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Synthesis and accumulation of nuclear and cytoplasmic proteins during interphase in mouse fibroblasts in vitro
Experimental
500
Cell Research 42, 500-511 (1966)
SYNTHESIS AND ACCUMULATION OF NUCLEAR AND CYTOPLASMIC PROTEINS DURING INTERPHASE IN MOUSE FIBROBLASTS
IN VITRO A. ZETTERBERG Institute for Cell Research, Karolinska
Institutet,
Stockholm, Sweden
Received November 22, 1965
previous studies, the growth of mouse tlbroblasts in vitro (cell line L-929) during interphase was investigated using cytophotometric [12, 281 and autoradiographic [29] techniques in combination with time lapse cinematography. The results obtained with these techniques showed that protein was synthesized and accumulated in the cell during the entire period of interphase. In these investigations, the whole cell was considered as a unit and no distinction was made between the cell nucleus and the cytoplasm. There are, however, reasons to believe that the synthesis and accumulation of protein in the cell nucleus differs from that in the cytoplasm during interphase growth. For example, in Paramecium aurelia the nuclear dry mass was shown to increase only during a part of the interphase [ 151 and in that respect differing from the dry mass increase of the cytoplasm [14]. It has also been shown that, in some mammalian cell lines in vitro, the predominant increase of the nuclear mass was confined to the second part of the interphase period [24], thus, contrasting to that of the whole cell [12, 281. This suggests that differences may also exist between the patterns of protein accumulation in the cell nucleus and in the cytoplasm of mammalian cells during interphase. The accumulation of protein material in the cell nucleus and the cytoplasm need not necessarily reflect a true synthesis in situ. Protein may be synthesized in either one of these two cell compartments and thereafter migrate to the other. In Amoeba proteus evidence of migration of protein from the site of synthesis in the cytoplasm to the cell nucleus has been reported [2, 31. However, it is not yet known whether this migration is of any quantitative significance for the accumulation of protein in the cell nucleus. The aim of the present investigation was to study simultaneously both the synthesis and the accumulation of protein in the cytoplasm and cell nucleus of growing L-cells in order to obtain more information about the interaction IN
Experimental
Cell Research 42
Nuclear
and cytoplasmic
proteins
during
interphase
in fibroblasts
501
between the cell nucleus and the cytoplasm during interphase growth. For this purpose a combination of cytophotometric, autoradiographic and time lapse cinematographic techniques was employed.
MATERIALS
AND
METHODS
Cell culture techniques-Mouse fibroblasts (cell line L-929) were cultivated directly on quartz slides as described previously [12]. The multiplying cell populations were photographed by time lapse cinematography every 2 min for about 40 hr under low power magnification. At the end of this period, the slide cultures were fixed in a mixture of absolute ethanol and acetone (1: 1) at 4°C for 24 hr before being mounted in redistilled glycerol (n = 1.455) for cytochemical analysis. The point in interphase development reached by the individual cells at the time of their fixation (cell age) was derived from the time lapse films [12, 291. The mean rates of cell multiplication of the fibroblast populations were obtained from cell counts which were also made from the time lapse films. Cytophotometric techniques.-Cells in the photographed area were analyzed individually for total cellular protein, nuclear protein and DNA content as represented by their total cellular dry mass, nuclear dry mass and their total extinction at 5460 A after Feulgen staining, respectively. The total cellular dry mass was determined by use of the rapid scanning microinterferometer [5, 6, 171, and the nuclear dry mass was determined in the recording microinterferometer [25] according to the procedure described below. The amount of Feulgen stain was determined by use of the rapid scanning microspectrophotometer [5, 6, 161. All measurements with the different instruments were made on the same individual cells. For the identification of cells in the time lapse filmed area, maps were drawn by use of a camera lucida (Zeiss). A more detailed description of certain aspects of the biophysical methods used in this work has been published elsewhere [4]. For the determination of the nuclear dry mass, detailed maps of each individual cell at a magnification of x 3000 were drawn by using a phase contrast microscope in conjunction with a camera lucida. All the scan lines recorded in the microinterferometer were drawn on the cell map. The part of the scan line projected over the nucleus was cut out from the remainder of the scan line together with its corresponding integral of the optical path difference as shown in Fig. 1. All these integrals of the parts of the different scan lines over the cell nucleus were summarized to give the surface integral of the optical path difference over the cell nucleus thus representing the nuclear dry mass. The cytoplasmic layers above and below the cell nucleus have been shown to be very thin in outgrown tissue culture cells [i9, 271. The mass that these two cytoplasmic layers added to the nuclear mass was, therefore, neglected. Autoradiographic techniques.-In two separate experiments, 3H-leucine (nn-leucine-4, 5-T, specific activity 151 mC/mM was added to the growth medium at a final concentration of 40 &/ml or 20 &/ml 4 min or 100 min before fixation of the cells, respectively. The stripping film method [7, 261 was used as described previously [29]. The autoradiographs were mounted in glycerol. Grain counts were made under oil immersion at a magnification of x 1200. Experimental Cell Research 42
502
A. Zetterberg
The autoradiographs were prepared after completion of all cytophotometric measurements. The grain counts, therefore, were based on radioactive proteins remaining in the cell after the Feulgen hydrolysis in N HCl for 8 min at 60°C. The mass lost during the Feulgen hydrolysis has previously been shown to be approximately 26 per cent [29]. Cytoplasmic specific activity.-To study the incorporation of labeled amino acids into the cytoplasm, tritium had to be used as the source of radioactivity in order to obtain an autoradiographic image with sufficient resolution. However, due to the extensive absorption of the tritium radiation within the cell, it was necessary to make the grain counts over areas of the cytoplasm which were uniform in respect to absorption of the tritium B-particles. If grain counts are made over parts of the cell which are “infinitely thick” (i.e., the thickness which for practical purposes absorbs all of the tritium radiation), the number of grains per unit area will represent the concentration of the radioactive isotope in the specimen (hereafter referred to as specific activity). In order to obtain cytoplasmic areas large enough to make grain counts with confidence, the thickness of the cytoplasm which absorbed more than 90 per cent of the tritium radiation was, in the present study, considered as being “infinitely thick”. Grain counts were therefore made over the part of the cytoplasm thicker than 0.05 mg/cm2 (corresponding to a cytoplasmic thickness of 1.8 ,u) giving an absorption of more than 90 per cent of the tritium radiation [18]. The cytoplasmic thickness adjacent to the nuclear membrane was 0.11 mg/cm2 (corresponding to 4.0 ,u), absorbing slightly less than 100 per cent of the tritium radiation [18]. Thus, the grain counts were made over cytoplasmic areas absorbing from 90 per cent to slightly less than 100 per cent of the tritium radiation. The average thickness of the cytoplasmic area considered was approximately 0.08 mg/cm2, giving an average tritium absorption of approximately 95 per cent [18]. The part of the cytoplasm having a thickness of 0.05 mg/cm2 was determined, prior to the autoradiographic procedure, by viewing the cell in air through an interference microscope. After first setting the analyser at zero and the background for the color transition from blue to red (which is most sensitive to the eye), the analyzer was turned 70”. Seventy degrees corresponds to a thickness of 0.05 mg/cm2, according to the following formula:
c( =the angle through which the analyzer is turned (in degrees), R =1.54 (the refractive index of the cell) [1], n, =l.OO (the refractive index of the embedding medium; air), m, = the thickness in g/cm2 corresponding to IX, 1 =546 x lo-’ cm (the wavelength of the light at the blue-red transition Q =1.3 g/cm3 (the density of the fixed cell material) [l].
