Synthetic activity of isolated rat liver nuclei

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form rrswced

Experimental Cell Research79 (1973) 391-403

SYNTHETIC

ACTIVITY

OF ISOLATED

RAT LIVER NUCLEI

II. Evidence for Protein Formation M. LAVAL and M. BOUTEILLE Institut de Recherches Scient(fiques sur le Cancer, 94800 Villejuif,

France

SUMMARY The incorporation of labelled amino acids into hot TCA-insoluble material was used as a measure of protein elaboration in nuclear fractions and compared with the kinetics and distribution of labelling by means of electronmicroscope autoradiography. The method of isolation and the various controls were designed in order to exclude reasonably that this incorporation could be due to cytoplasmic contamination. The pattern of incorporation was found to be characteristic, as compared with microsomal systems. It was insensitive to RNAse and did not require a cytoplasmic pH 5 fraction or an exogenous energy-yielding system. The localization of the activity in the autoradiographs was not random, but clearly associated with definite regions of the nucleus. The nucleolus was 3 times as radioactive as the rest of the nucleus. These results can be interpreted in favor of the concept that protein synthesis occurs in the nucleus. The theoretical limitations of this conclusion are discussed.

Data which have been presented in the literature suggest that protein synthesis does occur in the nucleus although it is now widely accepted that the main bulk of the nuclear proteins are synthesized in the cytoplasm [14]. Many investigators have reported the ability of isolated nuclei to elaborate proteins in vitro [l-5, 10, 11, 13, 16, 25-30, 34-36, 401. However, the main site of incorporation in the nucleus is still under discussion: nucleolus [6, 17, 24, 401,nuclear ribosomes [12, 23, 371, chromatin [15, 23, 361. Furthermore, there is no agreement about the effect of potential inhibitors, the nature of the proteins synthesized and their biological role. Therefore, the present situation concerning intranuclear protein synthesis seems quite controversial. On the one hand, studies on 26731802

isolated nuclear fractions with biochemical procedures alone can never exclude the presence of cytoplasmic ribosomes in socalled ‘pure’ preparations. On the other hand, autoradiographic studies on whole cells cannot distinguish between the site of synthesis and the site of accumulation of the end product, if one assumesa very rapid migration of proteins from the cytoplasm to the nucleus, which seemsto take place [7]. The only possible way to solve this problem is to carry out high resolution autoradiography on purified nuclear fractions, in parallel with biochemical analysis of their synthesizing capacity. However, one pitfall of this procedure is the possible loss of enzyme systems from the purified nuclei, leading to partial or total loss of synthesis which might naturally take place in situ. Exptl Cell Res 79 (1973)

