Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Factors influencing the heat shock response of Xenopus laevis embryos

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 degrees C), whereas others are synthesized only at higher temperatures (i.e., above 37 degrees C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 degrees C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.     

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.     

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.     

A nuclear protein associated with lethal heat shock of HL-60 cells.

The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.     

A comprehensive analysis of heat shock protein synthesis in human peripheral lymphocytes: the effect of penicillin/streptomycin.

A reliable experimental procedure is described for the simultaneous characterisation of a comprehensive range of heat shock proteins (hsps) in human peripheral lymphocytes. In this system, a mild heat shock from 37 to 42 degrees C for 1 h induced the synthesis of hsps 105, 90, 70, 60, 57, 47, 40, 27 and 16. Densitometric analyses of 35[S]-methionine labelled protein gels indicated that levels of these hsps peaked at 3 to 4 h, following post-heat shock recovery at 37 degrees C. The presence of penicillin and streptomycin in the cell culture medium, appeared to have little effect on the kinetics of hsp synthesis. The present method can be used for relatively small blood samples and its relative ease of application and reproducibility make it appropriate for screening the expression of hsps in human lymphocytes from a range of individuals.     

Regulation of heat-shock protein synthesis in chicken muscle culture during recovery from heat shock

Exposure of chick myotube cultures to a temperature (45 degrees C) higher than their normal growing temperature (37 degrees C) caused extensive synthesis of three major polypeptides of Mr = 25 000, 65 000 and 81 000 referred to as 'heat-shock polypeptides' (hsps). When these cells were allowed to recover from heat-shock treatment at 37 degrees C for 6-8 h, the rate of accumulation of isotope into the 65 000-Mr and 81 000-Mr hsps declined to levels comparable to those in control cultures maintained at 37 degrees C. However, incorporation of isotope in the 25 000-Mr hsp continued at an elevated rate for a longer period than the 65 000-Mr and 81 000-Mr hsps. When heat-shocked cells were allowed to recover at 37 degrees C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.     

Cryoprotection provided by heat shock treatment in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells exposed to 43 degrees C (normal being 30 degrees C) exhibit the synthesis of heat shock proteins (hsps). Time course studies indicated that the major hsps (97 kDa, 85 kDa and 70 kDa family) are induced within 10 min. of heat shock and attain maximum amount with two hours of treatment. The viability of cells decreased by 99% when directly frozen into liquid nitrogen. However, a prior heat shock (2 hours) increased the cell survival by 20-30 fold. Such an effect of prior heat shock treatment could be supported by light and electron microscopical studies. Differential scanning calorimetric analysis of whole cells revealed that heat shock treatment decreases the denaturation (delta H) of total cellular proteins. A direct correlation between the degree of hsp inducibility and protection against freezing and thawing injury was observed. Cycloheximide treatment curtailed the synthesis of hsps as well as protection against subsequent freezing. This suggests that prior heat shock treatment protects the cells from freezing injury and, furthermore, that hsps can act as biological cryoprotectants.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

The effect of dormancy on the heat shock response in gladiolus cormels

Cormels of Gladiolus X gandavensis Van Houtte respond to heat shock by an induced synthesis of heat shock proteins. Synthesis of some of the non-heat shock proteins is concomitantly reduced. The ability of dormant cormels to synthesize heat shock proteins (hsps) and to repress the synthesis of non-hsps is greater than that of nondormant ones. A hsp of apparent molecular weight 68 kilodaltons is synthesized only in dormant cormels or in cormels that lost their dormancy after long storage at 25°C. The synthesis of hsps at 40°C, but not at 25°C, is promoted by abscisic acid in nondormant cormels. Methionine incorporation into hsps declines after a 4-hour incubation period at 40°C. Induction of hsps is stronger if exposure to extreme temperature is done gradually.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.     

Identification of a cold-sensitive step in the mechanism of modeccin action

Modeccin is a toxic lectin that arrests protein synthesis in mammalian cells by catalytically inactivating 60 S ribosomes. To interact with 60 S ribosomes, the catalytic subunit of modeccin must pass through a membrane and enter the cytosol. Two known steps in the mechanism of modeccin action are the receptor-mediated internalization of the toxin into vesicles and a second step that requires a low pH within the vesicles. We report here another step required for modeccin to arrest protein synthesis, identified because this step was blocked at 15 degrees C. Modeccin traveling from cell surface receptors to the cytosol at 37 degrees C passed the low pH step within vesicles in a minimum time of 15 min after endocytosis and reached the cold-sensitive step 15 min later. There was no effect on protein synthesis until about 45 min after modeccin had passed the cold-sensitive step, suggesting that the toxin was still within vesicles at the time of the cold-sensitive event. The low temperature at which modeccin failed to reach the cytosol correlated with an apparent low temperature block in the transfer of endocytosed modeccin to lysosomes. The possibility is discussed that modeccin does not penetrate to the cytosol directly from endocytic vesicles.     

