results. We have previously determined that these analysis parameters identify truly reproducible results with the strong potential to be validated by other techniques (36, 38). We have also demonstrated the suitability of differential expression thresholds for the Drosophila 7k2 array platform (34). Using these parameters, we identified lists of genes whose expression was significantly different in the direct comparisons made in our study (see Supplemental Tables 1–3; available at the Physiological Genomics web site).1 Intersecting the dp and hsf 4 gene lists revealed a common group of 16 genes that were significantly affected by heat shock in both strains (Fig. 2A, purple intersected area). Of the 92 genes significantly affected by heat shock in only the dp strain (Fig. 2A, blue area, and Supplemental Table 1), 80 were upregulated, and 28 of the 32 heat-affected genes in hsf 4 larvae were upregulated (Fig. 2A, orange area, and Supplemental Table 2). In nonshocked animals, 135 genes were differentially expressed between the two strains, of which 50 were more abundant in the hsf 4 strain than in the dp strain (Supplemental Table 3). The above gene lists were queried against genome ontology terms (http://www.godatabase.org) to identify genes whose products might be involved in either thermotolerance or syn1 The Supplemental Material (Supplemental Tables 1–3 and Supplemental Fig. 1) for this article is available online at http://physiolgenomics.physiology. org/cgi/content/full/00195.2005/DC1. www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 SDS-PAGE and immunoblot analysis. Proteins were separated on 10% (wt/vol) polyacrylamide gels by SDS-PAGE (41), and standard immunoblot analysis was performed (48). Briefly, proteins were transferred to BioTrace NT pure nitrocellulose membranes (Pall; Mississauga, ON, Canada) using a Bio-Rad Trans-Blot Cell. Blocked membranes were incubated for 1 h with the following primary antisera: mouse monoclonal anti-Hsp70 (3A3, Affinity BioReagents; Golden, CO), rat monoclonal anti-Hsp70 (56) (7Fb, a gift from Dr. S. Lindquist, Whitehead Institute, MIT, Cambridge, MA), rabbit polyclonal anti-Hsp83 (9) and affinity-purified anti-DnaJ (27) (a gift from Dr. C. Wu). Blots were washed before incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody, either goat anti-rabbit IgG (Dako Cytomation; Mississauga, ON, Canada), goat anti-mouse IgG ⫹ IgM (Jackson ImmunoResearch Laboratories; West Grove, PA), or goat anti-rat IgG (Jackson ImmunoResearch Laboratories). Signals were detected with Enhanced Chemi-Luminescence Plus reagent (Amersham) on a Storm 840 Gel and Blot Imaging System (Amersham). Densitometry was performed using Storm software, and fold changes were calculated from the band densities. Blots were stained with Ponceau S reagent (Sigma) after detection to ensure that proteins had been transferred evenly. 495 496 THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT aptic transmission. A Pearson cluster was generated using the log-transformed microarray ratios for these genes (Fig. 2B). A complete Pearson cluster of all 140 heat-affected genes is also available (Supplemental Fig. 1, a and b). Most hsps showed reduced (i.e., hsp70Ab) or no induction (i.e., hsc70Cb) in hsf 4 larvae compared with dp larvae after heat shock. Several genes, including ebony and companion of reaper, were more upregulated in hsf 4 larvae than in dp larvae; however, in the case of ebony, a strong bias in relative transcript abundance existed between the two strains. One group of genes stood out because their induction by heat was strong and apparently strain independent. This group included three genes with known funcPhysiol Genomics • VOL 25 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 Fig. 2. Microarray analysis reveals candidate chaperone genes that are not affected by the temperature-sensitive mutation in HSF. A: a Venn diagram was constructed to show the intersection between the dp strain heat-affected gene list (blue) and the hsf 4 strain heat-affected gene list (orange). The intersection (purple) contains 16 genes. The original gene lists were generated by the Statistical Analysis of Microarray program and were subsequently filtered to include only those values that demonstrated a minimum 1.5-fold change and a coefficient of variance of ⬍100%. B: a Pearson cluster was formed using data from 37 differentially regulated genes with known or inferred functions in the stress and/or defense response or in neurotransmission. Columns represent hybridizations, whereas each row corresponds to the log2-normalized ratio of a single