Heat shock protection against cold stress of Drosophila melanogaster.

Heat shock protein synthesis can be induced during recovery from cold treatment of Drosophila melanogaster larvae. Survival of larvae after a cold treatment is dramatically improved by a mild heat shock just before the cold shock. The conditions which induce tolerance to cold are similar to those which confer tolerance to heat.

Heat shock induces a decrease in incorporation of 8-azidoadenosine 5'-triphosphate into a 42 kilodalton protein in Drosophila salivary glands.

The ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (8N3ATP) was used to identify changes which occur in ATP binding proteins in Drosophila salivary glands following heat shock. Photolabeling experiments were done on salivary gland homogenates. Photoincorporation of 8N3ATP was observed in several proteins in both 25 degrees C control and 35 degrees C heat-shocked samples. A 42 kDa protein showed a decrease in the level of photoincorporation observed at saturation with the analogue following heat shock. A 2 min heat shock is enough to induce the effect. Protection against photolabeling was observed with low concentrations (5 microM) of ATP, while excess GTP did not protect, demonstrating that the nucleotide binding site is specific for ATP. The change is rapid enough to suggest that it is one of the earliest cellular changes in response to heat shock.

forked proteins are components of fiber bundles present in developing bristles of Drosophila melanogaster.

The forked (f) gene of Drosophila melanogaster encodes six different transcripts 6.4, 5.6, 5.4, 2.5, 1.9, and 1.1 kb long. These transcripts arise by the use of alternative promoters. A polyclonal antibody raised against a domain common to all of the forked-encoded products has been used to identify forked proteins on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and in Drosophila pupal tissues. The antibody stains fiber bundles present in bristle cells for about 15 hr during normal pupal development. Electron microscopy shows that these fibers are present from 40 to 53 hr in bristles of wild-type flies but are absent in the null f36a mutant. The forked protein(s) thus appear to be an essential part of the bristle fibers. The phenotype of the f36a mutation can be rescued by a 13-kb fragment of the forked locus containing the coding regions for the 2.5, 1.9, and 1.1-kb transcripts, suggesting that the proteins encoded by the three large forked RNAs are dispensable during bristle development. Increasing the copy number of a P[w+,f+] construct containing the 13-kb fragment induces a hypermorphic bristle phenotype whose severity correlates with the number of copies of P[w+,f+] present. These results indicate that alterations in the ratios among the forked proteins, or between forked products and other components of the fiber, result in abnormal assembly of the fibrillar cytoplasmic structures necessary for bristle morphogenesis.

Induced thermal tolerance and heat shock protein synthesis in Chinese hamster ovary cells.

We have performed experiments to determine the kinetics of induction of thermal tolerance in Chinese hamster HA-1 cells, and the effects of heat treatments on the recovery of protein synthesis, with particular attention to whether heat induces specific proteins, perhaps the heat shock proteins (HSP). The kinetics of the development of thermal tolerance were measured by increases in cellular survival. In parallel experiments, the effects of heat treatment on the recovery of protein synthesis in HA-1 cells were examined. After heating (45 degrees, 20 minutes), some of these cells were immediately labeled with 35S-methionine (10 microCi/ml) for 1 hour at 37 degrees, while the others were incubated at 37 degrees for 1-8 hours and then labeled. The cell samples were prepared for electrophoresis on a gradient SDS gel. The incorporation of label into HA-1 cell proteins was drastically inhibited by the 45 degrees heat treatment, but recovered gradually during the 8-hour incubation period at 37 degrees C. A comparison of the proteins synthesized following heat shock with those synthesized by non-heated cells showed that the levels of synthesis of certain proteins were greatly enhanced following the 45 degrees treatment. By 8 hours, it was qualitatively apparent that three proteins, with molecular weights of 59K, 70K and 87K, were synthesized in greater amounts than in untreated cells. The kinetics of HSP synthesis were compared to the kinetics of thermal tolerance; these showed good correlation. Overall protein synthesis also increased during this time, although at a rate slower than the synthesis of the HSP. The question of whether the HSP play a causative role in the development of thermal tolerance and if so, what role might be, has not been answered.