border),
When the analyzer had been turned 70”, the border of color transition had moved from the background toward the center of the cell and consequently enclosed that area of the cell thicker than 0.05 mg/cm 2. This 70” border was then mapped for each individual cell using the camera lzzcida (Fig. 2A). After the autoradiographic procedure, the cells were viewed in the phase contrast microscope, under the same magnification as in the interference microscope (Fig. 2B). The maps of each cell Experimental Cell Research 42
Nuclear and cytoplasmic
proteins
during
interphase
in fibroblasts
503
drawn previously in the interference microscope (Fig. 2A) could be projected, by means of the camera lucida, on to the autoradiographic image of the cell (Fig. 2 C) and the individual silver grains, localized between the 70” border and the nuclear membrane (Fig. 2 D), could be marked. The number of grains over this particular cytoplasmic area was then counted and divided by the size of the area, thus giving the concentration of radioactivity in the cytoplasm (cytoplasmic specific activity).
‘. ;;.’ ,:;., ‘; .; _; ., : .( ‘, :: : ., “‘,..,, ;.: ‘,. ,. . . :. :... ., ‘., ..,’ : .“‘. ‘. 6 c
‘.
Fig. 1.
Fig. 2.
Fig. L-Recorded optical path difference (solid line) and integral of the optical path difference (broken line) of a scan line. The shaded areas illustrate the cell nucleus and the integrated optical path difference of the scan line projected over the cell nucleus. Fig. 2.-A, a map of the cell viewed in the interference microscope with the analyzer set at 0” and 70” (cf. text); B, the cell viewed in phase contrast microscope after the autoradiographic procedure; C, the map (A) of the cell projected on the autoradiographic image of the cell (B) by means of the camera Zucida; D, the autoradiographic grains marked on the map of the relevant cytoplasmic area of the cell.
The average number of grains over this cytoplasmic area was 55, and the sizes of the cytoplasmic areas were 212 k 93 pa (mean k standard deviation) corresponding to approximately 40 per cent of the total cytoplasmic area. Unfortunately, the nuclear activity cannot be obtained from grain counts over the cell nucleus in situ when using tritium due to the cytoplasmic layer between the cell nucleus and the autoradiographic emulsion. Although this layer is thin, the absorption of the weak tritium B-particles within it is nevertheless considerable. Furthermore, the tritium atoms localized in this thin cytoplasmic layer are, due to their proximity
to the emulsion,
very efficient
in producing
over the cell nucleus will not have much quantitative of the nuclear activity.
grains. Therefore,
grain counts
meaning in the determination Experimental
Cell Research 42
A. Zetterberg
504
XESULTS
The accumulation of mass in the cell nucleus and the cytoplasm during interphase.-The cytophotometrically measured cells were divided into seven groups according to relative cell age (fraction of interphase time). The first six of these age groups each represented a time interval of 15 per cent of the entire interphase period. The last age group consisted of cells of a relative cell age of more than 0.90 so that the mean relative cell age of that group was approximately equal to the mean generation time of the population (1 .O). The mean values of cellular DNA, total cellular mass and nuclear mass were calculated for each age group and plotted against the mean relative cell age of that group. The points obtained in this way thus describe the increase in the amounts of DNA, total cell mass and nuclear mass in the average cell of the growing cell population during the interphase period. Two different experiments, each of which showed the same results, were pooled. The generation times in these two experiments were 15 and 18 hr. Fig. 3 shows the typical time course of DNA synthesis where the G I, S and G 2 periods occupy approximately 35, 45 and 20 per cent of the total interphase period, respectively. The total increase in cellular mass during interphase is illustrated in Fig. 4.
0I
REKLATIVE CEUAGE Fig. 3.
Fig. 4.
Fig. 3.-Feulgen DNA content in relative units (mean and standard error of the mean) plotted against interphase time (n = 73). Fig. I.-Total cellular dry mass content (mean and standard error of the mean) plotted against interphase time (n = 61). Experimental
Cell Research 42
Nuclear and cytoplasmic
proteins
during
interphase
in fibroblasts
505
As has been previously shown in large cell samples [la, 281, the total mass of the cell accumulates at an increasing rate throughout the entire interphase ‘period. Though not necessarily exponential in type, it was shown, however, that the true mass curve was very similar to an exponential curve of the type m = e”jn 2,t, where m is the mass and t the relative cell age. Being mathematically well defined, this exponential curve was adjusted to the points in Fig. 4 and used for the derivation of the cytoplasmic mass (see below). The points in Fig. 5 describe the accumulation of nuclear mass during interphase in the average cell of the multiplying L-cell population. It is evident from this figure that the predominant nuclear mass increase occurs during the second half of interphase. However, a slight increase in the nuclear mass during the first half of interphase cannot be excluded. The increase in the nuclear mass is approximately twofold during interphase. Figs. 3 and 5 show that the cellular content of DNA and nuclear mass increase together at approximately the same time of interphase. However, a close comparison shows that the onset of nuclear mass increase might occur slightly after the onset of the DNA synthesis. In order to determine the accumulation of mass in the cytoplasm during
Fig. 6. Fig. 5.-Nuclear dry mass content phase time (n = 51).