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M. Lava1 & ikf. Bouteille

In the present study, nuclear fractions of a 0.25 M sucrose. 0.001 M M&I,, 0.002 M notassium phosphate at pk 6.8 and cekrii‘ugation; (4) washing high degree of purity were obtained as for 5 min in 0.14 M NaCI, 0.001 M MgCl,. 0.002 M described in a preceding paper [20] and potassium phosphate at PH 6.4 with-O.<“; Triton x-100. incubated with labelled amino acids. It was The last centrifugation at 1 200 g for 5 min profound that such preparations were able to vided the final nuclear pellet. incorporate amino acids. Because of the apparently complete removal of the outer Control of cytoplasmic contamination nuclear membrane with its attached ribo- Particular attention was paid to the possible presence of cytoplasmic residues between the isolated somes, this is interpreted as favoring the nuclei on the one hand, and of the outer nuclear existence of protein formation by isolated membrane with its attached ribosomes on the other. As electronmicroscopic autoradiography is a technuclei. que which deals with individual cells (or nuclei in However, as pointed out by Goldstein the present case), the contamination can be judged on each nucleus investigated. Each of them was [14] the question is not only whether nuclear photographed in the electron microscope and exfractions are able to elaborate proteins, but amined at an enlargement of 15 000 x . The control of purity was therefore as good as possible at the presif they do, whether this elaboration is ent time. As a special form of cytoplasmic contamspecific and different from the cytoplasmic ination could result from adding a cytoplasmic ‘pH 5’-fraction to the incubation medium [18], a protein synthesis. Therefore, in the present series of experiments with and without this fraction study, our attention was focused on such was carried out, the amount of incorporation was measured and the ultrastructural vizualisation of this characteristic differences. fraction was practised. On the one hand, we have found that the pattern of incorporation is characteristic as Incubation of nuclei for scintillation counting far as inhibitors and energy system re- Nuclei (l-2 rng of nuclear nrotein) were susuended in quirements are concerned. On the other 1 ml ok an kubation medium’ containing 0.1 M NaCl or NHLI. 0.01 M M&I,. 0.005 M mercantohand, the spatial distribution of labelled ethanol, 0.01 -M’Tris HCI, pk -/:5 [13] and incubated proteins throughout the nucleus does not with 1.5 ,&i of a 14C-amino acid mixture containing 15 amino acids. All the experiments were run in seem to be random, but is confined to duplicate. Zero time values were obtained as folcertain areas, as demonstrated by means of lows: samples were stopped immediately after addition of the radioactive amino acic$,with 3 ml of cold quantitative electronmicroscope autoradio- trichloroacetic acid (TCA) contammg 0.002 M nongraphy. Although observations on the distri- radioactive amino acids. The samples for incubation were left in contact with the radioactive amino acids bution of labelled proteins in situ are still at 37°C for various periods of time. The reaction was scarce [7], it is clear that a comparison of the stopped with 3 ml of the same cold 10% TCA and maintained for 30 min at 0°C. Then, the acid-indistribution of radioactivity in isolated nuclei soluble precipitates were centrifuged (3 000 g, 10 min) and the nellets were washed twice with cold 5 % and in nuclei in situ is of great interest. MATERIAL

AND METHODS

Isolation of nuclei Wistar rats, 3-4 months old, after fasting for 15 to 16 h were sacrificed by decapitation. Nuclei were prepared as described in a companion paper [20]. In short, minced livers were submitted to the following stepwise procedure: (1) homogenization in 0.25 M sucrose, 0.005 M MgC&, 0.001 M potassium phosphate at pH 6.8 and centrifugation at 1 200 g; (2) resuspension in 9 vol of 2.2 M sucrose, 0.005 M MgCl,, 0.001 M potassium phosphate at pH 6.8 and centrifugation at 50 000 g for 1 h; (3) resuspension in ExptI Cell Res 79 (1973)

TCA with 0.62 M non-radioactive amino acids, then heated for 20 min at 90°C in 10% TCA, centrifuged and finally washed with ethanol-&her (3: I). Pellets were transferred to Millinore filters (HA 25 0.45 urn) and washed with 5 % TCk The filteis were dried’and placed in counting vials with 10 ml of toluene mixture (toluene, 0.4 % PPO, 0.01 % POPOP). The radioactivity was counted in an Intertechnique liquid scintillation analyzer.

Incubation of nuclei for electron microscopic autoradiography The nuclei were incubated with a tritiated amino acid mixture containing 5 nmoles of *H-arginine, of SH-aspartic acid, of *H-leucine, and of ‘H-lysine in

Isolated nuclei, II 1 ml of incubation medium for 15, 30 and 60 min. In these exueriments. ATP. GTP, and ATP generating system- (pyruvatk kinase, phosphoenolpyruvate) and the ‘pH 5’-fraction obtained according to Hoagland et al. [18] and containing amino acid-activating enzymes and tRNA, were used because when we started the autoradiographic experiments, we had not yet realized that the& fractions were in fact not required. The incubation was stopped by addition of 3 ml of 4% paraformaldehyde in phosphate buffer, with 0.005 M non-radioactive amino acids and icechilling. The suspensions were centrifuged at 3 000 g for 10 min and the pellets were fixed in the same 4% paraformaldehyde for 4 h at O’C, with frequent changes of the fixative. After fixation the pellets were cut into small blocks and washed for 20 h at 4°C in nhosohate buffer with 0.005 M non-radioactive amino acids. The medium was changed frequently. After washing, the specimens were post-fixed for 1 h in osmium tetroxide at room temperature. Ultrathin sections of Epon-embedded material were obtained with a Porter Blum microtome at the same thickness as judged by the medium gold color (approx. 1 200 A) with every effort to reduce variations.

