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Synthesis of heat-shock proteins in developing sea urchins
DEVELOPMENTAL BIOLOGY 83, 173-177 (1981)
Synthesis of Heat-Shock Proteins in Developing Sea Urchins M A R I A CARMELA ROCCHERI, M A R I A G R A Z I A D I BERNARDO, AND GIOVANNI GIUDICE
Institute of Comparative Anatomy, The University of Palermo, Via Archirafi 22, Palermo, Italy Received June 12, 1980; accepted in revised form August 18, 1980 Heating sea urchin embryos at 31~ greatly reduces the synthesis of the bulk proteins, whereas it highly stimulates the synthesis of some new proteins, the main ones being two closely migrating proteins of about 70,000 daltons. The production of heat-shock proteins is obtained only if the embryos are heated after hatching. Stages which produce heat-shock proteins survive heating, whereas earlier stages, not producing heat-shock proteins, do not survive. Heatshock proteins are not produced in the presence of actinomycin D. INTRODUCTION
One of the main difficulties in the study of the regulation of protein synthesis during development, is that of the exceedingly high number of proteins that each cell synthesizes. Ritossa (1962) demonstrated that a brief heat treatment of t h e salivary glands of Drosophila larvae greatly modified the pattern of puffing by inducing the appearance of a few new characteristic puffs. It was later shown that this is accompanied by a great reduction in the number of proteins synthesized, and by the synthesis of a few new characteristic proteins, the so-called heat-shock proteins (Tissieres et al., 1974; Lewis et al., 1975; McKenzie et al., 1975). It would be of great interest to find out whether a heat treatment of entire embryos instead of single organs in an organism in which the problem of regulation of protein synthesis has been thoroughly investigated, as for example sea urchins, produces a reduction of the number of the proteins synthesized, and the production of heat-shock proteins: Preliminary experiments (Giudice et al., 1980) have clearly demonstrated that this is the case. It is the purpose of this paper to confirm and extend these observations, and to try a preliminary interpretation of the mechanism of production of heat-shock proteins in sea urchin embryos. MATERIALS AND METHODS
Embryonic cultures. Embryos of Paracentrotus lividus and of Arbacia lixula were cultured as previously described (Giudice and Mutolo, 1967) at 20~ and for shorter times at the temperatures indicated in the text. Embryo labeling. Embryos at the concentration of 5000/ml were exposed to 20 #Ci/ml of L-[3H]leucine (specific activity, 145 Ci/mmole) for 15 min at the temperatures indicated in the text.
Measurement of the protein synthesis rate. Embryos were labeled at the experimental temperatures for 15 min. After homogenization in 2% sodium dodecyl sulfate, samples were taken and dried onto filter paper disks, washed twice with cold 10% trichloroacetic acid (TCA) then with hot 5% TCA, alcohol,ether 2:1, ether, according to Mans and Novelli (1961), and counted in a liquid scintillation counter. Total proteins were measured by means of the procedure of Lowry et al. (1951). Gel electrophoresis. Embryos were homogenized in a Dounce homogenizer in 0.0625 M Tris, 5% ~-mercaptoethanol, 10% glycerol with the addition of 0.0025% bromophenol blue, and 2% sodium dodecyl sulate (SDS). Aliquots of 40 pl were run in 10% polyacrylamideSDS slab gels for 41/2 hr at room temperature; fluorography was then carried out by exposition to a Kodak RP Royal X-Omat (RP/R) film, after soaking in a 2,5diphenyloxazole in dimethyl sulfoxide solution and drying (Laskey and Mills, 1975). RESULTS
If one labels P. lividus gastrulas with radioactive leucine at their physiological temperature, i.e., 20~ and then looks at the autoradiography of the electrophoresis of their total proteins, one finds the expected complex pattern as reported in Fig. 1. If one exposes the embryos for 1 hr at a higher temperature and labels them during the last 15 min of the heat treatment, the pattern of protein synthesis observed after electrophoresis b e comes fainter as the temperature becomes higher, until at 37~ it completely disappears. However, at 31~ two closely migrating bands, with a molecular weight of about 70,000 daltons, become very heavily labeled in contrast with the highly reduced pattern of synthesis of all the other proteins (see Fig. 1). We have named
173 0012-1606/81/050173-05502.00/0 Copyright 9 1981by AcademicPress, Inc. All rights of reproductionin any form reserved.