(mean and standard
Fig. 6.-Cytoplasmic dry mass content interphase time (n = 51).
error of the mean) plotted
(mean and standard
against inter-
error of the mean) plotted
Experimenfal
against
Cell Research 42
506
A. Zetterberg
interphase, the calculated means of the nuclear mass and the corresponding standard errors of the means, at a given relative cell age, were subtracted. from a total mass value derived from the exponential curve in Fig. 4. The points and intervals thus obtained are illustrated in Fig. 6. By comparing Fig. 4, Fig. 5 and Fig. 6, it can be seen that of the total cellular mass, approximately half is accounted for by the mass of nucleus, the other half being cytoplasmic mass. Similar to the increase in nuclear mass, the increase in cytoplasmic mass appears to be approximately twofold during the interphase period. However, the cytoplasmic mass increased predominantly during the first half of interphase contrary to that of the nuclear mass. Incorporation
of
3H-leucine
in
the
cytoplasm
during
interphase.-Com-
plementary autoradiographic experiments were performed on the cells which had been previously analyzed cytophotometrically in order to find out whether the pattern of accumulation of protein in the cell nucleus and cytoplasm during interphase was a result only of the rate of protein synthesis in each of the two cell compartments or whether any migration of protein material between cytoplasm and cell nucleus occurred and resulted in a redistribution of mass within the cell. The amount of incorporated 3H-leucine in the cytoplasm after a pulse incubation, representing the rate of protein synthesis in the cytoplasm, was investigated on the cytophotometrically analyzed and age determined cells. The specific activity of the cytoplasm (amount of 3Hleucine per cytoplasmic mass unit) was determined by means of the quantitative autoradiographic methods (cf. methods). The growing cells were incubated with 3H-leucine for 2 different pulse lengths of 4 min and 100 min. The very short incubation time of 4 min was used as it was presumed that no significant amount of protein could be synthesized in the cytoplasm and then transported to the cell nucleus or vice versa during such a brief time. Radioactivity in the cytoplasm, after the short incubation pulse of 4 min, was therefore assumed to represent cytoplasmic protein synthesis in situ. The pulse length of 100 min, which has been shown previously to be suitable for investigations of this type [29], was here used for comparative purposes. In a manner similar to the one outlined above, the cells in which the cytoplasmic specific 3H-leucine activity had been determined were divided into seven age groups. The mean cytoplasmic activity and the mean relative cell age of each group was calculated. The results thus obtained are illustrated in Fig. 7. It is evident from both diagrams in Fig. 7 that the cytoplasmic specific activity, activity per unit cytoplasmic mass, remains practically constant throughout the whole interphase period. Consequently, the total 3H-leucine activity in the cytoplasm, representing the overall rate of protein synthesis in Experimental Cell Research 42
Nuclear and cytoplasmic
proteins
during
interphase
in fibroblasts
507
the cytoplasm, increases during interphase in proportion to the increase in cytoplasmic mass. In order to compare the results of the 4 min and 100 min experiments (top and bottom of Fig. 7), the cytoplasmic specific activity was expressed in number of grains per square micron produced per exposure day considering the concentration of 3H-leucine in the incubation medium.
1.0
a5
0 RELATIVE
CELL
AGE
Fig. 7.-Cytoplasmic specific activity (activity per unit cytoplasmic mass) of incorporated aHleucine (mean and standard error of the mean) plotted against interphase time (n = 134 and 148). The specific activity is calculated for a concentration of 1.0 PC per ,uM L-leucine per ml tissue culture medium and expressed in number of grains per square micron produced per exposure day.
By comparing the two results in Fig. 7, it can be seen that the average specific activity is approximately 16 times higher in the 100 min experiment than in the 4 min experiment, which is about 35 per cent less than would be expected on the basis of incubation time. DISCUSSION
The interferometric data presented here show that the patterns of accumulation of mass in the cytoplasm and in the cell nucleus during interphase are very different. Whereas, during the G 1 period the nuclear mass remained constant, or nearly so, the cytoplasmic mass increased markedly. At the time of the initiation of DNA synthesis or possibly slightly afterwards, the major increase in nuclear mass began and at the same time the rate of mass accumulation in the cytoplasm decreased. A part of this finding is in agreeExperimental
Cell Research 42
A. Zefterberg
508
ment with an earlier report [24], in which it was also shown that, in some mammalian cell lines in uitro, the predominant nuclear mass increase occurred during the second part of interphase together with the increase in the nuclear content of DNA. In the reorganization band of Euplotes, a doubling of the mass content [23] and the basic protein content [9] was shown to be associated with the synthesis of DNA. In isolated nuclei of Paramecium aurelia it has also been shown [15] that the contents of mass, DNA and UV,,,-absorbing material remained constant during the first part of interphase, whereafter all three parameters increased together. In contrast to this result on the isolated nuclei, the mass of the whole cell, predominantly cytoplasm, increased continuously throughout the entire interphase period [14] indicating differences in the nuclear and cytoplasmic growth similar to the findings presented in this paper. During the Gl period, most of the protein being synthesized in the cells is accumulated in the cytoplasm. The cellular G 1 growth may, therefore, be characterized as mainly cytoplasmic growth. In previous investigations on L-cells, it has been shown that a certain mass increase takes place during the Gl period before the initiation of DNA synthesis [12, 131. It may, therefore, be speculated that proteins are accumulating in the cytoplasm during the Gl period until a certain nuclear to cytoplasmic mass ratio is attained, whereupon the initiation of DNA synthesis occurs. The pattern of protein accumulation in L-cells during interphase, previously studied by microinterferometry [la, 281, agrees well with the pattern of de novo synthesis of proteins, studied by means of autoradiography [29]. These results suggest that there is neither an extensive turnover of protein during some specific period of interphase influencing the interferometric results nor are there any extensive changes in the immediate precursor pool during the interphase influencing the autoradiographic results. The extent of turnover of protein in tissue culture cells in the logarithmic phase of growth has, in addition, been shown to be quite small [8]. When comparing the rate of protein accumulation in the cytoplasm with the rate of protein synthesis in the cytoplasm, a discrepancy is found in contrast with the above mentioned results on the whole cell. The rate of protein synthesis per unit cytoplasmic mass is constant, i.e., the overall rate of cytoplasmic protein synthesis increases in proportion to the cytoplasmic mass throughout interphase. Despite the fact that the overall rate of protein synthesis in the cytoplasm is higher during the S period compared with the G 1 period less protein accumulates in the cytoplasm during the S period. This discrepancy between the rate of synthesis and the rate of accumulation of protein Experimental
