2007
The Journal of Experimental Biology 200, 2007–2015 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JEB0867
TISSUE-SPECIFIC VARIATION IN Hsp70 EXPRESSION AND THERMAL DAMAGE IN DROSOPHILA MELANOGASTER LARVAE 1Department
ROBERT A. KREBS1,* AND MARTIN E. FEDER1,2 of Organismal Biology and Anatomy and 2The Committee on Evolutionary Biology, The University of Chicago, 1027 East 57th Street, Chicago, IL 60637, USA Accepted 1 May 1997
Summary All tissues of larval Drosophila melanogaster express Hsp70, the major heat-shock protein of this species, after both mild (36 °C) and severe (38.5 °C) heat shock. We used Hsp70-specific immunofluorescence to compare the rate and intensity of Hsp70 expression in various tissues after these two heat-shock treatments, and to compare this with related differences in the intensity of Trypan Blue staining shown by the tissues. Trypan Blue is a marker of tissue damage. Hsp70 was rarely detectable before heat shock. Brain, salivary glands, imaginal disks and hindgut expressed Hsp70 within the first hour of heat shock, whereas gut tissues, fat body and Malpighian tubules did not express Hsp70 until 4–21 h after heat shock. Differences
in Hsp70 expression between tissues were more pronounced at the higher heat-shock temperature. Tissues that expressed Hsp70 slowly stained most intensely with Trypan Blue. Gut stained especially intensely, which suggests that its sensitivity to heat shock may limit larval thermotolerance. These patterns further suggest that some cells respond primarily to damage caused by heat shock rather than to elevated temperature per se and/or that Hsp70 expression is itself damaged by heat and requires time for recovery in some tissues. Key words: heat-shock proteins, stress, thermotolerance, vital dyes, Hsp70, Drosophila melanogaster.
Introduction In response to heat and other stresses, nearly all organisms express heat-shock proteins (Hsps), which promote stress tolerance by functioning as molecular chaperones (Lindquist, 1993). Recent years have witnessed enormous progress both in the elucidation of chaperone function at the biochemical level and in the demonstration that heat-shock proteins are responsible for a large component of organismal thermotolerance (Morimoto et al. 1994). Progress has not been as rapid, however, in establishing how the activities of Hsps at the cellular level enhance the thermotolerance of the individual (Hartl, 1996). At a more descriptive level, the way in which tissue-specific expression of Hsps is temporally and quantitatively related to the thermotolerance of the various tissues and the patterns of cell damage that ensue during and after heat shock are poorly understood. Accordingly, we have examined tissue-specific patterns of Hsp70 expression and cell damage in Drosophila melanogaster, the fruit fly. In D. melanogaster, Hsp70 is the primary inducible heat-shock protein (Lindquist, 1981) and is the product of 10–12 nearly identical genes at the 87A and 87C loci (Ish-Horowicz et al. 1979a,b). This protein is not expressed before stress and is very tightly autoregulated as Hsp70 concentrations increase during stress and/or recovery from stress (Lindquist, 1993). *e-mail:
[email protected].
Although Hsps are best known for their inducibility by heat, the presence of non-native proteins within cells is sufficient to induce their expression (Parsell and Lindquist, 1994). Hsp70 expression, which can be detected immunologically, may therefore be symptomatic both of a direct response to high temperatures and of damage to cells and tissues by hightemperature stress (Hofmann and Somero, 1995). To examine tissue damage directly, we stained with Trypan Blue, a dye that is excluded from intact cells but is rapidly absorbed by dead or dying cells. Hsp70 expression and Trypan Blue staining may pinpoint thermosensitive regions within an organism. We therefore examined the order in which tissues first expressed Hsp70 during or after heat shock, and related this to Trypan Blue staining. We performed this experiment in larvae, the life stage of D. melanogaster most likely to experience lethal heat stress in nature (Feder, 1996; Feder et al. 1997). Much of the extensive literature on Hsps in D. melanogaster and other insects has little bearing on the tissue specificity of Hsp expression and heat damage because it combines results for several different Hsps and/or tissues without distinguishing among them. Studies of whole D. melanogaster and cells in culture, which have been the most frequently used subjects of previous work, are not relevant to tissue-specific variation. All
