Abstract
The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.
THE heat-shock response, found in all living organisms, provides an effective defense against exposure to adverse environments. The distinctive feature of the heat-shock response is the synthesis of a set of conserved heat-shock proteins (Hsp's; Lindquist 1986; Lindquist and Craig 1988; Parsell and Lindquist 1993; Feder and Hofmann 1999). Hsp's can protect against a number of cellular stresses, including high temperatures, oxidative stress, and a variety of cytotoxins (Craig 1985; Lindquist 1986; Bond and Schlesinger 1987; Pauli et al. 1992; De Maio 1995; Morimoto et al. 1997; Feder and Hofmann 1999; Jaattela 1999; Kregel 2002). Convincing evidence for the protective function of Hsp's is derived from the induced thermotolerance test. The ability to survive severe heat stress is increased if an organism is first exposed to a mild heat treatment, but not if protein synthesis is blocked (McAlister and Finkelstein 1980; Plesofsky-Vig and Brambl 1985). Such mild heat treatments offer protection against a variety of stresses. The generally accepted interpretation for this phenomenon is that the synthesis of Hsp's that is triggered by the mild heat shock aids subsequent survival under more severe or alternative stresses.
The Hsp's and their constitutively synthesized relatives (termed heat-shock cognates, or Hsc proteins) form a diverse group of protein chaperones that can disaggregate proteins from large aggregates or assemblies, prevent aggregation of denatured proteins, aid the renaturation or folding of proteins to reach their proper conformation, direct proteins to degradative pathways, and bind proteins to restrain their function, making them available for ligand binding or allowing them to translocate across membranes (Glover and Tkach 2001; Houry 2001; Ryan and Pfanner 2001; Walter and Buchner 2002; Craig 2003; Newmyer et al. 2003; Pratt and Toft 2003). Although some of the classes of Hsp's clearly have distinct activities, they also exhibit overlapping functions (Sanchez et al. 1993), cooperate in their activities (Smith 1993; Ehrnsperger et al. 1997; Lee et al. 1997; Glover and Lindquist 1998; Veinger et al. 1998; Lee and Vierling 2000; Cashikar et al. 2005; Haslbeck et al. 2005), and may share proteins that act as cofactors, known as cochaperones (S. Chen et al. 1996; Glover and Lindquist 1998; Abbas-Terki et al. 2001).
Among Hsp's, Hsp70 is ubiquitous, with unusually high conservation in protein sequence and functional features (Boorstein et al. 1994). The chaperone functions of the Hsp70 family of proteins are well established (Skowyra et al., 1990; Flynn et al., 1991; Schroder et al., 1993; Hartl, 1996; Hartl and Hayer-Hartl, 2002; Mayer and Bukau 2005). Hsp70 and its relatives have several other roles as well. Although Hsp70 is not itself a protease, it is now known that cochaperones can control its activity to direct substrate proteins either to refold or to be degraded. CHIP (carboxyl-terminus of Hsc70 interacting protein) is a ubiquitin ligase that associates with Hsp70 and Hsp90 and their non-stress-induced cognates to direct substrate proteins to the proteasome (Ballinger et al. 1999; Connell et al. 2001; Höhfeld et al. 2001; Meacham et al. 2001; Murata et al., 2001; McDonough and Patterson 2003). CHIP is also involved in regulation of the heat-shock response (Dai et al. 2003). Other activities of Hsp70 family proteins are the regulation of apoptosis and eliciting innate and adaptive immunity (Gabai et al. 1997; Jaattela et al. 1998; Beere et al. 2000; Wadhwa et al. 2002; Wallin et al. 2002; Takayama et al. 2003; Gullo and Teoh 2004). Interest in the Hsp70 class of chaperones is growing because of the large variety of cellular processes in which they are involved, as well as their possible participation in aging, cancer, and several neurodegenerative genetic disorders.
The heat-shock response was first discovered in Drosophila, as a change in the puffing pattern of salivary gland polytene chromosomes in response to heat (Ritossa 1962). In Drosophila this response is particularly dramatic: following a shift to high temperature nearly all transcription and translation is devoted solely to expression of Hsp genes, while other genes are turned off (McKenzie et al. 1975; Spradling et al, 1975; Storti et al. 1980; Scott and Pardue 1981). Hsp70 is the major protein synthesized during this period. Although the synthesis of Hsp70 is nearly undetectable in Drosophila cells at the normal growth temperature of 25°, its expression is rapidly induced at least 1000-fold by raising the temperature to 37° (Velazquez et al. 1983). The prominent expression of Hsp70 suggests that it may play a large role in thermotolerance, and experimental evidence confirms this supposition. Solomon et al. (1991) found that Drosophila cells with extra copies of Hsp70 genes had increased survival after heat shock, but that interference with Hsp70 reduced their survival. Additionally, when cells carrying a metallothionein-controlled Hsp70 gene were treated with copper at normal temperature, and then shifted directly to severe temperature, a dramatic increase in survival was observed. Additional transgenic copies of Hsp70 provide Drosophila with enhanced thermotolerance (Welte et al. 1993; Feder et al. 1996; Krebs and Feder 1998). However, long-term survival is reduced by extra copies of Hsp70 (Krebs and Feder 1997, 1998). Thus, the role of Hsp70 in Drosophila thermotolerance is still not fully understood. As a counterpoint to the Drosophila results implicating Hsp70 in thermotolerance, both Saccharomyces cerevisiae and Escherichia coli rely mainly on the Hsp100 family to survive severe temperatures (Squires et al. 1991; Parsell and Lindquist 1993; Sanchez et al. 1993); however, Hsp100 has not been found in animals (Glover and Tkach 2001).