gene. The ratios are represented by the spectrum of colors from green (downregulated) to black (unchanged) to red (upregulated). The saturation threshold was set to the equivalent of a 3-fold change. tions in the stress response: glutathione-S-transferase E1 (gstE1), dnaJ-1, and hsp83. Two additional genes, glycoprotein 93 (Gp93) and cytochrome P-450 Cyp9b2, also clustered with this group but were not as strongly induced. The remaining genes whose expression were significantly affected by heat but had no obvious connections to the process of thermotolerance were analyzed using the program EASE (http://apps1.niaid.nih.gov/david/). This program considers the representation of functional categories from the Gene Ontology consortium for every gene on the array and calculates a statistic, the EASE score, to identify any classes that are significantly overrepresented in the gene list (19). In control animals, several classes of peptidases were more highly expressed in the dp larvae (data not shown). Genes involved in the biological processes of “stress response” and “response to biotic stimulus” were also overrepresented in the list of 135 genes that were differentially expressed in the two strains. The 32 hsf 4 heat shock-affected genes that did not intersect with the dp heat shock-affected gene list did not contain any overrepresented functional classes. Although a number of differentially expressed genes were identified, we focused our subsequent analysis on four genes, namely, hsp83, dnaJ-1, gstE1, and hsp70. Real-time RT-PCR was used (Fig. 3) to confirm the relative differences in expression that were first revealed by the microarray analysis. After the air heat shock regime, hsp70 transcripts were induced by ⬎210-fold in dp larvae and ⬃130-fold in hsf 4 larvae (Fig. 3A). In both cases, the detection of hsp70 transcripts in nonheatshocked samples was only slightly above the detection threshold. The relative inducibility of hsp70 between the two strains was consistent with the microarray results; however, the magnitude of the induction observed in hsf 4 larvae was unexpected. The real-time RT-PCR results obtained for hsp83, dnaJ-1, and gstE1 in larvae of both strains (Fig. 3B) were consistent with our microarray results, showing that these genes were in fact induced and that their induction was strain independent. Heat shock increased the relative transcript abundance by greater than sixfold, fourfold, and fivefold for hsp83, dnaJ-1, and gstE1, respectively, in dp and hsf 4 larvae. The magnitude of these inductions was similar to the ratios derived from the microarray experiments. Differential effects of heat shock conducted in air versus water. In light of the unexpected large induction of hsp70 transcripts in the hsf 4 strain, we referred to a number of previous studies where heat shock was administered by submerging the tightly sealed capsules containing the larvae in a heated water bath (14, 31, 46, 57). This method is in contrast to the method used in this and previous studies (21, 22) and in the original report on the hsf 4 mutant (20), where heat shocks were conducted in an air incubator or a forced-air hybridization oven. Whether differences in these two forms of heat shock administration could generate differences in the levels of gene and protein expression had not been previously tested. These experiments were anticipated to serve as controls to ensure that the two different methods of heat shock administration produced similar results. Surprisingly, for the dp strain, the water heat shock regime induced substantially greater expression of hsp70, hsp83, and dnaJ-1 gene transcripts (Fig. 4, A and B; compare with Fig. 3, A and B) but not for gstE1. However, the results for the hsf 4 strain were more in line with THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT 497 our original expectations with only a minor increase in hsp70 expression and the suppression of the induction of dnaJ-1 and gstE1 transcripts relative to that observed in dp larvae. An exception was noted for hsp83, where the induction of transcripts in the hsf 4 strain was similar between the treatments and not repressed by the water heat shock regime. Whether the differences observed in the extent of gene expression between the two treatments translated into similar differences in protein accumulation was investigated using immunoblot analyses (Fig. 5). Two HSP70 antibodies were used, one (3A3) detects several constitutively expressed Hsp70 cognates (Hscs) and the other (7Fb) detects only the inducible form of Hsp70. Little change was observed in the level of Hscs