Protein synthesis in Artemia salina embryos

DEVELOPMENTAL

Protein

BIOLOGY

17,

644-656

Synthesis

( 1968)

in Artemia

I. Studies

S&KY

Embryos

on Polyribosomesl

ALLYN GOLUB AND J. S. CLEGG Laboratory for Quantitative University of Miami, Accepted

Biology, Department Coral Gables, Florida January

of Biology, 33124

8, 1968

INTRODUCTION

During embryonic development of the crustacean Artemiu salina an encysted gastrula is produced ( Fautrez-Firlefyn, 1951) which undergoes essentially complete desiccation, resulting in the cessation of metabolism and development in the embryo (Clegg, 1967). Upon proper rehydration, metabolism is rapidly resumed (Muramatsu, 1960; Emerson, 1963; Clegg, 1964) and the encysted gastrula eventually differentiates into a partially formed nauplius larva (prenauplius) which emerges from the cyst. A remarkable feature of these embryos is that no cell division occurs throughout this period of preemergence development (Nakanishi et al., 1962). Presumably, the cells of the gastrula undergo appropriate rearrangement and differentiation as the prenauplius is formed during preemergence development. Consequently, the Atiemiu embryo provides a system in which the relationship between protein synthesis and differentiation can be examined in a metazoan embryo without the added complexity of concomitant cell division. Therefore, we have undertaken a study of protein synthesis in Artemia embryos. Previous work has shown that protein synthesis occurs throughout the period of preemergence development as judged from incorporation of ‘“CO, (Clegg, 1966). Because these embryos are impermeable to amino acids, we have utilized cell-free systems for further study. The ‘Supported by grant HD 03478 from the U.S. Public Health Service; A. G. received financial assistance from a NASA grant (NGR 10-007-010) as a predoctoral student. 644

I’OLYRIBOSOMES

IN

li?-t~?7Iil~

645

results presented here will show that protein synthesis occurring during preemergence development is carried out on conventional l~olyrihosomes, and that polyribosome formation occurs rapidly after dormancy ends in the encysted gastrula. hlATEHIALS

AND

hlETIIODS

Sowce and treatment of Artemia embryos. Dried encysted gastrular (brine shrimp eggs) of the Great Salt Lake variety were obtained from Longlife Fishfood Products, Harrison, New Jersey and stored at -20°C. Prior to use the embryos were hydrated and washed with i excess 0.5 M NaCl for 3 hours at 0°C. The embryos were then treated with excess ice-cold 5% sodium hypochlorite for 15-20 minutes with constant stirring and then washed repeatedly with ice-cold 0.5 M NaCl to remove hypochlorite. This treatment, like that used by Nakanishi et (11. (1962). removes the granular part of the shell and renders the preparation aseptic without damaging the embryos. If the temperature is maintained at 0°C during these operations, the embryos become fully hydrated but remain dormant. lL&ation jvroceduws. Dormancy was terminated by incubating these hydrated but still dormant gastrulae at 30°C in a metabolic, incubator with constant agitation to ensure adequate oxygen supply. Emergence of prenauplius larvae began in these populations after about 7 hours of incubation and about 70% of the embryos produced swimming nauplii after a total incubation period of 24 hours. The experiments to be described in the present paper involve the use of embryos incubated for 4 hours or less and, therefore, deal with populations of embryos that are in the Process of preemergence development, Hicarbon~lte-free artificial seawater (Robertson and \Vebb, 1939) was used as the incubation medium which was sterilized when the embrvon were to be incubated with NaH”C0,. In that case the flask was Hushed with oxygen, the NaH’CO,, was rapidly added to a final concentration of 10 hLC/ml, and the flasks were tightly stoppered. After incubation the embryos were collected on cloth filter supports ( Schleichrr and Schuell Co., No. 123), washed well with ice-cold distilled lvater. blotted free of excess fluid, weighed, and transferred to homogenizers. If the embryos were to be analyzed for incorporation of labeled bicarbonate, they were thoroughly homogenized with 5% trichloroacetic acid and the protein fraction was obtained as described previouslv ( Clegg, 1966). For sucrose gradient studies, the embrvos