In addition to its role in the Drosophila heat-shock response, Hsp70 and its cognates are clearly involved in non-heat-shock processes. A recent significant discovery is the finding that Hsp70 can modulate the effects of expressing polyglutamine disease genes. Polyglutamine (polyQ) diseases are a group of dominant inherited neurodegenerative disorders of humans, with the disease alleles characterized by expanded segments of CAG repeats encoding polyglutamine (Paulson et al. 2000). The pathogenic polyQ proteins are thought to self-associate to form insoluble aggregates inside cells, termed intracellular inclusions. When a segment of the human Machado–Joseph disease gene, which included a segment of CAG repeats, was expressed in the fly eye, developmental defects such as rough eyes and loss of pigment cells and photoreceptor neurons were observed (Warrick et al. 1998). It was further shown that Hsp70 was located in the aggregates, that overexpression of human Hsp70 suppressed the disease, and expression of a dominant negative mutant of a constitutively expressed Hsp70-cognate gene (Hsc4.k71) enhanced the degeneration (Warrick et al. 1999). Drosophila appears to be a very useful model organism to study human polyQ diseases and other human neurodegenerative diseases, and to uncover the role of Hsp70 in those pathologies (Feany 2000; Bonini and Fortini 2003).
In the work that we report here, we made use of Drosophila Hsp70 deletion mutants to study the role of Hsp70 in thermotolerance and the regulation of the heat-shock response, protein folding, and neurodegeneration. We find that the Hsp70 mutants affect all these processes.
MATERIALS AND METHODS
Drosophila strains and culture conditions:
The names of the Hsp70 deletion mutants (Gong and Golic 2004) have been altered slightly to conform to FlyBase convention (http://flybase.bio.indiana.edu/). Each homologous recombination event introduced a whs marker gene. The alleles carrying these whs markers were used in all experiments, except the polyQ experiments and the heat-shock puff analyses, in which the whs markers were removed by Cre-mediated recombination prior to the tests (Siegal and Hartl 1996). Fly lines bearing gmr-GAL4 and UAS-MJDtr-Q61 were provided by N. M. Bonini (Chan et al. 2000). Fly lines carrying Hsp70 transgenes were provided by M. E. Feder (Welte et al. 1993). The shi1 flies were obtained from Drosophila stock center (Bloomington, IN). Flies were raised at 25° on standard cornmeal–agar medium and crosses were carried out in standard vials or bottles.
Fertility tests:
To test fertility, 10 vials per genotype were started with two females and three males per vial. Flies were transferred to fresh food every day for approximately the first three weeks after the crosses were started, and thereafter every 1–6 days. Progeny eclosing from all vials were scored and summed as a measurement of lifetime fertility. The numbers reported reflect average lifetime fertility of 2 × 3 matings. The genotypes tested were w1118 (12 copy), w1118; Df(3R)Hsp70A Df(3R)Hsp70Ba304 (6 copy) and w1118; Df(3R)Hsp70A Df(3R)Hsp70B (Hsp70-null).
Heat-shock protocols:
The heat-shock protocols used in these experiments were empirically derived. For the adult heat-shock experiments, 0–1-day-old flies were anesthetized and grouped as 25 adult males or females per vial. On the next day, these 1–2-day-old adult flies were transferred to empty 25 × 75 mm glass vials, given a mild heat shock at 35° for 30 min, and then immediately transferred to 39°. Heat shocks were given by immersing the vials in a circulating water bath as described (Golic and Lindquist 1989). Every 10 min flies were checked under the microscope, then quickly returned to the 39° water bath. If flies did not move any parts of their bodies, even after vials were tapped, they were counted as paralyzed. The results for males and females were very similar, and so no distinction is made in reporting the results.