Cell Research 42
Nuclear and cytoplasmic
proteins during
interphase
in fibroblasts
509
in the cytoplasm during the S period most probably reflects a transportation to and an accumulation in the cell nucleus of protein material after synthesis in the cytoplasm. This interpretation is supported further by the finding of the rapid accumulation of protein in the cell nucleus during the same part of interphase. The magnitude of this migration of protein material from cytoplasm to cell nucleus during the S period cannot be calculated with any great degree of accuracy. However, some information may be obtained by comparing the expected accumulation of protein in the cytoplasm, deduced from the autoradiographic results, and the observed net increase of cytoplasmic mass during interphase. During the first half of interphase the cytoplasm increased by 6.7 mass units (Fig. 6) which corresponds to an overall rate of protein synthesis in the cytoplasm of 14.0 x C relative rate units-the mean cytoplasmic mass during the first half of interphase times the rate of protein synthesis per unit cytoplasmic mass, which was constant ( = C) throughout the whole of interphase (Fig. 7). During the second half of interphase, the overall rate of protein synthesis in the cytoplasm was 17.8 x C relative rate units. This rate should accordingly result in a mass increase of (17.8 x C/14.0 x C) X 6.7 = 8.5 mass units in the cytoplasm. However, a mass increase of only 3.3 units (Fig. 6) was observed in the cytoplasm during this time suggesting a transportation of cytoplasmic material equivalent to 8.5-3.3 = 5.2 mass units. During the same period of time, the cell nucleus increased with 8.0 mass units (Fig. 5), thus suggesting that 65 per cent of the nuclear mass was synthesized in the cytoplasm and transported to the cell nucleus during the second half of interphase. In Amoeba proteus evidence for a migration of protein between cell nucleus and cytoplasm has been reported [2. 3, 10, 11, 20, 211. A group of proteins specific for the cell nucleus was found to exist, among which two categories could be distinguished, viz., one shuttling between the cell nucleus and the other the cytoplasm in a non-random fashion, called cytonucleoprotein; being non-migratory in type and always localized in the cell nucleus. Both of these two types of proteins seemed to be synthesized, at least in part, in the cytoplasm. The results on the L-cells presented in this paper agree well with these findings on Amoeba proteus. Among the nuclear proteins two categories can be distinguished: the proteins which are components of the metaphase chromosomes and the remainder of the nuclear proteins present as part of the nuclear mass during interphase. The latter group, the non-chromosomal nuclear proteins, has been shown to make up more than 50 per cent of the nuclear mass in similar types of cells [22]. The above described migration to the cell nucleus of proteins Experimental
Cell Research 42
510
A. Zetterberg
synthesized in the cytoplasm, therefore, most likely demonstrates the hehavior of at least a great part of the non-chromosomal proteins. Whether the behavior of the chromosomal proteins follow the pattern of the non-chromosomal proteins cannot be evaluated at the present stage since a minor protein synthesis may go on within the cell nucleus itself.
SUMMARY
The synthesis and accumulation of protein in the cell nucleus and cytoplasm during interphase growth of the mouse fibroblasts in vitro (L-cells) has been studied by a combination of quantitative cytophotometric, autoradiographic and time lapse cinematographic methods. The nuclear dry mass of the average cell of the population remained constant or nearly so during the Gl period and increased predominantly during the S and G2 periods by a factor of approximately 2. The onset of this increase occurred at the same time or slightly after the initiation of the DNA synthesis in the cell. The cytoplasmic mass increased, contrary to that of the cell nucleus, predominantly during the Gl period. However, a slight increase of cytoplasmic mass also occurred towards the end of interphase. The rate of protein synthesis in the cytoplasm increased during the whole of interphase. Despite the fact that the rate of protein synthesis was higher in the cytoplasm during the S period than in the G 1 period, less protein was accumulated in the cytoplasm at that time than during the G 1 period. These results demonstrate a synthesis of nuclear proteins in the cytoplasm and a transportation of these proteins to the cell nucleus during the S period. These studies were supported by grants from the Swedish Cancer Society and from Reservationsanslaget at Karolinska Institutet. The biophysical portion of the work was supported by grants from the Swedish Medical Research Council (Project 12x-531-01) and the Swedish Natural Science Research Council (Grant 8-U).
REFERENCES 1. BARER, 2. BYERS,
3. __
R., in The Barer Interference Microscope, 2nd ed. Suppl. London and Croydon, T. J., PLATT, D. B. and GOLDSTEIN, L., J. Cell Biol. 19, 453 (1963).
1956.
ibid. 19, 467 (1963).
4. CASPERSSSON,
T., FARBER,
S., FOLEY,
G. E. and
KILLANDER,
D., Exptl
Cell Res. 32, 529
(19631. 5. 6. 7. 8.
CASPE~SSO~, T. and LOMAKKA, G., N. Y. Acad. Sci. 97, 449 (1962). CASPERSSON, T., LOMAKKA, G. and CASPERSSON, O., Biochem. Pharmaeol. 4, 113 (1960). DONIACH, I. and PELC, S. FL, Brif. J. Radial. 23, 184 (1950). EAGLE, H., PIEZ, K. A., FLEISCHMAN, R. and OYAMA, V. I., J. Biol. Chem. 234, 592 (1959).
Experimental
Cell Research 42
Nuclear and cytoplasmic proteins during 9. 10. 11. 12.
13. 14. 15.
16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27.
28. 29.
GALL, J. G., GOLDSTEIN, in R.J. KILLANDER,
J. Biophys.
interphase
in fibroblasts
511
Biochem. Cytol. 5, 295 (1959).