174
DEVELOPMENTAL BIOLOGY 20~
25 o
1
31 ~
|
VOLUME83, 1981
1
20~ e
31o
Mes. b l .
these two bands heat-shock proteins. They do not seem to correspond to any of the proteins synthesized at physiological temperature. The appearance of the 70,000-dalton proteins at stages following hatching after heat shock is a constant and highly reproducible phenomenon; also reproducible is the decrease of the overall protein synthesis; somewhat more variably, however, the appearance of at least seven minor heat-shock proteins has been observed at these stages. Because of their minor representation we will not describe them further in this paper, and will refer only to the two 70,000-dalton proteins as heatshock proteins. The reduction of [aH]leucine incorporation into the total proteins after heat shock at the gastrula stage is of 52.4 ( + 2.9)% (value calculated from 11 experiments). One has to consider that the incorporation of [~H]leucine into the total protein of the heat-treated embryos includes that into the heat-shock proteins and that no corrections for possible variations of permeability to amino acids and of the pool have been made; these variations would, however, most probably affect to the same extent [SH]leucine incorporation into the total protein and into the heat-shock proteins. If one treats gastrulas of a different sea urchin species, i.e., A. lixula, at 31~ similar results as in Paracentrotus are obtained: a sharp reduction of the synthesis of the bulk proteins and the appearance of one sharp band of incorporation in the region of 70,000 molecular weight. It has already been found (Giudice et al., 1980) that if the treatment at high temperatures is made at the 32-blastomere stage, instead of at the gastrula stage, the autoradiographic pattern of protein synthesis progressively fades with the increase of the temperature,
H.bl.
!
m
2o~
FIG. 1. Effect of heat t r e a t m e n t at different temperatures on the pattern of protein synthesis of Paracentrotus lividus gastrulas.
E.M.
32 bl.
37 ~
Gastr.
~
PI.
+
I!1' i FIG. 2. Effect on the p a t t e r n of protein synthesis of heating at 31~ at different developmental stages. Fert. egg., fertilized eggs; 32 bl., 32 blastomeres; E. bl., early blastulas; H. bl., hatched blastulas; Mes. bl., mesenchyme blastulas; Gastr., gastrulas; PI., plutei.
until complete disappearance, but no heat-shock proteins are ever synthesized. We tested here the effect of heat shock at various developmental stages between fertilization and pluteus. As shown in Fig. 2, the increase of temperature always produces a drastic decrease of the overall protein synthesis at all stages, but heat-shock proteins are produced only in the stages following the hatching blastula. They have the same electrophoretic mobility at all the responsive stages. How long does the heat-shock effect on the pattern of protein synthesis last? In order to answer this question we have returned the heat-treated gastrulas to normal temperature and then, after various lengths of time, labeled them for 15 min with [SH]leucine, and analyzed the protein synthesis pattern. The results of such experiments, reported in Fig. 3, demonstrate that the pattern of protein synthesis remains that of the heattreated gastrulas until at least 2 hr after return to normal temperature; after 5 hr, however, this pattern has practically completely reverted to normality. This result is essentially the same irrespective of whether the preheating has been protracted for 1, 2, or 4 hr.
BRIEF NOTES
FIG. 3. Reversal of the pattern of protein synthesis at various time lengths after 1 hr of heating of Paracentrotus gastrulas. C, control, nonheated embryos; O, 1 hr of heating; 1 hr, 2 hr, 4 hr, and 8 hr, 1 hr of heating followed by 1, 2, 4, and 8 hr, respectively, at normal temperature. The s t a n d a r d length of h e a t t r e a t m e n t which we have used in o r d e r to obtain the described effect on p r o t e i n synthesis at the responsive stages has been 45 to 60 min of p r e h e a t i n g followed by 15 min of radioactive labeling. We have, however, also checked the effect of various time lengths of p r e h e a t i n g at the responsive as well as at the nonresponsive stages, i.e., a f t e r h a t c h i n g and before hatching, respectively. F i g u r e 4 shows t h a t 15 min of p r e h e a t i n g at the g a s t r u l a stage is enough to elicit the a p p e a r a n c e of the two h e a t - s h o c k p r o t e i n s and
175