For the larval thermotolerance experiments, food that contained larvae was immersed in 0.7% NaCl to induce larvae to leave the food. Third instar larvae were collected manually and transferred to a drop of yeast paste on a coverslip, which was then placed into a new vial with fresh food at a concentration of 40 larvae per vial. The vials were heat-shocked at 35° for 30 min, then immediately shifted to 39° for 45 min. The number of eclosing flies was normalized to that without heat-shock treatment. In a second heat-shock protocol third instar larvae were treated at 37° for 1 hr, with or without a 30-min 35° pre-heat shock.
In the shi1 experiments 0–1-day-old adult flies were anesthetized. On the next day these 1–2-day-old adult flies were transferred to empty glass vials, given a pretreatment at 35° for 30 min, and then heat-shocked at 38° for 40 min. At 15-min intervals, for 2 hr after return to room temperature, recovery from paralysis was scored. If a fly could stand after the vial was tapped it was scored as having recovered. Flies were then transferred to the vials with food, and survival was scored the next day.
Cytology:
Third instar larvae were heat-shocked at 37° for 25 min. Then at various times after return to room temperature salivary gland polytene chromosomes were prepared as described (Lefevre 1976). For each nucleus, we examined the heat-shock puffs at 63B, 67B, 93D and 95D, representing the major heat-shock-inducible loci: Hsp83, the small Hsp genes, Hsrω and Hsp68. When no heat-shock puffing was observed at any of the four loci a nucleus was scored as showing no puffing; if any of the heat-shock puffs were visible it was scored as exhibiting heat-shock puffing.
Statistics:
Statistical analyses were performed using Graphpad Prism and Instat software for Macintosh. The lifetime fertility tests were analyzed using the Mann-Whitney test, with the lifetime production from each 2 × 3 mating treated as a single datum, grouped by genotype. The thermotolerance tests of Figure 1A were analyzed by two-factor ANOVA. The results presented in Figures 1B and 2, A–C, were analyzed using the Mann-Whitney test. In the adult tests a pair of vials (consisting of 25 flies of each sex for a total of 50 flies) was treated as a separate datum at each time point. In the larval tests each vial of 40 larvae was treated as a separate datum. Results were grouped by genotype for analysis. The error bars in Figures 1, 2, and 4 represent ±1 SEM. Error bars are not visible for many of the points on the line graphs because they are smaller than the symbols used to represent the data points. The Hsp gene repression experiment of Figure 3A was analyzed using a paired t-test.
RESULTS
The Hsp70 deletion genotypes:
Drosophila melanogaster carry six copies of Hsp70 per haploid genome, situated at two closely linked loci on chromosome 3. Deletion mutants were generated by homologous recombination (Gong and Golic 2004). The deletion of the two Hsp70 genes at the 87A locus is called Df(3R)Hsp70A, the single gene Hsp70Ba deletion allele is called Hsp70Ba304, and the four-gene deletion of all Hsp70B genes is called Df(3R)Hsp70B. By combining Df(3R)Hsp70A with Hsp70Ba304 we produced a chromosome lacking three of the six copies normally found on that chromosome; combining Df(3R)Hsp70A with Df(3R)Hsp70B produced a chromosome completely lacking Hsp70. All the mutant combinations were viable and fertile as homozygotes, including the Hsp70A Hsp70B recombinant that completely eliminates Hsp70, and all had similar developmental times (Gong and Golic 2004; not shown).
In a measurement of lifetime fertility, by crossing inter se, we found no significant difference between the 12-copy w1118 controls and the 6-copy flies (353 progeny vs. 284 progeny, P = 0.25). The Hsp70-null flies did produce fewer progeny in our tests (105, P < 0.002). The reduced fertility of flies lacking Hsp70 appears to be attributable primarily to a reduction in the progeny produced by females, with male fertility unaffected (not shown).
Reduced thermotolerance of Hsp70 mutants:
We first determined whether Hsp70 deficiencies had an effect on thermotolerance of adult flies. A standard thermotolerance assay is to first give flies a mild heat shock to allow the synthesis of Hsps, then test the ability of the pretreated flies to tolerate extreme temperatures. Flies were pretreated at 35° for 30 min, then moved to 39° and assayed for their resistance to the paralysis that is a consequence of exposure to high temperatures. We found that a reduction of Hsp70 copy number makes flies more susceptible to heat paralysis (Figure 1A). Wild-type flies, having 12 copies of Hsp70, can withstand 50–60 min at 39° before half of the flies are paralyzed; flies with only eight copies reach this point after ∼40 min of exposure (P < 0.0001); and flies with six copies require only ∼30 min to reach the same level of paralysis (P < 0.0001 in comparison with wild-type; P < 0.0001 in comparison with eight-copy flies).
Adult thermotolerance. (A) The rates at which flies of different genotypes succumb to heat shock are shown. The numbers in parentheses indicate the Hsp70 copy number of each genotype. (B) Overnight survival of flies with different doses of Hsp70 genes. Heat-shock conditions are discussed in the text. The sample size for each genotype was 300–500 flies. Genotypes are as indicated, except that all flies also carried the w1118 mutation on their X chromosomes.