646

GOLUB AND CLEGG

were placed into homogenizers containing ice-cold homogenizing medium and housed in ice. Preparation of extracts. Unless stated otherwise, the embryos were homogenized (lo-12 gentle passes) in all-glass Dounce tissue grinders equipped with large-clearance pestles ( Kontes). The homogenizing buffer consisted of 0.05 M Tris-HCl pH 7.8, 0.01 M MgCl, and 0.1 it4 KC1 (TMK), and 200 mg wet weight of embryos were homogenized per milliliter of this buffer. These operations were carried out between 0 and 4°C. Homogenates were centrifuged at 14,000 g for 30 minutes and the supernatants were carefully removed for layering on sucrose gradients, or for further centrifugation to pellet ribosomes and polyribosomes (100 minutes at 40,000 rpm in a Spinco 40.3 rotor at 4°C in a Model L-2). Cell-free amino acid incorporation. The pellet from high speed centrifugation was gently resuspended in one-fourth the original volume of TMK (containing 0.001 M 2-mercaptoethanol) and 0.1 ml of this preparation (about 0.5 mg of protein) was added to tubes containing the following reaction mixture: 7 pmoles of MgCl,; 28 pmoles of KCl; 48 pmoles of Tris-HCI, pH 7.8; 3 pmoles of 2-mercaptoethanol; 1 pmole of GTP, 1.5 pmole of ATP, 8 pmoles of creatine phosphate, and 50 pg of creatine kinase as the ATP-generating system; 50 ,ug of yeast sRNA; 0.6 Kpmoles of uniformly labeled L-leucine-W (0.15 PC), and 0.02 ml of the high-speed supernatant, in a total volume of 0.7 ml. Complete reaction mixtures, minus ribosomes, were incubated as controls for incorporation not due to ribosomes which were less than 25 cpm. Further details on the incorporating system will be presented in the second paper in this series. After incubation at 30°C an aliquot of the reaction mixture was layered on sucrose gradients. In some cases the mixture was treated with ribonuclease (either 1 or 5 pg/ml for 30 minutes at 0°C) prior to layering on the gradients. Sucrose density gradient fractionation. The 14,000 g supernatant, or reaction mixture described above, was layered onto linear sucrose gradients (1550%) p re p ared with TMK using a Technicon autoanalyzer pump. The tubes were spun in a Spinco SW 41 rotor for 80 minutes at 39,000 rpm and 4°C. Gradients were analyzed by means of an Isco gradient fractionator with continuous recording of absorbancy at 254 mp, and 1 ml fractions were collected into tubes containing 1 ml of 10%trichloroacetic acid for determination of radioactivity. The tubes

POLYRIBOSOMES

IN

647

Artemiu

were heated for 20 minutes at 95°C and the contents were transferred onto Whatman GF/C glass paper pads (2.4 cm) and washed with about 100 ml of 5% trichloroacetic acid containing 0.01 M leucine-l’C followed by 30 ml of 95% ethanol, The dried pads were glued to planchets, and radioactivity was determined with a low-background G-M counter (1.6 cpm). Source of chemicals. Uniformly labeled L-leucine-‘C (248 mC/ were obtained from New mmole) and NaHl’CO, (4.1 mC/mmole) England Nuclear Corp. and ribonuclease (Hirs component A) from Calbiochem. Sucrose ( ribonuclease-free) and sodium deoxycholate were purchased from Mann Research Laboratories. Yeast sRNA was purchased from Schwartz BioResearch, and the sodium salts of GTP, ATP. and creatine kinase from Sigma Chemical Corp. RESULTS Onset of Protein Synthesis during the Transition Dormancy to Metabolism

from

Studies on protein synthesis in these embryos are greatly hampered by their impermeability to appropriate labeled precursors and inhibitors. However, some information can be obtained by the use of 1”CO, (Clegg, 1966), and we have examined the onset of protein synthesis gastrulae were in intact embryos on this basis. Hydrated-dormant incubated with NaH%O, at 30°C for varying periods of time and the total protein fraction was obtained and analyzed for radioactivity (Fig. 1) . These results show that incorporation into protein occurs within the first 15 minutes of incubation. Unfortunately, it is not possible to do the appropriate “pulse” experiments to evaluate critically whether or not there is a lag in the onset of protein synthesis following dormancy. However, if such a lag exists, it clearly is not of long duration. We have examined this question further by the use of sucrose gradients. Polyribosome