Induction of the heat shock response and translational thermotolerance in day 15 ovine trophectoderm.

The objectives of this study were to determine the ability of trophectoderm from preimplantation ovine embryos to synthesize hsp70 in response to heat shock and to identify conditions which induce translational thermotolerance in this tissue. Day 15 embryos were collected, and proteins synthesized in 1.5-mm sections of trophectoderm were radioactively labeled with (35)S-methionine. One-dimensional SDS-PAGE gels, two-dimensional gel electrophoresis and Western blots were utilized to characterize the heat shock response and to examine the induction of translational thermotolerance. Increased synthesis of the 70 kDa heat shock proteins and a protein with an approximate molecular weight of 15 to 20 kDa was observed with heat shock (> or = 42 degrees C). Total protein synthesis decreased (P < 0.05) with increased intensity of heat shock. At 45 degrees C, protein synthesis was suppressed with little or no synthesis of all proteins including hsp70. Recovery of protein synthesis following a severe heat shock (45 degrees C for 20 min) occurred faster (P < 0.05) in trophectoderm pretreated with a mild heat shock (42 degrees C for 30 min) than trophectoderm not pretreated with mild heat. In summary, trophoblastic tissue obtained from ovine embryos exhibit the characteristic "heatshock" response similar to that described for other mammalian systems. In addition, a sublethal heat shock induced the ability of the tissue to resume protein synthesis following severe heat stress. Since maintaining protein synthesis is crucial to embryonic survival, manipulation of the heat-shock response may provide a method to enhance embryonic survival.

Effects of heat and chemical stress on development.

Similarities in the means by which developmental defects are induced in vertebrates and Drosophila suggest that some kinds of defects may be induced by similar mechanisms. The similarities include the fact that heat and a group of chemicals that induce synthesis of heat-shock proteins induce defects in mammals, chickens, and flies. Different kinds of defects are even produced in one type of animal, depending on the precise timing of the environmental insult. The effectiveness of the environmental treatment in inducing defects depends on the genetic background of the animal as well as on past exposure to chemicals and heat. Developmental defects induced by heat in mice, rats, and flies can all be prevented by thermotolerance-inducing treatments. The basis for these effects has been studied at the molecular level in Drosophila, and the evidence indicates that these teratogens and the thermotolerance-inducing treatments affect the level or timing of expression of specific genes during critical periods in the developmental program.

The induction of a multiple wing hair phenocopy by heat shock in mutant heterozygotes.

Phenocopies are developmental defects induced by environmental treatments during differentiation. Because of their resemblance to mutant phenotypes it has been suggested that phenocopies are due to environmental effects on the expression of specific genes during development. In this paper we describe the heat shock (40.8 degrees C) induction of a multiple wing hair phenocopy in the mutant heterozygote (mwh/+). The mwh phenocopy is only induced in heterozygotes of the recessive mutant during a short sensitive period which appears to be the time of expression of the multiple wing hair gene. We suggest that this phenocopy is due to failure of mwh gene expression and that phenocopy sensitive periods may be useful in identifying expression periods for particular genes during development. Furthermore we have been able to demonstrate that a 35 degrees C pretreatment will prevent the induction of the multiple wing hair phenocopy. A similar 35 degrees C pretreatment prevents induction of several different phenocopies by heat in wild-type flies (N. S. Petersen and H. K. Mitchell (1985). In "Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. X, Biochemistry." Pergamon, New York). This indicates a common molecular mechanism for both the induction and the prevention of heat-induced phenocopies.

Spontaneous fragmentation of several proteins in Drosophila pupae.