to severely reduce the overall protein synthesis. This effect is m o r e p r o n o u n c e d a f t e r 75 min of p r e h e a t i n g . I n c r e a s i n g the length of p r e h e a t i n g up to 21/2 h r does not s u b s t a n t i a l l y m o d i f y this response. I n c r e a s i n g the p r e h e a t i n g length to 4 hr, however, seems to produce a lower effect t h a n s h o r t e r t r e a t m e n t s , and a f t e r 8 h r of p r e h e a t i n g a c e r t a i n reversal of the h e a t - s h o c k effect is observed so t h a t an a l m o s t complete recovery of the radioactive leucine i n c o r p o r a t i o n into the total p r o t e i n is achieved, and the a u t o r a d i o g r a p h i c p a t t e r n looks quite similar to t h a t of the control embryos. P r e h e a t i n g s of c o m p a r a b l e lengths conducted a t a nonresponsive stage (two b l a s t o m e r e s ) always fail to elicit the a p p e a r a n c e of the h e a t - s h o c k proteins, resuiting only in a v e r y severe inhibition of protein synthesis, which is completely suppressed if p r e h e a t i n g is p r o t r a c t e d for 4 hr. W h a t is the effect of h e a t shock on d e v e l o p m e n t ? The a n s w e r to this question is quite d i f f e r e n t if the h e a t shock is o p e r a t e d a t p r e h a t c h i n g or a t p o s t h a t c h i n g stages, i.e., stages which do not or do produce heatshock proteins, respectively. One h o u r of t r e a t m e n t a t 31~ is enough to completely a r r e s t d e v e l o p m e n t if h e a t i n g is conducted in p r e h a t c h i n g stages. Only v e r y few e m b r y o s (less t h a n 1% ) are f o u n d to develop u n d e r these conditions. If, on the o t h e r hand, h e a t i n g at 31~ is o p e r a t e d at p o s t h a t c h i n g stages, an a l m o s t complete survival of the embryos, accompanied b y quite r e g u l a r TABLE 1 EFFECT ON DEVELOPMENT OF HEATING AT 31~ EMBRYONIC STAGES
Stage of treatment
Duration of heating (hr)
Fertilized eggs
1
2 Blastomeres
1
32 Blastomeres
1
Early blastulas
1
Hatched blastulas
1
AT DIFFERENT
36 hr after fertilization 50% Degeneration 50% Irregular morulas 50% Degeneration 25% Irregular morulas 25% Irregular blastulas 44% Degeneration 55% Irregular blastulas 1% Gastrulas 34% Degeneration 65% Irregular blastulas 1% Gastrulas 99% Plutei 1% Degeneration
Mesenchime
FIG. 4. Effect on the pattern of protein synthesis of heating gastrulas at 31~ for various time lengths. (The times of heating indicated are to be added to 15 rain of exposure to the isotope at the same temperature).
blastulas Gastrulas Prisms Gastrulas Gastrulas Gastrulas Control embryos
1 1 1 4 8 Continuous None
100% Plutei 100% Plutei 100% Plutei 100% Plueti 100% Plueti 100% Plueti 100% Pleuti
176
DEVELOPMENTAL BIOLOGY
development, is observed even if the embryos are continuously kept at 31~ The details of such an experiment are reported in Table 1 which clearly shows a complete capacity of developmental recovery from the heat shock of the posthatching stages, which sharply contrasts with the absolute lack of such a capacity in the prehatching stages. An important question is whether the synthesis of heat-shock proteins in sea urchin embryos is due to the synthesis of new specific messenger RNAs. In a preliminary attempt to answer such a question, we have conducted the heat shock of Paracentrotus gastrulas in the presence of actinomycin D. The results, reported in Fig. 5, show that actinomycin at 12 and 25, and especially at 50~g/ml, markedly reduces the synthesis of the heatshock proteins, whereas, as expected, the synthesis of the bulk proteins at normal temperature is little affected by the presence of actinomycin D. DISCUSSION
The phenomenon of the production of heat-shock proteins in Drosophila salivary glands, as well as in other materials (Ashburner and Bonner, 1979), has raised the question of whether or not these proteins have a function possibly in the defense against high temperature. The fact that sea urchin embryos also produce such proteins gives some more support to this possibility. It would be interesting to know if the 70,000-dalton proteins produced by sea urchins are the same as those produced by Drosophila. Since plasmids containing the heat-shock protein genes of Drosophila are available, experiments are in progress aimed to ascertain this hypothesis. These experiments might also provide an answer as to whether the synthesis of heat-shock proteins in sea urchins is operated through the start of the synthesis or the increase of the synthesis of specific messenger RNAs. The actinomycin experiments re-
L FIG. 5. Effect of actinomycin D on the production of heat-shock proteins.