Formation

after Dormancy

Figure 2 shows the absorbancy profiles of sucrose gradients using 14,000 g supematants. Extracts of hydrated-dormant gastrulae do not show typical polyribosome peaks (Fig. 2a). However, some ultravioletabsorbing material, which is sensitive to ribonuclease (5 pg/ml for 30 minutes at O(C), sediments below the ribosomes (Fig. 2b). Conceiv-

648

GOLUB AND CLEGG

3oa

z 2

200,

INCUBATION

PERIOD

(min)

FIG. 1. Incorporation of radioactivity from NaHl*CO, into protein by hydrated-dormant gastrulae when incubated at 30°C in bicarbonate-free sterile seawater. Each point represents the average of 5 experiments, and the vertical lines show standard deviations.

ably, this material might consist of polyribosomes. It should also be pointed out that the amount of this material is small, and close to the limits of resolution of our system. When hydrated dormant gastrulae are incubated at 30°C, polyribosome peaks appear (Fig. 2c), and as incubation proceeds (Fig. 2d,e), the amount of polyribosomes increases. The resolution of discrete polyribosome peaks is quite good, particularly in preparations from 4-hour embryos (Fig. 2e). We have examined the onset of polyribosome formation more carefully, and the results are shown in Fig. 3. After only 5 minutes of

. h.S

e

FIG. 2. Sucrose gradient profiles of 14,000 g supernatants obtained from (a) hydrated dormant and aftw their incubation at 30’~; for (c) 30 minutes: (d) 2 hours; and (e) 4 hours. gastrulae, from hydrated-dormant ‘IlIe profile shown in part ( b) \vas obtained lw treatment of the suprmatant gastrlllae \vitll rikonuclraw ( j ,‘g/ml for SO minutes at 0°C) prior to layering 011 the gradirnt. The tops of the gradients are at the left.

.5a

b

650

GOLUB

AND

CLEGG

0.2 1a 0.4.

FIG. 3. The onset of polyribosome formation following dormancy in hydrateddormant gastrulae. The conditions are the same as those given in Fig. 2 except for the duration of incubation at 30% prior to homogenization for (a) 5 minutes, (b) 10 minutes; (c) 15 minutes; (d) 30 minutes. The tops of the gradients are at the left; the tops of the 81 S ribosome peaks are not shown.

incubation, small polyribosomes can be detected (Fig. 3a). It is particularly interesting that the increase in polyribosomes that occurs as incubation proceeds (Fig. 3b-d) is due chiefly to an increase in heavier polyribosomes. Treatment of all these preparations (Figs. 2 and 3) with ribonuclease (1 @g/ml for 30 minutes at 0%) results in the essentially complete removal of these polyribosomes, producing a profile like that shown in Fig. 2b. In addition to polyribosomes, and soluble material located at the top of the gradient, these preparations also contain large amounts of single ribosomes (about 81 S) and two lighter particles (about 61 S and 36 S ) . Sedimentation coefficients for these particles were estimated by comparing their distance of sedimentation with that of Escherichia coli ribosomes (70 S ). Obviously, the values for Artemia preparations should be viewed as preliminary. We suspect that the “61 s” and “36 S” particles are ribosomal subunits since their aborption spectra are typical of nucleoproteins, and since the use of low Mg2+ concentration (0.1 mM) results in their formation from “81 s” ribosomes. Amino Acid Incorporation

on Polyribosomes in Vitro

Having established that the intact encysted gastrula begins to synthesize proteins within the first 15 minutes of metabolism at 30”

POLYRIBOSOMES

IN

651

Artemia

(Fig. 1 ), and that polyribosome formation occurs even more rapidly (Fig. 3), we next examined the ability of these polyribosomes to incorporate amino acids. High-speed pellets obtained from embryos which had undergone 4 hours of incubation at 30°C were incubated in vitro with leucine-“C

ID MIN.