L., Exptl Cell Res. 15, 635 (1958). C. HARRIS (ed.), Cell Growth and Cell Division. Academic Press, New York, 1963. D. and ZETTERBERG, A., Exptl Cell Res. 38, 272 (1965). ibid. 40, 12 (1965). KIMBALL, R. F., CASPERSSON, T., SVENSON, G. and CARLSON, L., Exptl Cell Res. 17,160 (1959). KIMBALL, R. F., VOGT-K~HNE, L. and CASPERSSON, T., ibid. 20, 368 (1960). LOMAKKA, G., Acta histochem. Suppl. VI, 47 (1965). __ ibid. Suppl. VI, 393 (1965). MAURER, W. and PRIMBSCH, D., Exptl Cell Res. 33, 8 (1964). PERRY, R. P., ERRERA, M., HELL, A. and D~~RWALD, H., J. Biophys. Biochem. Cytol. 11, 1 (1961). PRESCOTT, D. M., in R. J. C. Harris (ed.), Cell Growth and Cell Division. Academic Press, New York, 1963. PRESCOTT, D. M. and BENDER, M. A., J. Cell Comp. Physiol. 62, Suppl. 1, 175 (1963). RICHARDS, B. M., in The Cell Nucleus. Butterworths, London, 1950. RINGERTZ, N. R. and HOSKINS, G. C., Exptl Cell Res. 38, 160 (1965). SEED, J., Proc. Roy. Sot. B. 156, 41 (1962). SVENSSON, G., Exptl Cell Res. Suppl. 4, 165 (1957). TAYLOR, J. H., Physical Techniques in Biol. Res. 3, 545 (1965). WALKER, D. M. B. and YATES, H. B., Proc. Roy. Sot. B. 140,274 (1952). ZETTERBERG, A. and KILLANDER, D., ExPtl Cell Res. 39, 22 (1965). -ibid. 40, 1 (1965).
34 - 661805
Experimental
Cell Research 42
500
Cell Research 42, 500-511 (1966)
SYNTHESIS AND ACCUMULATION OF NUCLEAR AND CYTOPLASMIC PROTEINS DURING INTERPHASE IN MOUSE FIBROBLASTS
IN VITRO A. ZETTERBERG Institute for Cell Research, Karolinska
Institutet,
Stockholm, Sweden
Received November 22, 1965
previous studies, the growth of mouse tlbroblasts in vitro (cell line L-929) during interphase was investigated using cytophotometric [12, 281 and autoradiographic [29] techniques in combination with time lapse cinematography. The results obtained with these techniques showed that protein was synthesized and accumulated in the cell during the entire period of interphase. In these investigations, the whole cell was considered as a unit and no distinction was made between the cell nucleus and the cytoplasm. There are, however, reasons to believe that the synthesis and accumulation of protein in the cell nucleus differs from that in the cytoplasm during interphase growth. For example, in Paramecium aurelia the nuclear dry mass was shown to increase only during a part of the interphase [ 151 and in that respect differing from the dry mass increase of the cytoplasm [14]. It has also been shown that, in some mammalian cell lines in vitro, the predominant increase of the nuclear mass was confined to the second part of the interphase period [24], thus, contrasting to that of the whole cell [12, 281. This suggests that differences may also exist between the patterns of protein accumulation in the cell nucleus and in the cytoplasm of mammalian cells during interphase. The accumulation of protein material in the cell nucleus and the cytoplasm need not necessarily reflect a true synthesis in situ. Protein may be synthesized in either one of these two cell compartments and thereafter migrate to the other. In Amoeba proteus evidence of migration of protein from the site of synthesis in the cytoplasm to the cell nucleus has been reported [2, 31. However, it is not yet known whether this migration is of any quantitative significance for the accumulation of protein in the cell nucleus. The aim of the present investigation was to study simultaneously both the synthesis and the accumulation of protein in the cytoplasm and cell nucleus of growing L-cells in order to obtain more information about the interaction IN
Experimental
Cell Research 42
Nuclear
and cytoplasmic
proteins
during
interphase
in fibroblasts
501
between the cell nucleus and the cytoplasm during interphase growth. For this purpose a combination of cytophotometric, autoradiographic and time lapse cinematographic techniques was employed.
MATERIALS
AND
METHODS
Cell culture techniques-Mouse fibroblasts (cell line L-929) were cultivated directly on quartz slides as described previously [12]. The multiplying cell populations were photographed by time lapse cinematography every 2 min for about 40 hr under low power magnification. At the end of this period, the slide cultures were fixed in a mixture of absolute ethanol and acetone (1: 1) at 4°C for 24 hr before being mounted in redistilled glycerol (n = 1.455) for cytochemical analysis. The point in interphase development reached by the individual cells at the time of their fixation (cell age) was derived from the time lapse films [12, 291. The mean rates of cell multiplication of the fibroblast populations were obtained from cell counts which were also made from the time lapse films. Cytophotometric techniques.-Cells in the photographed area were analyzed individually for total cellular protein, nuclear protein and DNA content as represented by their total cellular dry mass, nuclear dry mass and their total extinction at 5460 A after Feulgen staining, respectively. The total cellular dry mass was determined by use of the rapid scanning microinterferometer [5, 6, 171, and the nuclear dry mass was determined in the recording microinterferometer [25] according to the procedure described below. The amount of Feulgen stain was determined by use of the rapid scanning microspectrophotometer [5, 6, 161. All measurements with the different instruments were made on the same individual cells. For the identification of cells in the time lapse filmed area, maps were drawn by use of a camera lucida (Zeiss). A more detailed description of certain aspects of the biophysical methods used in this work has been published elsewhere [4]. For the determination of the nuclear dry mass, detailed maps of each individual cell at a magnification of x 3000 were drawn by using a phase contrast microscope in conjunction with a camera lucida. All the scan lines recorded in the microinterferometer were drawn on the cell map. The part of the scan line projected over the nucleus was cut out from the remainder of the scan line together with its corresponding integral of the optical path difference as shown in Fig. 1. All these integrals of the parts of the different scan lines over the cell nucleus were summarized to give the surface integral of the optical path difference over the cell nucleus thus representing the nuclear dry mass. The cytoplasmic layers above and below the cell nucleus have been shown to be very thin in outgrown tissue culture cells [i9, 271. The mass that these two cytoplasmic layers added to the nuclear mass was, therefore, neglected. Autoradiographic techniques.-In two separate experiments, 3H-leucine (nn-leucine-4, 5-T, specific activity 151 mC/mM was added to the growth medium at a final concentration of 40 &/ml or 20 &/ml 4 min or 100 min before fixation of the cells, respectively. The stripping film method [7, 261 was used as described previously [29]. The autoradiographs were mounted in glycerol. Grain counts were made under oil immersion at a magnification of x 1200. Experimental Cell Research 42