Autoradiography The autoradiographic procedure was the same as in previous studies [7]. Ilford L4 emulsion was applied on single grids using the gold interference colored zone of a platinum loop [15]. After 3 months of exposure, the autoradiographs were developed after 5 min of gold latensification [31, 391in a Phenidon-containing developer [21]. The autoradiograms were stained, after completion of the autoradiographic procedure, with 5 % uranyl acetate for 10 min, then dried, and post-stained with lead citrate for 5 min.

Quantitation For each pulse period, at least 30 (but up to 50) nucleolus-containing nuclei were photographed in the electronmicroscope in sections from three different blocks. The number of grains covering the nucleolus and the extranucleolar area of the nucleus was counted on electronmicrographs. The surface area of each region was calculated using a planimeter (Filotecnica, Milan, Italy). The mean number of grains per unit area (grain density) was plotted against time with the standard error of the mean.

393

isolated rat liver nuclei was used as a measure of total protein synthesis and the kinetics of incorporation were determined (fig. 1). The rate of incorporation of l*C-amino acid mixture increased almost linearly with time during the first 30 min and diminished only slightly in the next 30 min. Therefore, in the following experiments a 30 min incubation was adopted as a measure of incorporation unless otherwise stated. It was established that the phenomenon was a true incorporation [2] by the fact that the labelled amino acids were not lost by exchange with non radioactive molecules. Incorporation of 14Camino acids mixture was allowed to proceed for 30 min. At that time, two samples were precipitated with TCA in order to measurethe 30 min incorporation. In two other samples, a 500-fold excessof 12C-amino acids mixture was added and incubation was continued for another 30 min. The specific activity of the nuclear proteins was found to remain constant after addition of the i2C-amino acid mixture. It follows that the labelled amino acids, once incorporated, cannot be diluted out and that the incorporation into nuclei is essentially irreversible for 30 min at least. Controls

As the incorporation in isolated nuclei is far below that in cytoplasmic cell-free systems, particular attention was paid to controls. The zero time value was obtained as described under Methods and was found Protein determination to be usually 60 cpm/mg of protein. In Protein was determined by the method of Lowry et samples left in contact with radioactive al. [22] with serum albumin as a standard. amino acids at 0°C the labelling was twice this value. When nuclei were incubated at 37°C in the radioactive amino acid solution, RESULTS but diluted with a 500-fold excess of nonAmino acid incorporation radioactive amino acids, the labelling was The incorporation of a mixture of 14C-amino equal to the zero time value as described acids into hot TCA insoluble material by above. In contrast, in the samples treated Expti Cell Res 79 (1973)

394 M. Lava1 & M. Bouteille

100 750 I

. .

500

5c

250.

C

I 15

/

/'

I 30

0.4

60

0.8

1.2

1.6

duration of incubation; ordinate: % of maximum activity. Curve of activity of nuclear fractions after incubation with a 14C-amino acid mixture, as observed by scintillation counting. Fig. 2. Abscissa: nuclear concentration (mg) of protein: ordinate: total radioactivity (cpm). Effect of nuclear concentration upon activity. For each point, the nuclei were incubated in a 1 ml vol with 1.5 &i of W-amino-acid mixture. Fig. 3. Abscissa: duration of incubation of nuclei; ordinate: number of silver grains per 100 pm2 (density). Curve of activity in nuclear fractions after incubation with SH-amino acids, as calculated from electron microscope autoradiograms. Total: density in the whole nucleus; Nu, density in the nucleolus. The curve of total activy is similar to that of fig. 1. The nucleolar density is about 3 times as high as the total density at all times of the incubation. Fig. 1. Abscissa:

3 150

100

50

15'