VOLUME83, 1981
ported in this paper already speak in favor of such a hypothesis, but they do not distinguish whether the inhibitory effect of actinomycin on the production of heatshock proteins, is exerted by directly inhibiting the production of the messenger RNA coding for heat-shock proteins, or through the inhibition of some other RNA which in turn is necessary for the synthesis of heatshock proteins. These experiments also demonstrate that neither RNA synthesis nor heat-shock protein synthesis are needed for the inhibition of the overall protein synthesis by heat shock to occur. That heat-shock protein production is not needed for the inhibition of the overall protein synthesis by heat is also suggested by the experiments of heating at prehatching stages. That the production of heat-shock protein does not have a trivial explanation, like that of denaturation of proteins that are produced at normal temperature, is already suggested by experiments of heating the embryos at prehatching stages in which the heat-shock proteins do not appear, and is demonstrated by experiments in which gastrulas have been exposed to radioactive leucine at normal temperature and heated at 31~ only after homogenization. In this case the electrofluorograms are absolutely identical to those of normal embryos, and no heat-shock proteins are observed. The different sensitivity of embryos in pre- or posthatching stages to the heat shock is very intriguing because it inversely correlates with the ability to produce heat-shock proteins, thus suggesting again a role for these proteins in the protection of the cells from high temperatures. In agreement with such a hypothesis, McAlister and Finkelstein (1980) have recently described a good correlation between resistance to heat of yeast cells and cellular level of heat-shock proteins. Moreover, Drosophila embryos, if heated at any stage preceding the migration of the nuclei to the periphery of the egg, fail to produce heat-shock proteins and stop cleaving, whereas if heated at later stages, produce heat-shock proteins and continue developing (Graziosi, et al., 1980). How do the heat-shock proteins protect the sea urchin embryos? This is not known at present. What we can say is that it is enough of a wave of production of heatshock proteins for these to produce their effect: in posthatching stages in fact the pattern of protein synthesis reverts to normality after a few hours of heating. It is interesting to recall here that sea urchin embryos are also able to develop until the hatching blastula stage in the absence of synthesis of new messenger RNAs, whereas this synthesis is necessary in order for development to proceed beyond this stage (Giudice et al., 1968; Giudice, 1973). How the latter observations correlate with the ability to produce heat-shock proteins is still a matter of speculation; the suggestion can be
BRIEF NOTES
made, however, that some genes cannot be switched on before certain developmental stages. The expert technical assistance of Mr. D. Cascino is acknowledged. This work was supported in part by the research project on reproduction of the Italian National Research Council, Contract 79.1155.85. REFERENCES ASH~Um~ER,M., and BO~ER, I. I. (1979). The induction of gene activity in Drosophila by heat shock. Cell 17, 241-254. GIUDICE, G. (1973). "Developmental Biology of the Sea Urchin Embryos." Academic Press, New York/London. GIUDICE, G., and MUTOLO,V. (1967). Synthesis of ribosomal RNA during sea urchin development. Biochim. Biophy& Acta 138, 276285. GIUDICE, G., MUTOLO,V., and DONATUTI,G. (1968). Gene expression in sea urchin development. Wilhelm Roux Arch. 161, 118-128. GIUDICE,G., ROCCHERI,M. C., and DI BERNARDO,M. G. (1980). Synthesis of "heat shock" proteins in sea urchin embryos. Cell BioL Int. Rep. 4, 69-74. GRAZIOSI,G., MICALI,F., MARZARI,R., DE CRISTINI,F., and SAVOINI, A. (1980). Variability of response of early Drosophila embryos to heat shock. J. Exp. Zool. 214, 141-145.
177
LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ~ and uC in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341. LEWIS, M., HELMSING, P. I., and ASHBURNER, M. (1975). Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc. Nat. Acad, Sci. USA 72, 36043608. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL,R. J. (1951). Protein measurement with the Folin phenol reagen. J. Biol. Chem. 193, 265-2"/5. MCALISTER,L., and FINKELSTEIN,V. (1980). Heat shock proteins and thermal resistance in yeast. Biochem. Biophys. Res. Commun. 93, 819-824. MANS, R. J., and NOVELLI, G. D. (1961). Measurement of the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch. Bioc~m. Biophya 94, 48-53. MCKENZIE,S. L., HENIKOFF,S., and MESELSON,M. (1975). Localization of RNA from heat induced polysomes at puff sites in Drosophila melanogaster. Proc. Nat. Acad, Sci. USA 72, 1117-1121. RITOSSA, F. (1962). A new puffing pattern induced by temperature shock and DNP in Drosophila. E~erientia 18, 571-573. TISSIERES, A., MITCHELL, H. K., and TRAcY,U. M. (1974). Protein synthesis in salivary glands of Drosophila melanogaster: Relations to chromosome puffs. J. MoL Biol. 84, 389-398.