Surprisingly, flies that completely lacked Hsp70 were just as resistant as the flies with six copies of Hsp70. However, further examination revealed that having six copies of Hsp70 clearly provided a survival advantage compared to flies without Hsp70. We heat-shocked adult flies at 35° for 30 min, then at 39° for 40 min, and scored their overnight survival after being returned to 25° (Figure 1B). The flies with 12 or six copies of Hsp70 showed similar rates of survival (81% and 72% respectively, P = 0.18), but the flies without Hsp70 had a greatly reduced survival (32%; P < 0.0001). We note that this result also indicates that the majority of six-copy flies that showed paralysis at the 40 min time point in Figure 1A will recover and survive. Hsp70 appears to have two roles in adult thermotolerance: a high copy number is needed to provide rapid resistance to the effects of severe heat shock, but a lower copy number is sufficient for long-term survival.
To determine whether reduced Hsp70 copy number also affects larval thermotolerance we measured larva-to-adult survival after applying a 35° 30-min pretreatment followed immediately by a 39° 45-min heat shock to third instar larvae. The survival rates were determined by normalizing the frequency of eclosion to that obtained without heat shock. The results were similar to those for adult survival (Figure 2A). A small but significant reduction in viability was observed for flies with only six copies of Hsp70 (P < 0.0001), while Hsp70-null flies rarely survived this treatment (P < 0.0001). To show that this lack of thermotolerance was specifically attributable to Hsp70 we added 12 transgenic copies of Hsp70 (Welte et al. 1993) to the six-copy flies and to the Hsp70-null flies. Wild-type or near-wild-type levels of thermotolerance were restored, indicating that the heat-shock lethality results from the Hsp70 deficiency.
Larval thermotolerance. Third instar larvae of the indicated genotypes were heat-shocked as described in the text, then scored for survival to adulthood (eclosion). The numbers in parentheses represent Hsp70 copy number. Each bar represents between 360 and 640 larvae. (A) Survival following a 39° heat shock, with (PT) or without (NO PT) pretreatment. P[Hsp70+] indicates a P element carrying 6 copies of Hsp70+, adding 12 copies when homozygous, as is the case here. The NO PT genotypes tested here were Hsp70+ and Df(3R)Hsp70A Df(3R)Hsp70B (the left and right NO PT bars, respectively). (B) Survival following a 37° heat shock. (C) Length of development (egg to adult) in response to a heat shock given to third instar larvae. The control represents developmental time for non-heat-shocked flies. All genotypes are as indicated, except that all flies carried either w or w1118 on their X chromosomes.
Hsp70 is not absolutely required to survive heat shock. We subjected larvae to a less severe 37° 60-min heat shock, and found very little difference in survival between wild-type and Hsp70-null flies (Figure 2B), regardless of whether the larvae were given a 35° pretreatment (P = 0.05) or not (P = 0.05). However, the 37° heat shock did cause a significant developmental delay (Figure 2C) for both wild-type (P < 0.0001) and Hsp70-null (P < 0.0001) larvae, and this delay was longer for the Hsp70-null than for the wild-type larvae (P = 0.014). A pretreatment of 35° for 30 min nearly eliminated this delay in wild-type larvae, but had no effect on the delay in the Hsp70-null larvae, identifying Hsp70 as a crucial component in eliminating developmental delay. This result confirms and complements the previous finding of Welte et al. (1993), who showed that extra copies of Hsp70 could alleviate a heat-shock-induced delay in embryonic development. Although Hsp70 is not vital at the reduced heat shock used here, it clearly still serves an important function.
The observation that pretreatment had essentially no effect on the survival or developmental delay of Hsp70-null larvae heat-shocked at 37° for 60 min led us to ask whether acquired thermotolerance, the increase in stress resistance produced in response to the pretreatment, was entirely dependent on Hsp70. To make this determination we gave third instar larvae the same 39° 45-min heat shock as before, but without pretreatment (Figure 2A). Only approximately one-third (34.2%) of the 12-copy larvae survived this heat shock, clearly showing that the pretreatment, with 72.3% survival, provided a large benefit to Hsp70+ larvae. The same heat shock caused almost complete lethality to Hsp70-null larvae (0.8% survival). Although the pretreated larvae showed some benefit from pretreatment, their survival was still quite low (7.1%). We conclude that the remaining Hsp's can provide some degree of acquired thermotolerance on their own, but in the absence of Hsp70 their contribution is quite small.