502
A. Zetterberg
The autoradiographs were prepared after completion of all cytophotometric measurements. The grain counts, therefore, were based on radioactive proteins remaining in the cell after the Feulgen hydrolysis in N HCl for 8 min at 60°C. The mass lost during the Feulgen hydrolysis has previously been shown to be approximately 26 per cent [29]. Cytoplasmic specific activity.-To study the incorporation of labeled amino acids into the cytoplasm, tritium had to be used as the source of radioactivity in order to obtain an autoradiographic image with sufficient resolution. However, due to the extensive absorption of the tritium radiation within the cell, it was necessary to make the grain counts over areas of the cytoplasm which were uniform in respect to absorption of the tritium B-particles. If grain counts are made over parts of the cell which are “infinitely thick” (i.e., the thickness which for practical purposes absorbs all of the tritium radiation), the number of grains per unit area will represent the concentration of the radioactive isotope in the specimen (hereafter referred to as specific activity). In order to obtain cytoplasmic areas large enough to make grain counts with confidence, the thickness of the cytoplasm which absorbed more than 90 per cent of the tritium radiation was, in the present study, considered as being “infinitely thick”. Grain counts were therefore made over the part of the cytoplasm thicker than 0.05 mg/cm2 (corresponding to a cytoplasmic thickness of 1.8 ,u) giving an absorption of more than 90 per cent of the tritium radiation [18]. The cytoplasmic thickness adjacent to the nuclear membrane was 0.11 mg/cm2 (corresponding to 4.0 ,u), absorbing slightly less than 100 per cent of the tritium radiation [18]. Thus, the grain counts were made over cytoplasmic areas absorbing from 90 per cent to slightly less than 100 per cent of the tritium radiation. The average thickness of the cytoplasmic area considered was approximately 0.08 mg/cm2, giving an average tritium absorption of approximately 95 per cent [18]. The part of the cytoplasm having a thickness of 0.05 mg/cm2 was determined, prior to the autoradiographic procedure, by viewing the cell in air through an interference microscope. After first setting the analyser at zero and the background for the color transition from blue to red (which is most sensitive to the eye), the analyzer was turned 70”. Seventy degrees corresponds to a thickness of 0.05 mg/cm2, according to the following formula:
c( =the angle through which the analyzer is turned (in degrees), R =1.54 (the refractive index of the cell) [1], n, =l.OO (the refractive index of the embedding medium; air), m, = the thickness in g/cm2 corresponding to IX, 1 =546 x lo-’ cm (the wavelength of the light at the blue-red transition Q =1.3 g/cm3 (the density of the fixed cell material) [l].
border),
When the analyzer had been turned 70”, the border of color transition had moved from the background toward the center of the cell and consequently enclosed that area of the cell thicker than 0.05 mg/cm 2. This 70” border was then mapped for each individual cell using the camera lzzcida (Fig. 2A). After the autoradiographic procedure, the cells were viewed in the phase contrast microscope, under the same magnification as in the interference microscope (Fig. 2B). The maps of each cell Experimental Cell Research 42
Nuclear and cytoplasmic
proteins
during
interphase
in fibroblasts
503
drawn previously in the interference microscope (Fig. 2A) could be projected, by means of the camera lucida, on to the autoradiographic image of the cell (Fig. 2 C) and the individual silver grains, localized between the 70” border and the nuclear membrane (Fig. 2 D), could be marked. The number of grains over this particular cytoplasmic area was then counted and divided by the size of the area, thus giving the concentration of radioactivity in the cytoplasm (cytoplasmic specific activity).
‘. ;;.’ ,:;., ‘; .; _; ., : .( ‘, :: : ., “‘,..,, ;.: ‘,. ,. . . :. :... ., ‘., ..,’ : .“‘. ‘. 6 c
‘.
Fig. 1.
Fig. 2.
Fig. L-Recorded optical path difference (solid line) and integral of the optical path difference (broken line) of a scan line. The shaded areas illustrate the cell nucleus and the integrated optical path difference of the scan line projected over the cell nucleus. Fig. 2.-A, a map of the cell viewed in the interference microscope with the analyzer set at 0” and 70” (cf. text); B, the cell viewed in phase contrast microscope after the autoradiographic procedure; C, the map (A) of the cell projected on the autoradiographic image of the cell (B) by means of the camera Zucida; D, the autoradiographic grains marked on the map of the relevant cytoplasmic area of the cell.
The average number of grains over this cytoplasmic area was 55, and the sizes of the cytoplasmic areas were 212 k 93 pa (mean k standard deviation) corresponding to approximately 40 per cent of the total cytoplasmic area. Unfortunately, the nuclear activity cannot be obtained from grain counts over the cell nucleus in situ when using tritium due to the cytoplasmic layer between the cell nucleus and the autoradiographic emulsion. Although this layer is thin, the absorption of the weak tritium B-particles within it is nevertheless considerable. Furthermore, the tritium atoms localized in this thin cytoplasmic layer are, due to their proximity
to the emulsion,
very efficient
in producing
over the cell nucleus will not have much quantitative of the nuclear activity.
grains. Therefore,
grain counts
meaning in the determination Experimental
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504
XESULTS
The accumulation of mass in the cell nucleus and the cytoplasm during interphase.-The cytophotometrically measured cells were divided into seven groups according to relative cell age (fraction of interphase time). The first six of these age groups each represented a time interval of 15 per cent of the entire interphase period. The last age group consisted of cells of a relative cell age of more than 0.90 so that the mean relative cell age of that group was approximately equal to the mean generation time of the population (1 .O). The mean values of cellular DNA, total cellular mass and nuclear mass were calculated for each age group and plotted against the mean relative cell age of that group. The points obtained in this way thus describe the increase in the amounts of DNA, total cell mass and nuclear mass in the average cell of the growing cell population during the interphase period. Two different experiments, each of which showed the same results, were pooled. The generation times in these two experiments were 15 and 18 hr. Fig. 3 shows the typical time course of DNA synthesis where the G I, S and G 2 periods occupy approximately 35, 45 and 20 per cent of the total interphase period, respectively. The total increase in cellular mass during interphase is illustrated in Fig. 4.