after heat shock in air (maximum 1.3-fold increase; Fig. 5A), but a stronger induction occurred as a result of water heat shock (maximum 1.8-fold increase; Fig. 5B). The changes in Hsc abundance as a result of heat shock were strain independent. The inducible form of Hsp70, however, showed a strong strain-dependent result (Fig. 5, A and B). This form of the protein only accumulated in heat-shocked dp larvae. The increase in inducible Hsp70 levels could not be determined because the basal protein levels were indistinguishable from the background and thus we also could not assess the effect of the treatment on the accumulation of this protein. Hsp83 and DnaJ-1 accumulation in heat-shocked larvae was also assayed by Western blot analyses (Fig. 5, C and D). Air heat shock gave rise to a 1.6-fold increase in Hsp83 in hsf 4 larvae but only a 1.2-fold increase in dp larvae (Fig. 5C). The induction of DnaJ-1 was similar between the two strains (maximum 1.3-fold increase) after air heat shock. When the water heat-shocked larvae were evaluated (Fig. 5D), a greater increase in Hsp83 abundance was observed for both strains (maximum 1.9-fold increase), although the relative difference in the induction between the strains was less (0.2-fold difference in water vs. 0.4-fold difference in air). Water heat shock generated a marked increase in DnaJ-1 (2.5-fold) in dp larvae, whereas its induction in hsf 4 larvae was the same as its air heat shock-induced levels (1.2-fold). Despite the large differential effects of the two treatments on transcription, much smaller changes were observed at the protein level. The 130-fold induction of hsp70 transcripts in air heat-shocked hsf 4 larvae did not appear to lead to any accumulation of inducible Hsp70 in these animals. hsp83 transcripts were induced to similar levels by both heat shock treatments, although a treatment effect was observed in the Western blots. A strain difference in Hsp83 accumulation was also apparent in larvae that were heat shocked in air, although the transcript induction was similar between the strains. For dnaJ-1, a 50-fold increase in transcripts in water heat-shocked dp larvae gave rise to only a 2.5-fold increase in the amount of protein, although a 20 –30% increase in protein was observed when the abundance of transcripts increased only 1.3- to 4-fold. With the use of reverse dot blots, Marchler and Wu (27) showed a 12-fold increase in dnaJ-1 transcripts after a 30-min heat shock in SL2 cells, but this only translated into a 2-fold increase in protein abundance. This lack of correlation between Fig. 4. Transcript levels of chaperone genes after water heat shock. Total RNA was isolated from dp or hsf 4 larvae that were heat shocked for 60 min at 36°C in a circulating water bath and allowed to recover for 30 min at 25°C. RNA was also isolated from nonshocked control larvae from each strain. A: the relative abundance of hsp70 transcripts was determined by real-time RT-PCR. Fluorescence signals from duplicate reactions were normalized to the fluorescence signal of amplified actin 5C transcripts from the same biological samples. The mean ratios from 3 independent biological samples are shown with SE bars on a logarithmic axis. B: results for dnaJ-1, hsp83, and gstE1. Physiol Genomics • VOL 25 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 Fig. 3. Transcript levels of chaperone genes after air heat shock. Total RNA was isolated from dp or hsf 4 larvae that were heat shocked for 60 min at 36°C in an air incubator and allowed to recover for 30 min at 25°C. RNA was also isolated from unshocked control larvae from each strain. A: the relative abundance of heat shock protein hsp70 transcripts was determined by real-time RT-PCR. Fluorescence signals from duplicate reactions were normalized to the fluorescence signal of amplified actin 5C transcripts from the same biological samples. The mean ratios from 3 independent biological samples are shown with SE bars. B: results for dnaJ-1, hsp83, and glutathione-S-transferase gstE1. 