30

normally for incorporation, the labelling was 8 to 12 times the zero time value, approx. 600 cpm/mg protein. Concentration of nuclei The amount of incorporation was observed to vary with the concentration of the nuclei (fig. 2). However, this variation was not directly proportional. The reason might be that adding more nuclei would alter the general properties of the preparation. It was therefore decided to compare each experiment with a control at the same concentration. Energy system requirements All attempts to increase the labelling by addition of a cytoplasmic pH 5 fraction, of ATP-GTP and/or an ATP generating system Exptl CeN Res 79 (1973)

were unsuccessful, suggesting that the energy necessaryto carry out incorporation of amino acids is generated by a nuclear endogenous energy system. 2,4-Dinitrophenol, an inhibitor of oxidative phosphorylation, showed an inhibitory effect upon the labelling of the nuclei. The incorporation of amino acids was 40 % of the normal value at 30 min when 6 x 1O-4 M DNP was added (table 1) Similarly 8 x 1O-3 M potassium cyanide allowed incorporation only to 10 “/o of the normal value. Potential inhibitors Table 1 shows the effects of inhibitors of protein synthesis at usual concentrations. Chloramphenicol inhibited strongly the incorporation, as in other studies in rat liver nuclei [4, 25, 29, 301,in tumour ascites nuclei

Isolated nuclei. II

[19], and polytene nuclei [16]. Puromycin, which is known to inhibit the transfer of amino acids from amino-acyl-tRNA to polypeptides on ribosomes, depressedmarkedly the incorporation. Cycloheximide reduced the activity to a lesser extent. Streptomycin (150 ,ug/ml) reduced the incorporation. Penicillin in bacteriocidal concentration did not affect incorporation. The addition of actinomycin D (10 y/ml) to the incubation medium had no effect. Therefore, new synthesis of RNA did not seem to be required for the in vitro incorporation of amino acids by nuclei. Enzymes

When nuclei were incubated with labelled amino acids in the presence of RNAse or DNAse or after preincubation with these enzymes, no inhibition of the incorporation was observed (table 1). The labelled product was sensitive to pronase since pronase digestion after precipitation of the acid insoluble material resulted in a complete loss of radioactivity. CytopIasmic contamination

No ribosomal particle or membrane could be seen between the nuclei at the end of the isolation procedure. As pointed out in the preceding paper [20], the nuclei were devoid of the outer nuclear membrane with its attached ribosomes. As a cytoplasmic ‘pH 5’ fraction was added to the incubation medium, an attempt was made to visualize this fraction electronmicroscopically. Only some amorphous residues, devoid of ribosomal particles and membranes, could be seen. It was assumed that this was the result of nuclear disruption, with release of nuclear proteins. To test this hypothesis, a series of experiments was performed without adding the ‘pH 5’ fraction to the incubation medium. Under these conditions, the amount of in-

395

Table 1. Effect of some inhibitors on incorporation ?b Control Inhibitor Chloramphenicol 3 x 1O-4 M Puromycin lo-$ M 1OWM Cycloheximide 1O-3 M Penicillin 100 y/ml Streptomycin 150 y/ml 2-4 Dinitrophenol 6 1O-4M Cyanide 8 lO-3 M Actinomycin D 10 y/ml RNAse 100 y/ml DNAse 100 y/ml

Average

Range

10

7-14

17 65

14-22 60-70

71

74-68

100 65 40

38-41

5 96 92

82-104

110

100-120

corporation was the same as with this fraction, when measured at the scintillation counter. Electronmicroscope autoradiography

The kinetics of labelling of the nuclei was determined by plotting the grain density (number of grains/unit area) against time after various periods of incubation with a mixture of tritiated amino acids. First, the total nuclear density was found to increase with time, so that a saturation curve similar to the curve obtained with scintillation counting was observed (fig. 3). Second, an attempt was made to investigate the distribution of the activity throughout the nucleus in order to make a comparison with the distribution in situ [7]. Unfortunately, the ultrastructural appearance Exptl Cell Res 79 (1973)