Autoproteolysis is an essential activity in the expression of the entire genomes of a number of viruses. That is, new viruses can be produced only after large polyprotein products translated from the genome or from subgenomic mRNA degrade themselves to the polypeptides necessary for RNA replication or for the construction of new virus particles. We have recently shown that the major heat shock protein of Drosophila and a mouse cell line (70 kDa) also undergoes autoproteolysis with the production of specific patterns of smaller polypeptides. We show now that many other proteins in eucaryotic tissues also have a potential for self-degradation. We suggest that special coding regions in many genes may have important roles in both protein turnover and in the production of regulatory peptides.

Effects of heat shock on protein processing and turnover in developing Drosophila wings.

Developmental defects called phenocopies can be induced by heating Drosophila melanogaster pupae at specific developmental stages. The induction of the defects is thought to be a result of interference with gene expression at some level (Petersen and Mitchell, Dev Biol 1987; 121:335-341, 1987). Here we look at protein turnover in developing 52-hour wings and at the effect of heat on the proteolytic processing of three proteins that normally turn over rapidly. The effect of the heat treatment itself on the turnover of each protein is different. However, all of the proteins appear to be stabilized at 25 degrees C during recovery from severe heat shocks.

Epithelial differentiation in Drosophila pupae.

The construction of cell hairs on the wings in developing pupae of Drosophila provides a unique system for studies of the regulation of differentiation in the absence of cell division. Early steps in hair construction are the extrusion of cell hairs and the deposition of the external impervious layer called "cuticulin." Some properties of six of the most abundant proteins that are present during the early stages of hair construction are described. These proteins make up about 40% of the total protein of the preparation.

Yeast mutant, rna 1, affects the entry into polysomes of ribosomal RNA as well as messenger RNA.

The entry of newly labeled ribosomal subunits and mRNA into polysomes was examined in the yeast mutant rna1. The entry of both types of RNA into polysomes is inhibited rapidly at the restrictive temperature. Analysis of the labeling of the ATP pool and the kinetics of synthesis and processing of mRNA at the restrictive temperature leads to the conclusion that the primary defect in the mutant affects transport of both ribosomes and messenger across the nuclear membrane.

Gradients of differentiation in wild-type and bithorax mutants of Drosophila.

We present evidence to show that differentiation in wing cells to produce hairs is synchronous over the distal 90% of the wing surface (approximately 28,000 cells). In spite of this synchrony within such a large area a temporal gradient exists between zones (in general anterior to posterior) on the animal surface with rather sharp boundaries in between. In order to evaluate the basis for the gradient we studied two mutants which carry different combinations of the genes of the bithorax complex. These were examined with respect to the temporal aspects of sensitivity to heat shock induction of the multihair phenocopy on wings and the time of initiation of the program of protein synthesis that is related to hair formation. Results show that the gradient observed is based on predetermined properties within specific areas of tissue rather than on the position of the cells in the animal.

Recovery of protein synthesis after heat shock: prior heat treatment affects the ability of cells to translate mRNA.

A mild heat shock at 35 degrees C, which induces heat shock gene expression, greatly enhances survival and the recovery of protein synthesis in Drosophila cells after a higher temperature heat shock. The 35 degrees C treatment is also effective in preventing heat-induced developmental defects in pupae. We show here that the major larval mRNAs are present in approximately normal (25 degrees C) concentrations after a 40.1 degrees C heat shock whether or not the animals receive a pretreatment. This indicates that the pretreatment affects translation directly rather than messenger concentration. We also observe selective translation of heat shock messages and some 25 degrees C messages during recovery from heat shock.

The Drosophila forked protein induces the formation of actin fiber bundles in vertebrate cells.

The forked protein is an actin binding protein involved in the formation of large actin fiber bundles in developing Drosophila bristles. These are the largest example of a type of actin bundle characterized by parallel, hexagonally packed actin fibers, also found in intestinal microvilli, kidney proximal tubule microvilli, and stereocilia in the ear. Understanding how these structures are constructed and how that construction is regulated is an important question in cell and developmental biology. Because the timing of forked gene expression coincides with the formation of the actin fiber bundles, and since the forked protein is localized at the site of initiation of these bundles before they form, it has been proposed that the forked protein is an initiator of actin bundle formation. In this paper we show that the forked protein can induce the formation of bundles and increase actin polymerization in vertebrate cells. We use this system to identify regions of the forked protein which are essential for bundle formation and actin co-localization.