498 THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT transcription and translation serves to underscore the necessity for protein analysis in physiological studies. DISCUSSION This work was motivated by the observation that a prior heat shock confers thermotolerance to neuronal synapses in mutants that were not expected to produce induced Hsps. This finding prompted a screen for candidate proteins that may confer thermotolerance in the whole organism; the same proteins were inferred to confer synaptic thermotolerance based on our previous findings (21, 22). Many physiological studies have addressed the phenomenon of acquired thermotolerance in a variety of organisms, especially in invertebrates. Although some studies have tested specific hypotheses at the molecular level, such as assaying the effects of depleting or overexpressing certain Hsps, little work exists where the molecular events surrounding the acquisition of thermotolerance have been broadly surveyed. We utilized DNA microarrays and Western blot analyses to determine the transcript and translational changes after a brief heat shock in the Drosophila hsf4 mutant, which fails to induce HSP70. Our study identified several candidate genes, including two chaperone genes, that appear to be regulated independently of the specific DNA binding activity of HSF. Herein, we also discuss how these chaperones may contribute toward synaptic thermoprotection. Microarray analysis of the heat shock response. Microarray analysis of the heat shock response has been most extensively applied to other organisms such as yeast and the mouse (15, 17, 54). The Drosophila heat shock response has been evaluated in only a few previous microarray studies including those involving embryos (25) and adult flies (45), both of which produced comparable results to the present study with regard to the induction of known hsps. However, these studies focused primarily on wild-type organisms. With the use of microarrays to survey the heat-induced transcription profile in hsf 4 larvae, we did not observe a classic heat shock response. The transcription of fewer than half as many genes was affected by heat shock in this strain compared with the dp strain, and only 16 genes were commonly affected in both strains. Moreover, the transcripts of several hsps including the small hsps (hsp23, Physiol Genomics • VOL 25 • hsp26, hsp27, and CG32041) and Hsc70Cb were less abundant in the nonshocked mutant larvae (Fig. 2B). Differential effects of air and water heat treatments. After heat shock, the moderate induction of many hsps in hsf 4 larvae was not expected because of the temperature-sensitive mutation in HSF. One explanation could be that the air heat shock regime resulted in a slower shift to the test temperature, and thus HSF4 may have been partially active while the hsf 4 larvae were heating up to 36°C as well as during the 30-min recovery phase. In the initial report on the hsf 4 mutant (20), it was shown that Hsps could be induced during recovery from anoxic stress administered at 25°C. However, the abolishment of hsp70 transcript accumulation in water heat-shocked hsf 4 larvae (Fig. 4A) compared with air heat-shocked larvae (Fig. 3A) supports the notion that the HSF4 protein might be active during the initial temperature increase more so than during recovery. Despite the apparent leakiness of hsp70 transcription in hsf 4 larvae, neither heat treatment resulted in the accumulation of detectable amounts of Hsp70 protein (Fig. 5, A and B). Contrarily, Hsc70 accumulation was increased by the water treatment in both strains and may factor into the observed thermotolerance (8). Heat-induced gene expression in hsf4 mutant larvae. We proceeded to investigate in detail the genes whose transcripts increased to the same degree in both strains: hsp83, dnaJ-1, and gstE1. GstE1 is a member of the ⑀-class of GSTs, which, as a family, have known roles in the defense response to oxidative damage (42). ⑀-GSTs metabolize 4-hydroxynonenal, which is known to induce apoptosis (42). Although the induction of gstE1 transcripts was confirmed by real-time RT-PCR, the similarity between the proteins in the 10-member family would have made it extremely difficult to be certain of the identity of any species detected by immunoblot analysis. The candidate genes hsp83 and dnaJ-1 could have many possible roles in the acquired thermotolerance of hsf 4 larvae. Although the upregulation of dnaJ-1 was abrogated in water heat-shocked larvae, the thermotolerance testing was performed on air heat-shocked larvae, and thus its contribution to the observed phenotype must be considered. hsp83 was the only gene to be induced regardless of the mode of treatment in www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 Fig. 5. Increases in chaperone protein levels accompany the upregulation of their transcripts in dp and hsf 4 larvae. Protein extracts were prepared from larvae that were heat shocked for 60 min at 36°C (in air or water) and allowed to recover for 30 min at 25°C and from control larvae that were maintained at 25°C. Total protein (10 ␮g) was separated on 10% (vol/vol) polyacrylamide gels by SDS-PAGE and blotted onto nitrocellulose