0I
REKLATIVE CEUAGE Fig. 3.
Fig. 4.
Fig. 3.-Feulgen DNA content in relative units (mean and standard error of the mean) plotted against interphase time (n = 73). Fig. I.-Total cellular dry mass content (mean and standard error of the mean) plotted against interphase time (n = 61). Experimental
Cell Research 42
Nuclear and cytoplasmic
proteins
during
interphase
in fibroblasts
505
As has been previously shown in large cell samples [la, 281, the total mass of the cell accumulates at an increasing rate throughout the entire interphase ‘period. Though not necessarily exponential in type, it was shown, however, that the true mass curve was very similar to an exponential curve of the type m = e”jn 2,t, where m is the mass and t the relative cell age. Being mathematically well defined, this exponential curve was adjusted to the points in Fig. 4 and used for the derivation of the cytoplasmic mass (see below). The points in Fig. 5 describe the accumulation of nuclear mass during interphase in the average cell of the multiplying L-cell population. It is evident from this figure that the predominant nuclear mass increase occurs during the second half of interphase. However, a slight increase in the nuclear mass during the first half of interphase cannot be excluded. The increase in the nuclear mass is approximately twofold during interphase. Figs. 3 and 5 show that the cellular content of DNA and nuclear mass increase together at approximately the same time of interphase. However, a close comparison shows that the onset of nuclear mass increase might occur slightly after the onset of the DNA synthesis. In order to determine the accumulation of mass in the cytoplasm during
Fig. 6. Fig. 5.-Nuclear dry mass content phase time (n = 51).
(mean and standard
Fig. 6.-Cytoplasmic dry mass content interphase time (n = 51).
error of the mean) plotted
(mean and standard
against inter-
error of the mean) plotted
Experimenfal
against
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interphase, the calculated means of the nuclear mass and the corresponding standard errors of the means, at a given relative cell age, were subtracted. from a total mass value derived from the exponential curve in Fig. 4. The points and intervals thus obtained are illustrated in Fig. 6. By comparing Fig. 4, Fig. 5 and Fig. 6, it can be seen that of the total cellular mass, approximately half is accounted for by the mass of nucleus, the other half being cytoplasmic mass. Similar to the increase in nuclear mass, the increase in cytoplasmic mass appears to be approximately twofold during the interphase period. However, the cytoplasmic mass increased predominantly during the first half of interphase contrary to that of the nuclear mass. Incorporation
of
3H-leucine
in
the
cytoplasm
during
interphase.-Com-
plementary autoradiographic experiments were performed on the cells which had been previously analyzed cytophotometrically in order to find out whether the pattern of accumulation of protein in the cell nucleus and cytoplasm during interphase was a result only of the rate of protein synthesis in each of the two cell compartments or whether any migration of protein material between cytoplasm and cell nucleus occurred and resulted in a redistribution of mass within the cell. The amount of incorporated 3H-leucine in the cytoplasm after a pulse incubation, representing the rate of protein synthesis in the cytoplasm, was investigated on the cytophotometrically analyzed and age determined cells. The specific activity of the cytoplasm (amount of 3Hleucine per cytoplasmic mass unit) was determined by means of the quantitative autoradiographic methods (cf. methods). The growing cells were incubated with 3H-leucine for 2 different pulse lengths of 4 min and 100 min. The very short incubation time of 4 min was used as it was presumed that no significant amount of protein could be synthesized in the cytoplasm and then transported to the cell nucleus or vice versa during such a brief time. Radioactivity in the cytoplasm, after the short incubation pulse of 4 min, was therefore assumed to represent cytoplasmic protein synthesis in situ. The pulse length of 100 min, which has been shown previously to be suitable for investigations of this type [29], was here used for comparative purposes. In a manner similar to the one outlined above, the cells in which the cytoplasmic specific 3H-leucine activity had been determined were divided into seven age groups. The mean cytoplasmic activity and the mean relative cell age of each group was calculated. The results thus obtained are illustrated in Fig. 7. It is evident from both diagrams in Fig. 7 that the cytoplasmic specific activity, activity per unit cytoplasmic mass, remains practically constant throughout the whole interphase period. Consequently, the total 3H-leucine activity in the cytoplasm, representing the overall rate of protein synthesis in Experimental Cell Research 42
Nuclear and cytoplasmic
proteins
during
interphase
in fibroblasts
507
the cytoplasm, increases during interphase in proportion to the increase in cytoplasmic mass. In order to compare the results of the 4 min and 100 min experiments (top and bottom of Fig. 7), the cytoplasmic specific activity was expressed in number of grains per square micron produced per exposure day considering the concentration of 3H-leucine in the incubation medium.
1.0
a5
0 RELATIVE
CELL
AGE
Fig. 7.-Cytoplasmic specific activity (activity per unit cytoplasmic mass) of incorporated aHleucine (mean and standard error of the mean) plotted against interphase time (n = 134 and 148). The specific activity is calculated for a concentration of 1.0 PC per ,uM L-leucine per ml tissue culture medium and expressed in number of grains per square micron produced per exposure day.