Stages of cell hair construction in Drosophila.

The construction of cell hairs (trichomes) on the wings of Drosophila occurs in synchrony on 30,000 cells over a period of about 20 hr. Changes in both morphology and patterns of protein synthesis occur rapidly during this time period. In this report we describe the use of stress-induced (heat shock) abnormalities in morphogenesis to provide further details on the stepwise processes of differentiation within single wing cells. A cartoon summary of the overall process and a discussion of some possible mechanisms is included.

Self-degradation of heat shock proteins.

The 70-kDa heat shock protein of Drosophila decays in vivo at a much faster rate than other abundantly labeled proteins. Degradation also occurs in vitro, even during electrophoresis. It appears that this degradation is not mediated by a general protease and that the 70-kDa heat shock protein has a slow proteolytic action upon itself. Heat-induced proteins in CHO cells and a mouse cell line also degrade spontaneously in vitro, as do certain non-heat shock proteins from Drosophila tissues as well as the cell lines.

Heat shock response of germinating embryos of wheat : effects of imbibition time and seed vigor.

Seeds frequently face a hostile environment during early germination. In order to determine whether seeds have evolved unique mechanisms to deal with such environments, a survey of the heat shock response in isolated embryos of wheat (Triticum aestivum L.) was undertaken. Embryos simultaneously heat shocked and labeled following several different periods of prior imbibition up to 12 hours synthesized many groups of heat shock proteins (hsps) typical of other plant and animal systems. Also, five developmentally dependent hsps, present only in treatments imbibed less than 6 hours prior to heat shock, were detected. These proteins have relative molecular masses of 14, 40, 46, 58, and 60 kilodaltons. One of the developmentally dependent hsps is among the most highly labeled hsps found in early imbibed embryos. The possibility that this protein is the E(m) protein is discussed. The hypothesis that the capacity for hsp synthesis is affected by seed vigor was also tested. The heat shock responses of embryos from two high and two low vigor seed lots were compared using one- and two-dimensional electrophoresis of labelled protein extracts. The results indicate that both of the low vigor lots tested had weaker heat shock responses than their high vigor counterparts overall. Not all hsps were relatively less abundant in low vigor embryos. The developmentally dependent hsps showed little relationship to vigor. Some of the developmentally dependent hsps were actually made in greater amounts, relative to other proteins, in the low vigor seed lots. The results presented here demonstrate that imbibing embryos are capable of expressing an enhanced heat shock response, and that this response is related to seed vigor.

Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos.

Heat shock has a dramatic effect on the organization of the cytoplasm, causing the intermediate filament cytoskeleton to aggregate at the nucleus. This has previously been shown in cultured Drosophila and mammalian cells. In this paper we analyze the heat lability of the intermediate filament cytoskeleton in early Drosophila embryos by indirect immunofluorescence. At all stages of embryogenesis tested, the intermediate filament cytoskeleton, which is maternally provided, is severely disturbed by 30 min heat shock at 37 degrees C. After the nuclei have migrated to the subcortical cytoplasm, it collapses around them. Nuclei in all heat-shocked embryos are considerably enlarged and become displaced. Embryos before cellular blastoderm stage, in which heat shock protein synthesis is not inducible, are irreversibly arrested in development by heat shock. Embryos at or after cellular blastoderm, which do synthesize heat shock proteins in response to stress, are also immediately arrested in development but continue development when returned to 25 degrees C. We discuss the possibility that cytoplasmic events such as the intermediate filament cytoskeleton rearrangement may be involved in heat shock-mediated phenocopy induction.