membranes. Proteins were detected with specific primary antibodies (1:10,000 3A3, 1:50,000 7Fb, 1:10,000 303, and 1:100 anti-DnaJ) coupled to the appropriate secondary antibody [3A3: 1:20,000 horseradish peroxidase (HRP)-anti-mouse; 7Fb: 1:20,000 HRP-anti-rat; 303 and anti-DnaJ: 1:100 HRP-anti-rabbit]. Western blots were performed on protein samples from 3 independent experiments, and a representative blot is shown. Band intensities were quantified using ImageQuant (Amersham). Where possible, the relative intensities of heat shock samples to their controls were calculated and are indicated on the blots. Detection of Hsp70 was performed for larvae that were heat shocked in an air incubator (A) or in a circulating water bath (B). Expression of Hsp83 and DnaJ-1 in larvae that were heat shocked in an air incubator (C) or in a circulating water bath (D) was also examined. THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT Physiol Genomics • VOL 25 • transcripts’ 3⬘-UTRs, which prevent their degradation by both maternal and zygotic degradation machinery (4). It has recently been shown that the decay of Hsp83 mRNAs in Drosophila embryos is mediated by Smaug, which recruits the CCR4/ POP2/NOT deadenylase complex to these transcripts, resulting in their degradation (44). Thus, because of the known mechanisms regulating Hsp83 transcript stability during early development, it is reasonable to speculate that its levels during heat shock might also be regulated in this manner. For example, if the recruitment of the deadenylase complex was inhibited during heat shock and/or the protection mechanism was enhanced, Hsp83 transcript levels would increase in the absence of de novo transcription. Further experimentation is required to test this hypothesis. Chaperones that may afford synaptic thermotolerance in hsf4 mutants. Previous work has shown in Drosophila that a prior heat shock affords thermotolerance to larval neuromuscular junctions (NMJs) with the extent of thermoprotection corresponding to the levels of HSP70 expressed in the organism (21, 22). However, overexpression of Hsp70 was shown to enhance performance presynaptically but not postsynaptically (21). Using hsf 4 mutants, we attempted to further substantiate a role for HSP70 by testing the hypothesis that reduced HSP70 levels result in diminished synaptic thermoprotection. Contrary to our hypothesis, we found substantial synaptic thermoprotection after heat shock in these mutants (Fig. 1), indicating that additional factors other than HSP70 afford thermotolerance. In rabbit motor neurons, HSP70 expression is not detected (28), and others have failed to detect increases in HSP70 expression in the brain (1). However, the gene products of two candidate genes from our microarray screen, hsp83 and dnaJ-1, have been shown to play a functional role at synapses (7, 16). HSP90 has been found to be involved in mediating postsynaptic receptor trafficking (13) and paired-pulse facilitation at cultured rat hippocampal synapses (16). In synaptosomes, HSP90 is reported to form a chaperone complex with cysteine string proteins (CSPs) and HSC70 (2, 8), and this complex binds to the Rab3A-specific inhibitor ␣-GDP dissociation inhibitor to potentially regulate the synaptic vesicle cycle (40). At Drosophila larval NMJs, antibody labeling reveals Hsp83 to be primarily localized in muscle with the highest intensity of staining near postsynaptic regions after heat shock (S. Karunanithi, unpublished observations). HSP40 is shown to be localized at postsynaptic sites in the rat brain (51); however, it is yet to be demonstrated whether its ortholog, DnaJ-1, is localized at Drosophila larval NMJs. CSP is found attached to synaptic vesicles and contains a J domain that could potentially bind DnaJ-1 (7). CSP is shown to have multiple presynaptic functions at Drosophila larval NMJs, including exocytosis and calcium handling in presynaptic nerve terminals (8, 10). Deletion of the J domain results in compromised synaptic strength at elevated temperatures (7). Thus both Hsp83 and DnaJ-1 could be required for synaptic thermoprotection and, given that DnaJ-1 enhances HSP90 autophosphorylation (43), they could be working in concert. The overexpression of one or more Hsps is often sufficient to ensure thermoprotection in tissues (32). Here, we demonstrate that constitutively expressed Hsp83 is strongly upregulated after heat shock in a mutant that fails to accumulate inducible HSP70. Hsp83 has a proven role in the normal functioning of synapses, and a previous study (26) noted that www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.4 