10 MIN.+

WI I u

RNaSe

0 30MIN.

-90 .60

M L.

FIG. 4. Incorporation of “C-1eucine into hot trichloroacetic acid insoluble material by polyribosomes in z;itro for (a) 10 minutes; (b) 10 minutes followed by treatment with 1 pg of ribonuclease per milliliter for 30 minutes at O’C; (c) 30 minutes. The high-speed pellet was obtained from 4-hour embryos. The tops of the gradients are at the left.

as described, and after a designated period the reaction mixtures were layered on sucrose gradients and analyzed as usual. Figure 4 illustrates a typical result of such experiments. After incorporation in vitro for 10 minutes (Fig. 4a) most of the incorporated leucine is associated with the polyribosomes, and virtually all this radioactivity is displaced to the 81 S peak by mild ribonuclease treatment (1 pg/ml

652

GOLUB AND CLEGG

for 30 minutes at 0°C) as illustrated in Fig. 4b. The profile shown in Fig. 4c is the result of a 30-minute period in uitro. The observed increase in radioactivity associated with the “81 S” ribosomes relative to polyribosomes might result from polyribosome breakdown. Although some loss of absorbance in the polyribosome region can be seen between 10 and 30 minutes, it is clear that polyribosome breakdown occurring under these conditions is not extensive. We have some evidence that the heavy material not removed by ribonuclease treatment (Fig. 4b) is glycogen, which is present in large amounts in the pellet obtained by high-speed centrifugation. Using the methods outlined above we have also examined embryos incubated for 8 hours at 30°C prior to analysis. Although more polyribosomes were present, and more leucine-14C was incorporated, the results with S-hour embryos were essentially the same as those for 4-hour embryos (Fig. 4). Experiments of this type carried out with preparations from hydrated dormant gastrulae have not, thus far, yielded repeatable results, and their presentation would seem premature at the present time. Evaluation

of Methods Used for Polyribosome

Analysis

The data we have presented show that our methods are adequate for demonstrating functional polyribosomes in developing Artemia embryos. These methods were arrived at through an examination of those parameters known to affect polyribosomes: disruption procedure, ionic composition of the homogenizing medium (particularly Mg”) and involvement of endogenous ribonuclease activity. The method of choice for disrupting these embryos involves the use of Dounce homogenizers and pretreatment of the embryos with 5% sodium hypochlorite. However, even when the granular part of the shell is not removed and the embryos are severely sheared in closefitting Ten Broeck homogenizers, polyribosomes can still be observed. Figure 5 compares this severe type of disruption (a) with the gentle method usually utilized in our experiments (b) . Although severe disruption clearly results in extensive breakdown of heavy polyribosomes with concomitant increase in lighter ones, the results show that a substantial number of polyribosomes still remain. Consequently, we do not believe that the lack of typical polyribosome peaks in dormant gastrulae is the result of disruption. Relative to the concentrations of other ions present in TMK (and

POLYRIBOSOhiES

Iii Artemiu

65.3

FIG. 5. Comparison of (a) severe disruption with (b) mild disruption on sucrose density gradient profiles using l&O@0 g supernatants from d-hour embryos. The tops of the gradients are at the left.