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M. Lava1 & M. Bouteille

of the isolated nuclei when suspended in the incubation medium was strikingly different from that of the nuclei in situ, as discussed extensively in a preceding paper [20]. Particularly, the chromatin was observed in a highly condensed state (figs 4-10) so that no distinction could be safely made between heterochromatin and euchromatin. Similarly, the perichromatin region, as defined in another paper [7], could not be considered under the present conditions. Therefore, only the nucleolar and extranucleolar regions were quantitatively investigated. It was observed (fig. 3) that the nucleolar grain density was 3 times as high as the extranucleolar one after 1 h of incubation. The comparison of the curves made also clear that there was no delay in the nucleolar labelling as compared with the extranucleolar labelling. Although no further quantitative evaluation could be made it was observed by mere examination of the autoradiograms that some grains fell on the chromatin as well as on the extra chromatin regions (figs 4-10).

DISCUSSION The problem of protein synthesis in situ

It is realized that although incorporation is a necessary condition, it is not a demonstration of protein synthesis by itself. The possibilities of elongation of chains migrating from the cytoplasm, or of synthesis of small peptides, which have been ruled out in complete cells [9], cannot be excluded in cell subfractions. In addition, it is possible that ribosomal ribonucleoprotein precursors are able to incorporate amino acids in the rather artificial conditions of incubation, and this cannot be tested with the techniques available at the present time. Finally; it Exptl Cell Res 79 (1973)

remains to be demonstrated that the mechanism of nuclear protein synthesis occurs, not only in nuclear fractions, but in nuclei in situ when nucleocytoplasmic interactions are fully expressed. Therefore, it does not necessarily follow from the amino acid incorporation into isolated nuclei, that protein synthesis is a function of the nucleus in situ. However, our results, taken together with the data of the literature must be interpreted as evidence for labelled amino acid incorporation by isolated nuclei which are as free of cytoplasmic contamination as possible. No contamination occurred under the form of cytoplasmic residues, recognizable as such, between the nuclei, nor of the outer nuclear membrane with its attached ribosomes. This was ascertained by electronmicroscopic autoradiography, since this technique has the advantage of providing controls for each nucleus investigated. The control was not only made on the screen of the electron microscope, but on pictures at 15 000 magnification. Furthermore, the technique allows to test whether the silver grains are associated with the nuclei and not with extranuclear material. The possibility that ribosomal particles or subunits are adsorbed on to the nuclei during the isolation procedure has been excluded by Anderson et al. [4]. They have shown that prelabelled microsomes, when added to the homogenate, were not found to be associated with the nuclei. Our isolation procedure is similar. It can therefore be ascertained that in our system, the incorporation is not related with a cytoplasmic component, as judged from the most severecontrols available at the present time. As pointed out by Goldstein [14] more information is needed in order to establish the presence of protein synthesis in the nucleus itself. One approach is to analyse the proteins elaborated by the nuclear frac-

Isolated nuclei. II

397

Fig. 4. Low magnification of an autoradiogram, incubation for 30 min. All the activity is associated with nuclei in diametral or tangential arrows section. Some nucleoli (Nu) are more densely labelled than the surrounding nucleoplasm. Exptl Cell Res 79 (1973)

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Figs 5, 6. Incubation for 15 min. There is already a significant labelling of the nuclei. Exptl Cell Res 79 (1973)

Isolated nuclei. II

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Figs 7, 8. Incubation for 30 min. The activity has increased and the higher activity in the nucleolus becomes conspicuous. Exptl Cell Res 79 (1973)

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M. Lava1 & M. Bouteille

tions. This is under investigation in several laboratories [3, 4, 13, 401, and generally it is possible to demonstrate that the labelled nuclear proteins have a different polyacrylamide gel electrophoretic pattern than the microsomal proteins. A second point would be the demonstration of a different pattern of incorporation. A third one would be to establish that the localization of the elaborated proteins in the nucleus is also different. These two last points are discussed below.