Repression of the heat-shock response is delayed in Hsp70-null flies:
The developmental delay observed in Hsp70-null larvae indicates that Hsp70 aids in the recovery from heat shock to allow more rapid resumption of normal development. It has been previously proposed that Hsp70 is a major regulator of heat-shock response repression during recovery (DiDomenico et al. 1982a,b; Shi et al. 1998; Marchler and Wu 2001). To test whether the developmental delay observed in Hsp70-null larvae is a consequence of an extended heat-shock response we assayed the course of the transcriptional response by scoring the regression of heat-shock puffs, which reflect Hsp gene transcription, on salivary gland chromosomes. We heat-shocked third instar larvae at 37° for 25 min, and at various times afterwards we monitored the presence of heat-shock puffs. For each time point and each genotype >50 nuclei were scored. Nuclei were scored as positive if any active heat-shock puffs were observed. We found that the heat-shock puffs persist much longer in the Hsp70-null strain, with puffs disappearing in half of the nuclei of wild-type larvae by 40 min, but requiring over 70 min to reach the same point in the Hsp70-null larvae (P < 0.01, Figure 3).
The effect of Hsp70 on repression of the heat-shock response. The results of assaying heat-shock puff regression in Hsp70+ and Hsp70-null larvae at various times during recovery from heat shock are shown. (A) The percentage of nuclei exhibiting heat-shock puffing. (B) Representative puffing patterns at various times after heat shock. The cytological locations of other heat-shock puffs are indicated. Experimental details are given in the text. Genotypes are as indicated, except that all flies carried w1118 or w on their X chromosomes.
The extended transcription of heat-shock genes in this experiment is clearly insufficient to account for the 1.5-day delay in development in the previous experiment. Part of the explanation likely lies with the fact that we used a lesser heat shock in this experiment (25 min vs. 60 min previously), and the delay in recovery may well be longer with a stronger heat shock. It is also likely that the regression of heat-shock puffs tells only part of the story. The response to heat shock in Hsp70-null flies is clearly defective, and this is likely to have consequences for the animal for some time after the transcriptional response has been repressed (see discussion).
Loss of Hsp70 enhances a temperature-sensitive mutant phenotype:
If the major function of Hsp70 in response to heat shock is to aid the refolding of proteins that have been denatured by exposure to high temperatures, then it is likely that the reduced thermotolerance of Hsp70-null flies results from a loss of this ability. To test more directly whether loss of Hsp70 inhibits the recovery of function for proteins affected by heat shock, we examined flies carrying a temperature-sensitive (ts) mutation in shibire (shi), which encodes dynamin, a protein involved in synaptic vesicle recycling. The shi1 allele has a single amino acid substitution in the GTPase domain (van der Bliek and Meyerowitz 1991; Damke et al. 1995). Exposure to the nonpermissive temperature causes rapid and reversible paralysis (Grigliatti et al. 1973; Ozawa and Hagiwara 1976; Siddiqi and Benzer 1976; Kosaka and Ikeda 1983; Kawasaki et al. 2000). Because shi1 flies have an easily scored temperature-sensitive phenotype, and because this phenotype is reversible upon return to normal temperature, we thought this would be a good test of the participation of Hsp70 in facilitating the recovery of protein function after heat shock.
Flies were pretreated at 35° for 30 min and then given a 40-min heat shock at 38°. We scored recovery from paralysis at several time points during a 2-hr recovery at room temperature (Figure 4A). The Hsp70-null flies were unaffected by this heat treatment. The shi1 flies were initially paralyzed, but showed nearly complete recovery after ∼30 min at room temperature. The shi1 Hsp70-null double mutants were extremely sensitive, with the majority failing to recover within the 2-hr period. No further recovery was observed when these flies were examined again the next day (Figure 4B). Thus, without Hsp70, the effect of heat on shi1 is effectively irreversible. Because shi1 flies that do have Hsp70 recover rapidly after heat shock, it is unlikely that recovery of shi1 flies from heat shock requires new synthesis of shibire/dynamin. Instead, we consider it most likely that the preexisting dynamin reacquires a functional conformation, and that this process is strongly dependent on direct interaction with Hsp70 that facilitates refolding.
The effect of Hsp70 on the temperature-sensitive paralytic mutations, shi1. (A) Recovery from heat-shock-induced paralysis, scored at intervals following return to room temperature. (B) Survival after overnight recovery at room temperature. Experimental details are given in the text. Approximately 350 flies were scored for each genotype, with 23–29 of each sex in each vial. Genotypes are as indicated, except that shi+ flies carried w1118 on their X chromosomes and the shi1 flies carried w+.