derived from the embryos during extraction) a Mg” concentration ot 10 mhl has been found to be optimal for the demonstration of polyribosomes and ribosomes. The “81 S” ribosomes begin to dissociate in 1 miZl Mg”, and polyribosome breakdown and ribosome dissociation is complete in 0.1 mRi Mg”. Although we have not extensively examined the involvement of other ions, our results show that fluctuations in KC1 from 0.05 &I to 0.2 M (in TMK) do not alter the absorbancy profiles. Fortunately, endogenous ribonuclease activity is not a problem in Artemia preparations, as illustrated bv the stability of our in ,vitro incorporating system (Fig. 4). We ha\~, also examined the effects of commonly used ribonuclease inhibitors and feel their use in Artemiu preparations to be more of a liability than an asset. Thus, polyvinyl sulfate at a concentration of 0.1 mg/ml causes dissociation of “81 S” ribosomes, bentonite (1%) results in a considerable reduction in the yield of ribosomes and polyribosomes, and macaloid ( 1%) has little, if any, effect. We have also examined the possibility that polyribosomes were being lost in the initial centrifugation (14,000 g) by treating the homogenates with various concentrations of sodium deoxycholate (DOC ). The results of DOC treatment are interesting and complex in Artemiu preparations and will be reported in detail in a future publication. Suffice it to say here that although a small increase in yield of polyribosomes has been observed in developing embryo preparations, DOC treatment (0.5%) of hydrated-dormant embryo homogenates or 14,000 g supernatants caused extensive dissociation of “81 S” ribosomes.

654

GOLUB

AND

CLEGG

DISCUSSION

The Onset of Protein Synthesis after Dormancy The onset of general metabolic activity after the end of dormancy in encysted gastrulae is known to be extremely rapid as judged from oxygen consumption measurements (Muramatsu, 1966; Emerson, 1963; Clegg, 1964). Although the use of %O., cannot accurately determine the precise time at which protein synthesis begins, our results (Fig. 1) indicate that if a lag does exist in the onset of this activity, its duration is probably much shorter than 15 minutes. This is based on the fact that 14C0, must first be incorporated into amino acids [chiefly aspartic and glutamic (Clegg, 1966) ] and the specific activity of these must be quite low initially due to the relatively large pool of free amino acids in these embryos (Emerson, 1967). As a result, the occurrence of protein synthesis can be detected only after sufficient time has passed to allow the accumulation of enough radioactivity in the amino acid pool. Although we have not yet determined whether or not a lag exists, this question is not trivial since its answer will bear directly on the nature and mechanism of polyribosome formation in the gastrula immediately after dormancy ends. The Onset of Polyribosome

Formation

after

dormancy

We have been able to detect polyribosome formation within the first 5 minutes of incubation after the resumption of metabolic activity in the encysted gastrula (Fig. 3). The polyribosomes formed initially are, for the most part, small ones, probably composed of 5 or fewer ribosomes (Fig. 3a). These may be produced by the breakdown of larger polyribosomes, but since we have shown that our technique is adequate for the retention of large polyribosomes (Fig. 5b), this does not seem likely. Rather, we suggest that the initial formation of polyribosomes following dormancy might involve a sequential attachment of ribosomes to what must be assumed to be messenger RNA. This suggestion is supported by the progressive increase in the amount of heavy polyribosomes, relative to light ones, that occurs as incubation proceeds (Fig. 3b-d). Th e i d ea of sequential attachment of ribosomes to messenger RNA is not a new one, and has been supported by several lines of evidence in other systems (Yanofsky and Ito, 1966; Schaecter and McQuillen, 1966; Dresden and Hoagland, 1967). Based on in Vitro incorporation of leucine-14C the general nature of

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polyribosome function in preparations from developing embryos appears to be conventional (Fig. 4). We have examined the in vitro incorporating system in some detail and consider it further in the next paper of this series. However, a point worthy of notice here is the apparent stability of Artemia polyribosomes under in vitro amino acid incorporating conditions ( Fig. 4). Th is will permit more detailed study on the polyribosomes of Artemia in vitro. On the Stutus of Polyrihosomes

in H:gdrated Dormant

Gastrulae

We have not conclusively answered the question whether or not polyribosomes are present in extracts of hydrated dormant gastrulae. However, we have detected a small amount of ribonuclease-sensitive material sedimenting below the ribosomes (Fig. 2a,b), and it is possible that this material actually consists of polyribosomes. If such is the case, however, they must differ from those polyribosomes we have detected as discrete peaks in embryos which have metabolized for only 5 minutes ( Fig. 3a). LVe are currently making a detailed examination of the dormant gastrula in the hope of clarifying this situation and extending our current knowledge of the onset of polyribosome formation following dormancy. SUMMARY