By comparing the two results in Fig. 7, it can be seen that the average specific activity is approximately 16 times higher in the 100 min experiment than in the 4 min experiment, which is about 35 per cent less than would be expected on the basis of incubation time. DISCUSSION
The interferometric data presented here show that the patterns of accumulation of mass in the cytoplasm and in the cell nucleus during interphase are very different. Whereas, during the G 1 period the nuclear mass remained constant, or nearly so, the cytoplasmic mass increased markedly. At the time of the initiation of DNA synthesis or possibly slightly afterwards, the major increase in nuclear mass began and at the same time the rate of mass accumulation in the cytoplasm decreased. A part of this finding is in agreeExperimental
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508
ment with an earlier report [24], in which it was also shown that, in some mammalian cell lines in uitro, the predominant nuclear mass increase occurred during the second part of interphase together with the increase in the nuclear content of DNA. In the reorganization band of Euplotes, a doubling of the mass content [23] and the basic protein content [9] was shown to be associated with the synthesis of DNA. In isolated nuclei of Paramecium aurelia it has also been shown [15] that the contents of mass, DNA and UV,,,-absorbing material remained constant during the first part of interphase, whereafter all three parameters increased together. In contrast to this result on the isolated nuclei, the mass of the whole cell, predominantly cytoplasm, increased continuously throughout the entire interphase period [14] indicating differences in the nuclear and cytoplasmic growth similar to the findings presented in this paper. During the Gl period, most of the protein being synthesized in the cells is accumulated in the cytoplasm. The cellular G 1 growth may, therefore, be characterized as mainly cytoplasmic growth. In previous investigations on L-cells, it has been shown that a certain mass increase takes place during the Gl period before the initiation of DNA synthesis [12, 131. It may, therefore, be speculated that proteins are accumulating in the cytoplasm during the Gl period until a certain nuclear to cytoplasmic mass ratio is attained, whereupon the initiation of DNA synthesis occurs. The pattern of protein accumulation in L-cells during interphase, previously studied by microinterferometry [la, 281, agrees well with the pattern of de novo synthesis of proteins, studied by means of autoradiography [29]. These results suggest that there is neither an extensive turnover of protein during some specific period of interphase influencing the interferometric results nor are there any extensive changes in the immediate precursor pool during the interphase influencing the autoradiographic results. The extent of turnover of protein in tissue culture cells in the logarithmic phase of growth has, in addition, been shown to be quite small [8]. When comparing the rate of protein accumulation in the cytoplasm with the rate of protein synthesis in the cytoplasm, a discrepancy is found in contrast with the above mentioned results on the whole cell. The rate of protein synthesis per unit cytoplasmic mass is constant, i.e., the overall rate of cytoplasmic protein synthesis increases in proportion to the cytoplasmic mass throughout interphase. Despite the fact that the overall rate of protein synthesis in the cytoplasm is higher during the S period compared with the G 1 period less protein accumulates in the cytoplasm during the S period. This discrepancy between the rate of synthesis and the rate of accumulation of protein Experimental
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Nuclear and cytoplasmic
proteins during
interphase
in fibroblasts
509
in the cytoplasm during the S period most probably reflects a transportation to and an accumulation in the cell nucleus of protein material after synthesis in the cytoplasm. This interpretation is supported further by the finding of the rapid accumulation of protein in the cell nucleus during the same part of interphase. The magnitude of this migration of protein material from cytoplasm to cell nucleus during the S period cannot be calculated with any great degree of accuracy. However, some information may be obtained by comparing the expected accumulation of protein in the cytoplasm, deduced from the autoradiographic results, and the observed net increase of cytoplasmic mass during interphase. During the first half of interphase the cytoplasm increased by 6.7 mass units (Fig. 6) which corresponds to an overall rate of protein synthesis in the cytoplasm of 14.0 x C relative rate units-the mean cytoplasmic mass during the first half of interphase times the rate of protein synthesis per unit cytoplasmic mass, which was constant ( = C) throughout the whole of interphase (Fig. 7). During the second half of interphase, the overall rate of protein synthesis in the cytoplasm was 17.8 x C relative rate units. This rate should accordingly result in a mass increase of (17.8 x C/14.0 x C) X 6.7 = 8.5 mass units in the cytoplasm. However, a mass increase of only 3.3 units (Fig. 6) was observed in the cytoplasm during this time suggesting a transportation of cytoplasmic material equivalent to 8.5-3.3 = 5.2 mass units. During the same period of time, the cell nucleus increased with 8.0 mass units (Fig. 5), thus suggesting that 65 per cent of the nuclear mass was synthesized in the cytoplasm and transported to the cell nucleus during the second half of interphase. In Amoeba proteus evidence for a migration of protein between cell nucleus and cytoplasm has been reported [2. 3, 10, 11, 20, 211. A group of proteins specific for the cell nucleus was found to exist, among which two categories could be distinguished, viz., one shuttling between the cell nucleus and the other the cytoplasm in a non-random fashion, called cytonucleoprotein; being non-migratory in type and always localized in the cell nucleus. Both of these two types of proteins seemed to be synthesized, at least in part, in the cytoplasm. The results on the L-cells presented in this paper agree well with these findings on Amoeba proteus. Among the nuclear proteins two categories can be distinguished: the proteins which are components of the metaphase chromosomes and the remainder of the nuclear proteins present as part of the nuclear mass during interphase. The latter group, the non-chromosomal nuclear proteins, has been shown to make up more than 50 per cent of the nuclear mass in similar types of cells [22]. The above described migration to the cell nucleus of proteins Experimental
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A. Zetterberg
synthesized in the cytoplasm, therefore, most likely demonstrates the hehavior of at least a great part of the non-chromosomal proteins. Whether the behavior of the chromosomal proteins follow the pattern of the non-chromosomal proteins cannot be evaluated at the present stage since a minor protein synthesis may go on within the cell nucleus itself.
SUMMARY
The synthesis and accumulation of protein in the cell nucleus and cytoplasm during interphase growth of the mouse fibroblasts in vitro (L-cells) has been studied by a combination of quantitative cytophotometric, autoradiographic and time lapse cinematographic methods. The nuclear dry mass of the average cell of the population remained constant or nearly so during the Gl period and increased predominantly during the S and G2 periods by a factor of approximately 2. The onset of this increase occurred at the same time or slightly after the initiation of the DNA synthesis in the cell. The cytoplasmic mass increased, contrary to that of the cell nucleus, predominantly during the Gl period. However, a slight increase of cytoplasmic mass also occurred towards the end of interphase. The rate of protein synthesis in the cytoplasm increased during the whole of interphase. Despite the fact that the rate of protein synthesis was higher in the cytoplasm during the S period than in the G 1 period, less protein was accumulated in the cytoplasm at that time than during the G 1 period. These results demonstrate a synthesis of nuclear proteins in the cytoplasm and a transportation of these proteins to the cell nucleus during the S period. These studies were supported by grants from the Swedish Cancer Society and from Reservationsanslaget at Karolinska Institutet. The biophysical portion of the work was supported by grants from the Swedish Medical Research Council (Project 12x-531-01) and the Swedish Natural Science Research Council (Grant 8-U).
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