Hsp70 deficiencies enhance polyQ cellular degeneration:
Another experimental paradigm with strong involvement of Hsp70 chaperone function is the cell death brought about by expression of a human neurodegenerative disease protein (Bonini 2002). Defects in the human MJD1 gene are responsible for Machado–Joseph disease (MJD), or Spinocerebellar Ataxia type 3 (Kawaguchi et al. 1994). This gene has a polyQ tract, and alleles with polyQ expansions act dominantly to produce neurodegenerative phenotypes. Warrick et al. (1998) constructed transgenic flies for eye-specific expression of a truncated form of MJD1 (MJDtr) with an expanded polyQ repeat. Degenerative phenotypes such as rough eyes and loss of pigment cells were observed. Several studies have implicated chaperone function in such neurodegenerative diseases (Bonini and Fortini 2003). For instance, overexpression of Hsp70 in the Drosophila eye alleviates the degenerative phenotypes produced by MJDtr expression, and dominant negative forms of Hsc70, or of Hsp70 cochaperones, enhance the degenerative phenotypes.
To test whether reduction in Hsp70 dosage would affect the MJDtr phenotypes we combined the Hsp70 deletions with transgenes that expressed MJDtr. We chose to test a combination that produced a relatively mild neurodegenerative phenotype by itself because we expected that loss of Hsp70 would enhance the phenotype. We found that this was so, and the observed enhancement was roughly proportional to the number of copies that were deleted (Figure 5). In the combinations having the fewest copies of Hsp70 the phenotype was notably less severe in the anterior of the eye, likely reflecting the fact that cells differentiate later in the anterior than in the posterior and have expressed MJDtr for a shorter time than cells in the posterior. Our results confirm that the normal complement of wild-type Hsp70 genes plays a role in mitigating the damage that results from expression of an expanded polyQ disease protein.
The effect of Hsp70 on neurodegeneration produced by expression of a human disease gene. As shown here, a reduction in Hsp70 copy number enhances the cell death and pigment loss that is produced by expression of MJDtr in the eye. Representative examples of the phenotypes are shown. With the wild-type Hsp70 copy number, only slight pigment loss is observed (left), while a complete lack of Hsp70 strongly enhances the phenotype (right). In addition to the Hsp70 genotypes indicated, with the Hsp70 copy number indicated in parentheses, all flies were w; gmr-GAL4 UAS-MJDtr-Q61/SM1.
DISCUSSION
Flies with a reduction in Hsp70 copy number are viable, including flies with no copies of Hsp70. Similarly, mice that lack all Hsp70 genes are also viable (Hunt et al. 2004). However, mutations in several of the constitutively expressed homologues of Hsp70 in Drosophila do cause lethality (Elefant and Palter 1999; Burmester et al. 2000; http://flybase.bio.indiana.edu), indicating that the Hsc70 family of proteins carries out critical functions at normal temperatures. Furthermore, we found that Hsp70-null females, though fertile, have a significant reduction in fertility, indicating that the heat-inducible genes also have some role at normal temperature. Nonetheless, the fact that the Drosophila Hsp70 deletion strains are viable and fertile as homozygotes provided substantial versatility in characterizing the effects of Hsp70 dosage, and also allowed us to examine the phenotypes of Hsp70-null flies.
When multiple copies of a gene are present, the possibility that those genes have divergent functions must be considered. For instance, in yeast, the SSA1 and SSA2 genes, though encoding nearly identical Hsp70 proteins, differ in their interaction with the [URE3] prion (Schwimmer and Masison 2002). Lakhotia and Prasanth (2002) presented evidence that the two Hsp70 clusters of D. melanogaster are differentially regulated. However, Bettencourt and Feder (2002) concluded that the Hsp70 gene copies have not diverged, and likely all provide the same function. The proteins encoded by these six genes have between 98.6% and 100% identity, and 99.5% or greater similarity. In the work we present here, we have assumed that the deletions we produced are functionally distinguished only by copy number. Our experiments on thermotolerance and the effect of polyQ protein expression are easily interpreted strictly in terms of Hsp70 copy number. For instance, in the adult thermotolerance experiment, the two different six-copy genotypes were indistinguishable. Furthermore, when transgenic copies of Hsp70 were added back to the Df(3R)Hsp70A Hsp70Ba304 genotype, they complemented the larval thermotolerance defect, even though the Hsp70 transgenes represent genes that were not deleted in that genotype (Craig et al. 1979; Welte et al. 1993).
The two Hsp70 clusters differ by intergenic segments. Portions of the S-element transposon are found at both 87A and 87C, and the examination of sequence diversity suggests that these elements are maintained by selection (Maside et al. 2002). However, there are several other S-elements, including many complete elements, found throughout the genome, so it seems unlikely that these particular copies provide any function that is not also encoded elsewhere (Kaminker et al. 2002). The large intergenic region at 87C hosts the largest euchromatic cluster of transposons found in the genome (Kaminker et al. 2002), and includes heat-shock-transcribed repetitive DNA, the αβ and αγ repeats (Lis et al. 1978). It is not known whether these transcripts serve any function. Their heat-induced transcription at this location may simply be an accident of proximity to the massively induced Hsp70 genes. Copies of the same sequences found in centric heterochromatin are not induced by heat shock (Lis et al. 1981). Although we think it unlikely, we cannot rule out the possibility that deletion of these repetitive sequences plays a role in some of the phenotypes we described. To fully address such questions it may be necessary to introduce point mutations into individual genes, or to delete specific repetitive elements (Rong et al. 2002; Gong and Golic 2003).