A study of polyribosomes from encysted embryos of the crustacean Artemia salina has been carried out by sucrose density gradient centrifugation. Although no discrete polyribosome peaks were detected in extracts from hydrated-dormant gastrulae, a small amount of ribonuclease-sensitive material sedimenting below the ribosomes was present. Formation of small polyribosomes has been detected within 5 minutes after the dormant gastrulae were permitted to resume metabolism and development by incubation at 30°C. The increase in polyribosome content which occurred with further incubation appeared to result chiefly from the formation of heavier polyribosomes. Using NaH”CO,, as a precursor, protein synthesis has been detected in intact gastrulae within the first 15 minutes after the end of dormancy. Polyribosomes were shown to be functional by their in vitro incorporation of leucine-lC into hot trichloroacetic acid-insoluble material. An evaluation has been made of the method used for the study of polyribosomes in Artemia.

656 Note added in poof: related paper on Artemia (1968).

GOLUB AND CLEGG

Since this manuscript was accepted for publication a ribosomes has been published by Hultin and Morris REFERENCES

CLEGG, J. S. ( 1964). The control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cysts of Artemia salina. J. Exptl. Biol. 41, 871-892. CLEGG, J. S. ( 1966). Protein synthesis in the absence of cell division during the development of Artemia salinu embryos. Nature 212, 517-519. CLEGG, J. S. ( 1967). Metabolic studies of cryptobiosis in encysted embryos of Artemia s&w. Comp. Biochem. Playsiol. 20, 801-809. DRESDEN, M. H., and HOAGLAND, M. B. ( 1967). Polyribosomes of Escherichia coli. Re-formation during recovery from glucose starvation. J. Biol. Chem. 242, 1069-1073. EMERSON, D. N. ( 1963). The metabolism of hatching embryos of the brine shrimp, ATtemiu salina. Proc. S. Dakota Acad. Sci. 42, 131-135. EMERSON, D. N. ( 1967). Some aspects of free amino acid metabolism in developing encysted embryos of Artemiu saline, the brine shrimp. Comp. Biochem. Physiol. 20, 245-261. FAUTREZ-FIRLEFYN, N. ( 1951). Etude cytochimique des acides nuclkiques au tours de la gam&og&Gse et des premiers stades du developpement embryonnaire chez Artemiu salinu C. Arch. Bid. 62,391-438. HULTIN, T., and MORRIS, J. E. ( 1968). The ribosomes of encysted embryos of Artemiu sulina during cryptobiosis and the resumption of development. Develop. Biol. 17, 143-164. MURAMATSU, S. ( 1960). Studies on the physiology of Artemia embryos. I. Respiration and its main substrate during early development. Embryologia 5, 95-106. NAKANISHI, Y. H., IWASAKI, T., OKIGAKI, T., and KATO, H. (1962). Cytological studies of Artemia sulinu. I. Embryonic development without cell multiplication after the blastula stage in encysted dry eggs. Annotationes Zool. Japan. 35, 223-228. ROBERTSON, J. D., and WEBB, D. A. ( 1939). The micro-estimation of sodium, potassium, calcium, magnesium, chloride, and sulphate in sea water and the body fluids of marine animals. J. Exptl. Biol. 186, 155-177. SCHAECTER, M., and MCQUILLEN, K. (1966). Synthesis of messenger RNA and the assembly of polysomes in Bacillus megaterium infected with bacteriophage. J. Mol. Biol. 22, 22%23X YANOFSKY, C., and ITO, J, ( 1966). Nonsense codons and polarity in the tryptophan operon. 1. Mol. Biol. 21, 313-334.