on November 6, 2017 hsf 4 larvae. However, gp93, which encodes an Hsp83-related peptide, was also slightly upregulated in hsf 4 larvae. The microarray results for these two potentially functionally related genes clustered together, lending indirect support for their involvement in the thermotolerance of the hsf 4 strain. The upregulation of known Hsps was not observed in an earlier report involving this mutant (20); however, it is likely that our direct immunoblotting approach is more specific and potentially more sensitive than the 35S labeling of proteins employed at that time. In particular, a less than twofold increase in Hsp83 levels may not have been noted if the autoradiographs were not quantified, and DnaJ-1 is not observable in [35S]methionine labeling experiments (J. T. Westwood, personal observations). Alternative regulation of hsp83 and dnaJ-1 orthologs in other organisms. In yeast, the proteins Hsp82 and Ydj1 are orthologs of Hsp83 and DnaJ-1, respectively. The transcription of the respective genes in this organism is atypical and may relate to the fact that their promoters contain nonconsensus heat shock elements (HSEs) (52). Despite having only a single HSF, like Drosophila, a second NH2-terminal activation domain on this molecule controls the expression of genes with nonconsensus HSEs (18, 52). In avians, the basal expression of hsp90 and the induced expression of hsp90 and hdj2 are regulated by HSF3 (53), whereas other hsps are predominantly regulated by HSF1, as they are in most organisms. In Drosophila polytene chromosomes, the hsp83 gene locus is one of the only areas where HSF is specifically associated in nonshocked animals (58), and HSF has a fourfold higher affinity for the hsp83 promoter in vitro than for the promoters of other hsps (47). However, there did not appear to be any regions of specific HSF association in hsf 4 polytene chromosomes (J. P. Paraiso and J. T. Westwood, unpublished observations). Posttranscriptional mechanisms of Hsp regulation. The observation of increased hsp83 transcript levels in both air and water heat-shocked hsf 4 larvae suggests that a mechanism independent of HSF transcriptional activity, such as transcript stabilization, may be at work in these organisms. There is evidence for other hsps in Drosophila, namely, the inducible HSP70 genes, that posttranslational regulation of expression occurs. It is known that the preferential translation of Hsp70 mRNA during heat stress is controlled by cis-acting elements contained in the 5⬘-untranslated region (UTR); that, after heat shock, Hsp70 mRNA is deadenylated and destabilized; and that the rapid deadenylation of Hsp70 messages is controlled, at least in part, by sequences in the 3⬘-UTR (11, 30). It is thus unclear why we fail to observe increases in inducible HSP70 despite the accumulation of its transcripts. It is possible that a novel heat-inducible and/or heat-regulated factor is required for the initiation of translation via the known cis elements in the 5⬘-UTR of Hsp70 mRNAs. We have not identified a clear candidate for such a role in the present study, but we have presently only surveyed less than one-half of the predicted genes in the Drosophila genome in this study. Hsp83 mRNA stability. Hsp83 transcript stability has been studied extensively during early Drosophila development. In unfertilized eggs, the maternally deposited Hsp83 transcripts are uniformly distributed, but, upon fertilization, they are degraded during the first 4 h of development in all regions of the embryo except the posterior pole plasm (3, 4, 12). This spatiotemporal localization of Hsp83 transcripts is thought to be controlled in part by cis protection elements located in the 499 500 THERMOPROTECTION IN THE DROSOPHILA hsf 4 MUTANT thermotolerance immediately after heat shock was consistent with the contribution of Hsp83. Future studies will address the function of this protein in thermoprotection. ACKNOWLEDGMENTS The authors acknowledge the intellectual contributions of Dr. Ian R. Brown during the initial stages of this study. We further thank Jeff W. Barclay and Dr. R. Meldrum Robertson for the assistance in collecting the data presented in Fig. 1 and Rob DaCunha for the initial work on the Western blot experiments. For contributing valuable reagents, we thank Dr. S. Lindquist for the antiHsp70 antibody and Drs. G. Marchler and C. Wu for the anti-DroJ1 antibody. Present address of S. Karunanithi: Arizona Research Laboratories Div. of Neurobiology, Univ. of Arizona, 1040 E. 4th St., Tucson, AZ 85721. 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