Not surprisingly, we found that Hsp70 makes an important contribution to thermotolerance in Drosophila subjected to a severe 39° heat shock. Adult flies with reduced Hsp70 copy number succumb more quickly to lethal high temperatures than do flies with their full complement of Hsp70 genes, larvae are killed at a higher rate, and larvae lag in development in response to non-lethal heat shocks. Many previous studies that examined the effects of Hsp70 overexpression in cell lines or in whole animals, or underexpression in cell lines, also led to the conclusion that Hsp70 is an important component of thermotolerance (Solomon et al. 1991; Feder et al. 1996; Feder and Krebs 1997; Krebs and Feder 1998; Roberts et al. 2003). However, none of these prior studies were able to examine the effects of heat on flies that completely lacked Hsp70. Furthermore, some studies show that overexpression of Hsp70 is not always beneficial. Larvae carrying extra transgenic copies of Hsp70 have reduced survival following some heat-shock regimens (Krebs and Feder 1997, 1998). Females with extra copies of Hsp70 also show a reduction in fertility following heat shock (Silbermann and Tatar 2000). Such studies have led to the idea that the existing copy number of Hsp70 in D. melanogaster was produced by a balance between selection for its chaperone function under stress conditions and against its deleterious effects on growth, viability, and fecundity (Feder and Hofmann 1999). Thus, it was important to examine the phenotypes of Hsp70-null flies to test whether copy number reduction would also have a deleterious effect on thermotolerance. Our results with Hsp70 mutants confirm that Hsp70 plays a major role in thermotolerance in Drosophila and supports the hypothesis that Hsp70 copy number represents a balance arrived at by competition between positive and negative selection.
We were surprised to find that Hsp70 is not required to survive a slightly milder 37° 60-min heat shock, even though the temperature was only 2° less than a lethal heat shock. This phenotype of Hsp70 mutants in Drosophila is reminiscent of the phenotype of hsp104 mutants in S. cerevisiae. Hsp104 is required to tolerate extreme heat shocks, but a small degree of thermotolerance remains in hsp104 mutants, and at 37°, a temperature that induces the heat-shock response, the mutants grow as well as cells with a functional HSP104 gene (Sanchez and Lindquist 1990). The thermotolerance that remains in hsp104 mutants is attributable to Hsp70 (Sanchez et al. 1993). The Drosophila strains we characterized in this study still carry the closely related Hsp68 gene. It is quite possible that the function of Hsp70 is partly provided by Hsp68, much as overexpression of the Hsp70-encoding SSA1 gene can partially compensate for loss of Hsp104 in yeast (Sanchez et al. 1993). Indeed, by examining protein synthesis in embryos homozygous for large deficiencies that removed the Hsp70 genes (and many other genes as well), Ish-Horowicz et al. (1979) found that Hsp68 expression increased in the absence of Hsp70. When Hsp68 mutants become available it will be informative to combine them with the Hsp70 mutants to assess phenotypes in the complete absence of this class of heat-induced chaperones. The constitutively expressed forms of Hsp70 may also function at high temperatures to provide a substantial baseline level of thermotolerance. The amount of Hsp70 produced after heat shock is always less than the constitutively synthesized level of Hsc70 proteins (Palter et al. 1986), allowing for the possibility that the Hsc70 proteins contribute to thermotolerance, though their expression is not induced by heat shock. A role for Hsc70 genes in thermotolerance is suggested by results in several organisms (Ulmasov et al. 1992; Kampinga 1993; M. S. Chen et al. 1996; DiIorio et al. 1996).
The heat sensitivity of Hsp70 mutant flies likely results from the loss of Hsp70 chaperone function and the consequent reduction in the capacity of cells to refold proteins that were denatured by heat shock (Pelham 1986). Evidence for this mechanism is provided by our finding that shi1 temperature-sensitive paralytic flies, which normally recover rapidly upon return to normal temperature, recover quite poorly if they lack Hsp70. Feder and Krebs (1997) showed that Hsp70 overexpression helped to restore alcohol dehydrogenase activity to D. melanogaster larvae after heat shock. Using a luciferase reactivation assay, it has been shown that the Hsp70 homologues from other organisms are also involved in rescuing proteins after thermal denaturation (Schroder et al. 1993; Levy et al. 1995; Turman and Rosenfeld 1999; Lee and Vierling 2000). Hsp104 performs a similar function in yeast in cooperation with Hsp70 (Glover and Lindquist 1998), and the participation of the Hsp104 homolog in refolding denatured proteins is vital to produce thermotolerance in E. coli (Weibezahn et al. 2004).