Pattern of incorporation

The study of the effect of various inhibitors enables us to establish some specificity in their action. First, RNAse does not affect the incorporation. This would mean that the RNA concerned is insensitive or not accessible to RNAse. Second, neither the cytoplasmic ‘pH 5’-fraction, nor an exogenous energy-yielding system are required. These data are contradictory with some studies [13], but in agreement with the majority of the investigations [l-4, 16, 19, 25, 27-29, 30, 33, 35, 401. Therefore, there is no need for an exogenous energy system nor for the cytoplasmic enzyme system, which suggeststhat the nucleus has its own oxidative phosphorylation [I, 21 and the specific amino-acyl-tRNA synthetases. This is in agreement with some studies on the synthetic activity of nuclear subfractions [23]. In this respect, it is worth mentioning that puromycin inhibited the nuclear incorporation at higher concentrations than in cytoplasmic cell-free system. This seems to indicate a different accessibility of the inhibitor to the synthetic system. Although these points deserve more investigation, they suggest on the whole that the pattern of incorporation in nuclei may be different from that of cytoplasm. Exptl Cell Res 79 (1973)

Distribution

of the activity

The distribution of the activity throughout the nucleus can easily be investigated by means of electronmicroscope autoradiography. First of all it is interesting to observe that the kinetics of incorporation into the nuclei, as judged from the number of grains per unit area, is similar to the kinetics obtained by scintillation counting, This confirms that the incorporation is really associated with the individual nuclei, and not only with the nuclear fraction as a whole. The hypothesis of nuclear protein elaboration is supported by the fact that the distribution of radioactivity is not random, but localized in definite parts of the nucleus. This has not been reported previously, and argues strongly for the concept that the incorporation of amino acids into the nuclear fractions does not result from adsorption, but from a true biological and active process. The most striking incorporation is found to occur in the nucleolus, which is 3 times as active as the rest of the nucleus. This confirms the biochemical studies in which most of the activity was observed in the nucleolus [6, 8, 17, 19, 24, 26, 401as opposed to some other investigations [16, 25, 381. It is now known that a large amount of the cytoplasmic proteins which migrate into the nucleus is to be found in the nucleolus (see [7]). The present results suggest that the nucleolus is also able to incorporate amino acids. In this respect it is noteworthy that no delay is observed between the nuclear and the nucleolar labelling. On the contrary, nucleolar labelling occurs earlier and at a higher rate than the extranucleolar labelling. A nucleolar density comparable to the peak of extranucleolar activity is obtained 45 min earlier. This precludes a migration into an extranucleolar pool prior to migration into the nucleolar

Isolated nuclei. II

401

Figs 9,10. Incubation for 60 min. The activity has not increased significantly in the nucleoplasm, but the nut deoli are more heavily labelled than after 30 min. Exptl Cell Res 79 (i ‘9; 73)

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M. Lava1 & M. Bouteille

one. On the contrary, it argues for the idea that the nucleolus is responsible for the bulk of the nuclear labelling. However, this phenomenon might reflect only a difference in the rate of protein turnover which could only be investigated with pulse-chase experiments. The rest of the activity is observed in the extra-nucleolar region. In a previous study, we described a pattern of localization in the chromatin, perichromatin, and interchromatin regions of the nucleus after labelling of whole cells [7]. We expected to find a quite different distribution in isolated nuclei. This would have brought some additional evidence for nuclear protein synthesis. Unfortunately, the various regions of the isolated and incubated nuclei could not be identified as easily as in intact nuclei in situ. In addition, as discussed extensively in the companion paper [20], the ultrastructural definition of these regions might not be reliable under the conditions of isolation and incubation. Therefore, no quantitative investigation was attempted, although the extranucleolar activity seemsto be associated rather with the perichromatin region than with the chromatin itself (figs 4, 6). Nevertheless this method could be of interest for experiments where the activity in various parts of the nucleus is to be compared with the activity in various biochemical fractions, such as histones, acidic proteins, etc., or when the incorporation under different stimulations, hormonal for instance [3, 321 is investigated. The authors wish to express their gratitude to Drs W. Bernhard, B. Droz and J. P. Zalta for helpful suggestions, to A. Bernadac and M. J. Burglen for their help in performing the experiments, and to Ch. Taligault for preparation of the manuscript.

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Exptl Cell Res 79 (1973)