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or most individual tissues of D. melanogaster (Tissiéres et al. 1974; Mitchell et al. 1979) and other dipterans (Nath and Lakhotia, 1989; Joplin and Denlinger, 1990; Tiwari et al. 1995) clearly increase their expression of unspecified members of the major families of Hsps upon heat shock. Seldom, however, have the individual Hsps been identified [but see Palter et al. (1986) and Singh and Lakhotia (1995)] or have the tissues under study been systematically exposed to heat shocks of graded severity. Materials and methods We analyzed Hsp70 expression and tissue damage in the Chromosome II excision strain of Drosophila melanogaster (Welte et al. 1993). This strain, which was a control in some previous analyses of the effects of hsp70 copy number on thermotolerance, growth and Hsp70 expression (e.g. Krebs and Feder, 1997), contains a second-chromosome P-element insertion but expresses Hsp70 normally. Eggs were collected from, and larvae reared on, yeast–cornmeal–molasses–agar medium sprinkled with live yeast. Third-instar larvae (6–7 days post-laying) were separated from the medium in 3 mol l−1 NaCl (Ashburner, 1989) and transferred to 5 cm Petri dishes containing medium. This procedure does not affect Hsp70 concentration. To characterize the thermal sensitivity of Hsp70 expression in entire larvae, third-instar larvae were exposed to constant temperatures between 33 and 40 °C for 1 h, followed by 1 h at 25 °C, as a recovery period. Other larvae were treated at 38.5 °C for 1 h, and then placed at 25 °C for variable periods. Whole-body Hsp70 concentration was then determined by enzyme-linked immunosorbent assay (ELISA) and is presented relative to a standard concentration, that produced by Drosophila S2 cells treated for 1 h at 36.5 °C and 1 h at 25 °C (Welte et al. 1993; Feder et al. 1996). Fluorescent staining Larvae were immersed in PBS (phosphate-buffered saline), and cuticle and muscle tissue were peeled from the body cavity. Larvae were then placed in 4 g l−1 paraformaldehyde (Fisher, T-353) in PBS (pH 7.3) and rotated for 30 min at room temperature (23 °C), rinsed three times with PBS, washed with rotation in PBST [5 g l−1 bovine serum albumin (Sigma A6793), 5 g l−1 Triton X-100 (Sigma T-6878) in 1× PBS] for 30 min to permeabilize cells, and stored overnight at 4 °C in fresh PBST. Tissues were incubated for 1 h in 1:1000 anti-Hsp70 rat monoclonal antibody, 7FB, which is specific for the Drosophila melanogaster heat-inducible Hsp70 family member (Velazquez and Lindquist, 1984). The secondary antibody was FITC-conjugated goat anti-rat IgG, affinitypurified F(ab′)2 fragments (Cappel 55747) prepared according to the instructions of the manufacturer and diluted 1:300 in PBST. To remove any nonspecifically binding components of the secondary antibody, each 1 ml of this dilution was incubated at room temperature for 1 h with 3–5 heat-shocked
larvae that had not been treated with primary antibody. Then, Hoechst 33258 dye was added (10 µl ml−1 of a 1 mg ml−1 stock solution to secondary antibody) for coincident staining of nuclei. Samples were incubated with secondary antibody for 1 h, after which they were rinsed three times with PBS and washed for 30 min in PBST after addition of each antibody. Tissues were transferred to a glass slide and mounted in several drops of 1 mg ml−1 p-phenylenediamine in 70 % glycerol. Slides were examined with a Zeiss fluorescent microscope and photographed (2.5× camera lens) with 100 ASA Ektachrome film, using exposures of 5 s with a 10× objective and 40 s with a 4× objective. Comparisons of expression levels were possible for photographs taken with the same objective. All slides were scanned as Adobe Photoshop documents (Adobe Systems, Inc.), with assembly and text additions in PowerPoint (Microsoft Corp.). Trypan Blue staining Larvae were dissected as for Hsp70 analysis, a step requiring less than 10 min, immersed in 0.2 mg ml−1 Trypan Blue in PBS, and rotated for 30 min at room temperature to bring internal tissues into contact with dye. Groups of three larvae were then rinsed three times in PBS, washed for 30 min in PBS, and each group was immediately scored for Trypan Blue staining of tissues and cells. Scoring for these groups of larvae was based
Hsp70 expression (as percentage of standard)
2008 R. A. KREBS
100 A 90 80 8 70 60 5 50 40 30 8 20 5 4 8 10 1 1 100 0 90 32 33 34 35 36 37 38 39 40 Temperature (°C) 80 70 B 60 3 50 40 4 30 20 10 3 0 0 4 8 12 16 20 24 Recovery time at 25 °C (h)
Fig. 1. Expression of Hsp70 in whole-body lysates of Drosophila melanogaster larvae relative to a standard, the concentration in Drosophila S2 cells after 1 h at 36.5 °C and 1 h at 25 °C. (A) Hsp70 level determined by ELISA after 1 h at the indicated temperature followed by 1 h at 25 °C; (B) Hsp70 concentration after 1 h at 38.5 °C and at the indicated time at 25 °C. Values are means ± S.E.M.; values of N are given beside the data points.
Hsp70 expression and tissue damage 2009 on an average composite index per larva: no color, 0; any blue, 1; darkly stained nuclei, 2; large patches of darkly stained cells, 3; or complete staining of most cells in the tissue, 4. As these data are sequential categories, differences due to treatment effects (control versus heat shock) and due to recovery time (immediately after heat shock versus 21 h after heat shock) were tested by Mann–Whitney U-tests. For presentation, images were taken with tissues in PBS and recorded on a Wild microscope with a direct computer feed. Staining for Hsp70 and Trypan Blue was not possible in the same larva, as Trypan Blue leaches from the sample after fixation. Results In whole larvae, Hsp70 was undetectable in the absence of stress. Hsp70 level varies with temperature in third-instar larvae (Fig. 1A, F7,32=28.6, P