In the absence of Hsp70, proteins that have been unfolded by heat must be refolded by alternate pathways, either spontaneously or with the involvement of other chaperones. The loss of the Hsp70, the most highly expressed Hsp in Drosophila, may overload the remaining chaperone systems and delay recovery. Following a severe heat shock the remaining Hsp's are relatively ineffective, as shown by the only minimal increase in survival that is produced by a low-temperature pretreatment in Hsp70-null larvae. The shi1 experiment shows that the function of some proteins cannot be fully restored without Hsp70. It is likely that they must be synthesized anew to restore function, resulting in developmental delays or lethality. In some cases the sensitivity of genetic screens for temperature-sensitive mutants might be greatly improved by incorporating Hsp70 deficiencies.
It is conceivable that the temperature-sensitive dynamin encoded by shi1 may be exceptionally responsive to Hsp70 chaperone activity. The Hsp70 cognate, Hsc70-4, binds to clathrin and dynamin and participates in the assembly and disassembly of clathrin cages, with mutants showing defects in endocytosis and exocytosis (Schlossman et al. 1984; Bronk et al. 2001; Newmyer and Schmid 2001; Chang et al. 2002; Newmyer et al. 2003). In Hsp70+ flies, perhaps Hsp70 takes the part of Hsc70-4 and associates with clathrin and/or dynamin at high temperature. In shi1 animals these associations could maintain ts-dynamin in a configuration that allows it to resume its function when the temperature is lowered. In the absence of Hsp70, this association does not occur and a change in the conformation of the temperature-sensitive shi1 protein may be irreversible. In support of the idea that Hsp70 may substitute for Hsc70-4, Hsp70 is abnormally induced in Hsc70-4 mutant flies when it would otherwise be silent (Elefant and Palter 1999; Bronk et al., 2001). However, if this hypothesis were true it seems that Hsp70-null flies should exhibit paralysis under the same conditions that inactivate shi1, and they do not.
Hsp70 has been implicated as having a critical role in regulation of the heat-shock response in prokaryotes (Tilly et al. 1983) and in eukaryotes. Originally, a tight correlation between repression of Hsp70 mRNA translation and resumption of non-Hsp mRNA translation was observed (DiDomenico et al. 1982a,b). Subsequently, interference with Hsp70 expression was seen to cause a delay in repression of Hsp mRNA translation and resumption of normal translation (Solomon et al. 1991). In S. cerevisiae, the Hsp70-encoding SSA1 gene similarly carries out self-regulation (Stone and Craig 1990). Under nonstress conditions, Hsp70 participates in repressing the activity of HSF, the positive transcription factor for Hsp genes (Halladay and Craig 1995; Shi et al. 1998; Marchler and Wu 2001). It is believed that Hsp70, in cooperation with other Hsp's, sequesters HSF and restrains its activity. Under stress conditions the Hsp's are diverted to chaperoning other denatured proteins, freeing HSF to activate transcription of the Hsp genes (Morimoto 1998; Voellmy 2004). Our examination of the duration of heat-shock puffing in Hsp70 wild type and mutant flies confirms that Hsp70 is needed for normal regulation of heat-shock transcription because Hsp repression is delayed in Hsp70-null flies.
The functional analysis of Hsp's is certain to be facilitated by the availability of Hsp70 deficiencies. The absence of Hsp70 is likely to help reveal roles of the remaining Hsp's that were obscured in the presence of Hsp70. Phenotypic assessment of mutant combinations has been quite useful for revealing the roles of specific Hsp-encoding genes in yeast (see, for instance, Craig and Jacobsen 1984; Werner-Washburne et al. 1987; Sanchez et al. 1993; Parsell et al. 1994; Glover and Lindquist 1998; Cashikar et al. 2005; Haslbeck et al. 2005). Combining Hsp70 mutations with mutations in other Hsp genes is likely to be equally informative in Drosophila. As we showed, Hsp70-deficiencies also sensitize flies to at least one type of neurodegeneration, that resulting from the expression of a human polyQ disease gene. The use of Hsp70 mutants may facilitate the identification and analysis of other components that either prevent or contribute to such degeneration.
Acknowledgments
We thank N. Bonini for MJDtr-expressing flies and M. Feder for Hsp70 transgenic flies. We thank C. Diaz-Castillo for technical help. This work was supported by grant GM-065604 from the National Institutes of Health. Wei Gong was partially supported by a University of Utah Graduate Research Fellowship.
Footnotes
↵1 Present address: Stowers Institute for Medical Research, Kansas City, MO 64110.
Communicating editor: J. Tamkun
- Received July 26, 2005.
- Accepted September 21, 2005.
- Copyright © 2006 by the Genetics Society of America