Synthesis of Heat-Shock Proteins in Developing Sea Urchins M A R I A CARMELA ROCCHERI, M A R I A G R A Z I A D I BERNARDO, AND GIOVANNI GIUDICE
Institute of Comparative Anatomy, The University of Palermo, Via Archirafi 22, Palermo, Italy Received June 12, 1980; accepted in revised form August 18, 1980 Heating sea urchin embryos at 31~ greatly reduces the synthesis of the bulk proteins, whereas it highly stimulates the synthesis of some new proteins, the main ones being two closely migrating proteins of about 70,000 daltons. The production of heat-shock proteins is obtained only if the embryos are heated after hatching. Stages which produce heat-shock proteins survive heating, whereas earlier stages, not producing heat-shock proteins, do not survive. Heatshock proteins are not produced in the presence of actinomycin D. INTRODUCTION
One of the main difficulties in the study of the regulation of protein synthesis during development, is that of the exceedingly high number of proteins that each cell synthesizes. Ritossa (1962) demonstrated that a brief heat treatment of t h e salivary glands of Drosophila larvae greatly modified the pattern of puffing by inducing the appearance of a few new characteristic puffs. It was later shown that this is accompanied by a great reduction in the number of proteins synthesized, and by the synthesis of a few new characteristic proteins, the so-called heat-shock proteins (Tissieres et al., 1974; Lewis et al., 1975; McKenzie et al., 1975). It would be of great interest to find out whether a heat treatment of entire embryos instead of single organs in an organism in which the problem of regulation of protein synthesis has been thoroughly investigated, as for example sea urchins, produces a reduction of the number of the proteins synthesized, and the production of heat-shock proteins: Preliminary experiments (Giudice et al., 1980) have clearly demonstrated that this is the case. It is the purpose of this paper to confirm and extend these observations, and to try a preliminary interpretation of the mechanism of production of heat-shock proteins in sea urchin embryos. MATERIALS AND METHODS
Embryonic cultures. Embryos of Paracentrotus lividus and of Arbacia lixula were cultured as previously described (Giudice and Mutolo, 1967) at 20~ and for shorter times at the temperatures indicated in the text. Embryo labeling. Embryos at the concentration of 5000/ml were exposed to 20 #Ci/ml of L-[3H]leucine (specific activity, 145 Ci/mmole) for 15 min at the temperatures indicated in the text.
Measurement of the protein synthesis rate. Embryos were labeled at the experimental temperatures for 15 min. After homogenization in 2% sodium dodecyl sulfate, samples were taken and dried onto filter paper disks, washed twice with cold 10% trichloroacetic acid (TCA) then with hot 5% TCA, alcohol,ether 2:1, ether, according to Mans and Novelli (1961), and counted in a liquid scintillation counter. Total proteins were measured by means of the procedure of Lowry et al. (1951). Gel electrophoresis. Embryos were homogenized in a Dounce homogenizer in 0.0625 M Tris, 5% ~-mercaptoethanol, 10% glycerol with the addition of 0.0025% bromophenol blue, and 2% sodium dodecyl sulate (SDS). Aliquots of 40 pl were run in 10% polyacrylamideSDS slab gels for 41/2 hr at room temperature; fluorography was then carried out by exposition to a Kodak RP Royal X-Omat (RP/R) film, after soaking in a 2,5diphenyloxazole in dimethyl sulfoxide solution and drying (Laskey and Mills, 1975). RESULTS
If one labels P. lividus gastrulas with radioactive leucine at their physiological temperature, i.e., 20~ and then looks at the autoradiography of the electrophoresis of their total proteins, one finds the expected complex pattern as reported in Fig. 1. If one exposes the embryos for 1 hr at a higher temperature and labels them during the last 15 min of the heat treatment, the pattern of protein synthesis observed after electrophoresis b e comes fainter as the temperature becomes higher, until at 37~ it completely disappears. However, at 31~ two closely migrating bands, with a molecular weight of about 70,000 daltons, become very heavily labeled in contrast with the highly reduced pattern of synthesis of all the other proteins (see Fig. 1). We have named
173 0012-1606/81/050173-05502.00/0 Copyright 9 1981by AcademicPress, Inc. All rights of reproductionin any form reserved.