Heat shock protein induction and the acquisition of thermotolerance in the psychrotrophic yeastTrichosporon pullulans

Two-dimensional gel electrophoretic analysis of the heat shock response in the psychrotrophic yeastTrichosporon pullulans revealed the induction of 11 heat shock proteins (hsps) after a 5° to 21°C heat shock, 12 hsps after a 5° to 26°C heat shock, and 12 hsps after a 5° to 29°C heat shock. Heat shock from 5° to 26° or 29°C resulted in a statistically significant increase in thermotolerance to a lethal heat challenge at 45°C for 5 min. When the protein synthesis inhibitor, cycloheximide, was added prior to the heat shock, no statistically significant thermotolerance was acquired. To confirm the correlation between the synthesis of hsps and the acquisition of thermotolerance, protein extracts of cells that had been heat shocked in the presence or absence of cycloheximide were electrophoretically analyzed. Addition of the same concentration of cycloheximide that prevented the acquisition of thermotolerance also inhibited the synthesis of any hsps.     

Temperature ranges over which rainbow trout fibroblasts survive and synthesize heat-shock proteins

Cultures of the rainbow trout fibroblast cell line RTG-2 withstood temperatures from 0 degrees C to 28 degrees C. At 0 degrees C and 28 degrees C, no proliferation occurred, but cells persisted for at least 7 days. If the cultures were placed back at 22 degrees C, proliferation returned to normal in those that had been kept at 0 degrees C but was reduced in cultures that had been kept at 28 degrees C. Above 28 degrees C, cultures survived for only short periods. If RTG-2 cells that were grown routinely at 22 degrees C were shifted to 26, 28, and 30 degrees C, heat shock proteins (hsps) of 100, 87, 70, 68, 60, 39, 27, and 19 kilodaltons were synthesized. Synthesis was most pronounced at 28 degrees C, and at this temperature hsp synthesis was maximal by 2 hr and had returned to control levels by 36 hr. Individual hsps were synthesized maximally at slightly different times and temperatures, but under all conditions hsps 87 and 70 were most abundant. If cultures were shifted to 24 degrees C or 32 degrees C, hsp synthesis was not observed. Neither the placement of cultures at 5 degrees C nor the shift of cultures that had been maintained at 0 degrees C or 5 degrees C back to 22 degrees C induced the synthesis of hsps. However, cultures incubated at 5 degrees C for 24 hr did synthesize hsps at 26 degrees C, 28 degrees C, and 30 degrees C.     

Effects of temperature stress on bean-nodulating Rhizobium strains

High soil temperatures in tropical areas limit nodulation and dinitrogen fixation by strains of Rhizobium. Several heat-tolerant bean-nodulating Rhizobium strains have been isolated previously. However, the basis of their resistance to heat remains unknown. In this study, we compared the effects of heat on symbiotic nitrogen fixation, cell survival, amino acid uptake, and protein synthesis in a heat-tolerant (CIAT899) and a heat-sensitive (CNPAF512) bean-nodulating Rhizobium strain. Acetylene reduction activity of nodulated roots excised from unstressed plants was strongly diminished at 35 or 40 degrees C when plants were nodulated either by CIAT899 or by CNPAF512. When these strains were tested under free-living conditions, survival at 40 degrees C as well as the kinetics of l-[S]methionine uptake and protein synthesis at 35 and 40 degrees C indicated the higher tolerance of CIAT899 than of CNPAF512 to thermal stress. The synthesis of heat shock proteins was detected in both strains, although at different temperatures. Increased synthesis of 14 heat shock proteins in CNPAF512 and of 6 heat shock proteins in CIAT899 was observed at 40 and 45 degrees C, respectively. A heat shock protein of approximately 21 kDa, of which the synthesis was strongest in both Rhizobium strains upon a temperature shift up, was also conserved in several other bean-nodulating rhizobia. Acquired thermotolerance in CIAT899 was shown to depend on protein synthesis.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997