174
DEVELOPMENTAL BIOLOGY 20~
25 o
1
31 ~
|
VOLUME83, 1981
1
20~ e
31o
Mes. b l .
these two bands heat-shock proteins. They do not seem to correspond to any of the proteins synthesized at physiological temperature. The appearance of the 70,000-dalton proteins at stages following hatching after heat shock is a constant and highly reproducible phenomenon; also reproducible is the decrease of the overall protein synthesis; somewhat more variably, however, the appearance of at least seven minor heat-shock proteins has been observed at these stages. Because of their minor representation we will not describe them further in this paper, and will refer only to the two 70,000-dalton proteins as heatshock proteins. The reduction of [aH]leucine incorporation into the total proteins after heat shock at the gastrula stage is of 52.4 ( + 2.9)% (value calculated from 11 experiments). One has to consider that the incorporation of [~H]leucine into the total protein of the heat-treated embryos includes that into the heat-shock proteins and that no corrections for possible variations of permeability to amino acids and of the pool have been made; these variations would, however, most probably affect to the same extent [SH]leucine incorporation into the total protein and into the heat-shock proteins. If one treats gastrulas of a different sea urchin species, i.e., A. lixula, at 31~ similar results as in Paracentrotus are obtained: a sharp reduction of the synthesis of the bulk proteins and the appearance of one sharp band of incorporation in the region of 70,000 molecular weight. It has already been found (Giudice et al., 1980) that if the treatment at high temperatures is made at the 32-blastomere stage, instead of at the gastrula stage, the autoradiographic pattern of protein synthesis progressively fades with the increase of the temperature,
H.bl.
!
m
2o~
FIG. 1. Effect of heat t r e a t m e n t at different temperatures on the pattern of protein synthesis of Paracentrotus lividus gastrulas.
E.M.
32 bl.
37 ~
Gastr.
~
PI.
+
I!1' i FIG. 2. Effect on the p a t t e r n of protein synthesis of heating at 31~ at different developmental stages. Fert. egg., fertilized eggs; 32 bl., 32 blastomeres; E. bl., early blastulas; H. bl., hatched blastulas; Mes. bl., mesenchyme blastulas; Gastr., gastrulas; PI., plutei.
until complete disappearance, but no heat-shock proteins are ever synthesized. We tested here the effect of heat shock at various developmental stages between fertilization and pluteus. As shown in Fig. 2, the increase of temperature always produces a drastic decrease of the overall protein synthesis at all stages, but heat-shock proteins are produced only in the stages following the hatching blastula. They have the same electrophoretic mobility at all the responsive stages. How long does the heat-shock effect on the pattern of protein synthesis last? In order to answer this question we have returned the heat-treated gastrulas to normal temperature and then, after various lengths of time, labeled them for 15 min with [SH]leucine, and analyzed the protein synthesis pattern. The results of such experiments, reported in Fig. 3, demonstrate that the pattern of protein synthesis remains that of the heattreated gastrulas until at least 2 hr after return to normal temperature; after 5 hr, however, this pattern has practically completely reverted to normality. This result is essentially the same irrespective of whether the preheating has been protracted for 1, 2, or 4 hr.
BRIEF NOTES
FIG. 3. Reversal of the pattern of protein synthesis at various time lengths after 1 hr of heating of Paracentrotus gastrulas. C, control, nonheated embryos; O, 1 hr of heating; 1 hr, 2 hr, 4 hr, and 8 hr, 1 hr of heating followed by 1, 2, 4, and 8 hr, respectively, at normal temperature. The s t a n d a r d length of h e a t t r e a t m e n t which we have used in o r d e r to obtain the described effect on p r o t e i n synthesis at the responsive stages has been 45 to 60 min of p r e h e a t i n g followed by 15 min of radioactive labeling. We have, however, also checked the effect of various time lengths of p r e h e a t i n g at the responsive as well as at the nonresponsive stages, i.e., a f t e r h a t c h i n g and before hatching, respectively. F i g u r e 4 shows t h a t 15 min of p r e h e a t i n g at the g a s t r u l a stage is enough to elicit the a p p e a r a n c e of the two h e a t - s h o c k p r o t e i n s and
175
to severely reduce the overall protein synthesis. This effect is m o r e p r o n o u n c e d a f t e r 75 min of p r e h e a t i n g . I n c r e a s i n g the length of p r e h e a t i n g up to 21/2 h r does not s u b s t a n t i a l l y m o d i f y this response. I n c r e a s i n g the p r e h e a t i n g length to 4 hr, however, seems to produce a lower effect t h a n s h o r t e r t r e a t m e n t s , and a f t e r 8 h r of p r e h e a t i n g a c e r t a i n reversal of the h e a t - s h o c k effect is observed so t h a t an a l m o s t complete recovery of the radioactive leucine i n c o r p o r a t i o n into the total p r o t e i n is achieved, and the a u t o r a d i o g r a p h i c p a t t e r n looks quite similar to t h a t of the control embryos. P r e h e a t i n g s of c o m p a r a b l e lengths conducted a t a nonresponsive stage (two b l a s t o m e r e s ) always fail to elicit the a p p e a r a n c e of the h e a t - s h o c k proteins, resuiting only in a v e r y severe inhibition of protein synthesis, which is completely suppressed if p r e h e a t i n g is p r o t r a c t e d for 4 hr. W h a t is the effect of h e a t shock on d e v e l o p m e n t ? The a n s w e r to this question is quite d i f f e r e n t if the h e a t shock is o p e r a t e d a t p r e h a t c h i n g or a t p o s t h a t c h i n g stages, i.e., stages which do not or do produce heatshock proteins, respectively. One h o u r of t r e a t m e n t a t 31~ is enough to completely a r r e s t d e v e l o p m e n t if h e a t i n g is conducted in p r e h a t c h i n g stages. Only v e r y few e m b r y o s (less t h a n 1% ) are f o u n d to develop u n d e r these conditions. If, on the o t h e r hand, h e a t i n g at 31~ is o p e r a t e d at p o s t h a t c h i n g stages, an a l m o s t complete survival of the embryos, accompanied b y quite r e g u l a r TABLE 1 EFFECT ON DEVELOPMENT OF HEATING AT 31~ EMBRYONIC STAGES
Stage of treatment
Duration of heating (hr)
Fertilized eggs
1
2 Blastomeres
1
32 Blastomeres
1
Early blastulas
1
Hatched blastulas
1
AT DIFFERENT
36 hr after fertilization 50% Degeneration 50% Irregular morulas 50% Degeneration 25% Irregular morulas 25% Irregular blastulas 44% Degeneration 55% Irregular blastulas 1% Gastrulas 34% Degeneration 65% Irregular blastulas 1% Gastrulas 99% Plutei 1% Degeneration
Mesenchime
FIG. 4. Effect on the pattern of protein synthesis of heating gastrulas at 31~ for various time lengths. (The times of heating indicated are to be added to 15 rain of exposure to the isotope at the same temperature).
blastulas Gastrulas Prisms Gastrulas Gastrulas Gastrulas Control embryos
1 1 1 4 8 Continuous None
100% Plutei 100% Plutei 100% Plutei 100% Plueti 100% Plueti 100% Plueti 100% Pleuti
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DEVELOPMENTAL BIOLOGY
development, is observed even if the embryos are continuously kept at 31~ The details of such an experiment are reported in Table 1 which clearly shows a complete capacity of developmental recovery from the heat shock of the posthatching stages, which sharply contrasts with the absolute lack of such a capacity in the prehatching stages. An important question is whether the synthesis of heat-shock proteins in sea urchin embryos is due to the synthesis of new specific messenger RNAs. In a preliminary attempt to answer such a question, we have conducted the heat shock of Paracentrotus gastrulas in the presence of actinomycin D. The results, reported in Fig. 5, show that actinomycin at 12 and 25, and especially at 50~g/ml, markedly reduces the synthesis of the heatshock proteins, whereas, as expected, the synthesis of the bulk proteins at normal temperature is little affected by the presence of actinomycin D. DISCUSSION
The phenomenon of the production of heat-shock proteins in Drosophila salivary glands, as well as in other materials (Ashburner and Bonner, 1979), has raised the question of whether or not these proteins have a function possibly in the defense against high temperature. The fact that sea urchin embryos also produce such proteins gives some more support to this possibility. It would be interesting to know if the 70,000-dalton proteins produced by sea urchins are the same as those produced by Drosophila. Since plasmids containing the heat-shock protein genes of Drosophila are available, experiments are in progress aimed to ascertain this hypothesis. These experiments might also provide an answer as to whether the synthesis of heat-shock proteins in sea urchins is operated through the start of the synthesis or the increase of the synthesis of specific messenger RNAs. The actinomycin experiments re-
L FIG. 5. Effect of actinomycin D on the production of heat-shock proteins.
VOLUME83, 1981
ported in this paper already speak in favor of such a hypothesis, but they do not distinguish whether the inhibitory effect of actinomycin on the production of heatshock proteins, is exerted by directly inhibiting the production of the messenger RNA coding for heat-shock proteins, or through the inhibition of some other RNA which in turn is necessary for the synthesis of heatshock proteins. These experiments also demonstrate that neither RNA synthesis nor heat-shock protein synthesis are needed for the inhibition of the overall protein synthesis by heat shock to occur. That heat-shock protein production is not needed for the inhibition of the overall protein synthesis by heat is also suggested by the experiments of heating at prehatching stages. That the production of heat-shock protein does not have a trivial explanation, like that of denaturation of proteins that are produced at normal temperature, is already suggested by experiments of heating the embryos at prehatching stages in which the heat-shock proteins do not appear, and is demonstrated by experiments in which gastrulas have been exposed to radioactive leucine at normal temperature and heated at 31~ only after homogenization. In this case the electrofluorograms are absolutely identical to those of normal embryos, and no heat-shock proteins are observed. The different sensitivity of embryos in pre- or posthatching stages to the heat shock is very intriguing because it inversely correlates with the ability to produce heat-shock proteins, thus suggesting again a role for these proteins in the protection of the cells from high temperatures. In agreement with such a hypothesis, McAlister and Finkelstein (1980) have recently described a good correlation between resistance to heat of yeast cells and cellular level of heat-shock proteins. Moreover, Drosophila embryos, if heated at any stage preceding the migration of the nuclei to the periphery of the egg, fail to produce heat-shock proteins and stop cleaving, whereas if heated at later stages, produce heat-shock proteins and continue developing (Graziosi, et al., 1980). How do the heat-shock proteins protect the sea urchin embryos? This is not known at present. What we can say is that it is enough of a wave of production of heatshock proteins for these to produce their effect: in posthatching stages in fact the pattern of protein synthesis reverts to normality after a few hours of heating. It is interesting to recall here that sea urchin embryos are also able to develop until the hatching blastula stage in the absence of synthesis of new messenger RNAs, whereas this synthesis is necessary in order for development to proceed beyond this stage (Giudice et al., 1968; Giudice, 1973). How the latter observations correlate with the ability to produce heat-shock proteins is still a matter of speculation; the suggestion can be
BRIEF NOTES
made, however, that some genes cannot be switched on before certain developmental stages. The expert technical assistance of Mr. D. Cascino is acknowledged. This work was supported in part by the research project on reproduction of the Italian National Research Council, Contract 79.1155.85. REFERENCES ASH~Um~ER,M., and BO~ER, I. I. (1979). The induction of gene activity in Drosophila by heat shock. Cell 17, 241-254. GIUDICE, G. (1973). "Developmental Biology of the Sea Urchin Embryos." Academic Press, New York/London. GIUDICE, G., and MUTOLO,V. (1967). Synthesis of ribosomal RNA during sea urchin development. Biochim. Biophy& Acta 138, 276285. GIUDICE, G., MUTOLO,V., and DONATUTI,G. (1968). Gene expression in sea urchin development. Wilhelm Roux Arch. 161, 118-128. GIUDICE,G., ROCCHERI,M. C., and DI BERNARDO,M. G. (1980). Synthesis of "heat shock" proteins in sea urchin embryos. Cell BioL Int. Rep. 4, 69-74. GRAZIOSI,G., MICALI,F., MARZARI,R., DE CRISTINI,F., and SAVOINI, A. (1980). Variability of response of early Drosophila embryos to heat shock. J. Exp. Zool. 214, 141-145.
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LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ~ and uC in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341. LEWIS, M., HELMSING, P. I., and ASHBURNER, M. (1975). Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc. Nat. Acad, Sci. USA 72, 36043608. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL,R. J. (1951). Protein measurement with the Folin phenol reagen. J. Biol. Chem. 193, 265-2"/5. MCALISTER,L., and FINKELSTEIN,V. (1980). Heat shock proteins and thermal resistance in yeast. Biochem. Biophys. Res. Commun. 93, 819-824. MANS, R. J., and NOVELLI, G. D. (1961). Measurement of the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch. Bioc~m. Biophya 94, 48-53. MCKENZIE,S. L., HENIKOFF,S., and MESELSON,M. (1975). Localization of RNA from heat induced polysomes at puff sites in Drosophila melanogaster. Proc. Nat. Acad, Sci. USA 72, 1117-1121. RITOSSA, F. (1962). A new puffing pattern induced by temperature shock and DNP in Drosophila. E~erientia 18, 571-573. TISSIERES, A., MITCHELL, H. K., and TRAcY,U. M. (1974). Protein synthesis in salivary glands of Drosophila melanogaster: Relations to chromosome puffs. J. MoL Biol. 84, 389-398.