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Pattern of total protein content regulation in Trithigmostoma steini (Chilodonella steini)
Europ. J. Protistol. 31, 167-173 (1995) May 26,1995
European Journal of
PROTISTOLOGY
Pattern of Total Protein Content Regulation in Trithigmostoma steini (Chilodonella steinij Stefan Radzikowski, "Elzbleta Pleszczynska, Matgorzata Gotembiewska-Skoczylas, and Beata Sapetto-Rebow University of Warsaw, Institute of Zoology, Warsaw, Poland and *Institute of Computer Science of the Polish Academy of Science, Warsaw, Poland
SUMMARY A series of experiments on the ciliate Trithigmostoma steini was conducted in order to analyze interrelation of total protein contents between two daughter cells and their "adopted mother" (mother's sister) cell. We discuss the pattern of total proteins regulation in respect to the incremental model [1, 2,4]. The results allow to reject the hypothesis of constant protein increment in Trithigmostoma steini. Instead we suggest that the increment tends to diminish when the true mother's content of protein is large. We also noticed a negative correlation between the length of the cell cycle and the initial protein content of the cell. However, one cell cycle is not a sufficient period to investigate this effect. The regulation processes observed in Trithigmostoma steini seem to be similar to those of other ciliates and we believethat they are based on molecular control mechanisms known for other eukaryotes. Introduction In ciliates, the partition of the cell mass during division is not precise, and the newly formed sister cells differ in their protein content. This phenomenon is probably responsible for the striking individual variability in the cell total protein contents in the population. However, in Trithigmostoma, in spite of the great variability in the total protein contents, the mean values of this parameter remain constant for many generations at stable culture conditions. Moreover, the total protein contents of sister cells are always positively, strongly, and linearly codependent [25]. These findings suggest the existence of (a) special mechanism(s) which either regulate(s) the total protein level in the course ofthe cell cycle or operate(s) during cytokinesis and maintain(s) the differences between protein content of sister cells within certain limits
[2,25]. In the present study we analysed: - the interdependence of total protein contents between two daughter cells and their "adopted mother" (mother's sister) cell, and © 1995 by Gustav Fischer Verlag, Stuttgart
- the correlation between initial G 1 levels of total protein and the length of the cell cycle.
Material and Methods The study was based on a clone of Trithigmostoma steini that is nonmature for conjugation. In this paper we present experiments carried out on more than 450 cells. The methods of cultivation were as described earlier [24]. All experiments were conducted at 19 DC in constant light of 500 lux. We have used the "sister model" of experiment [5,9, 12,25]. The "sister model" was thoroughly checked with respect to different parameters. We accepted that the total protein levels of sister cellswere always positively and linearly codependent in all experiments. This relationship does not depend on the kind of the cell (prater or opisthe). In all experiments, the values of mean absolute differences, expressed as percentage of the mean value, between total protein content of sister cells never exceeded the values ofVC% of the total population. On the basis of the data described in Table 1, the mean absolute difference between sister cells is 10.6% of the mean value while VC% is equal to 17%. The mean absolute difference 0932-4739-95-0031-0167$3.50-0
168 . S. Radzikowski et aI. obtained for the same cells after accidental formation of sister pairs was 23.6%. To investigate the amount of total protein in the mother cell and its progeny we employed the following experimental protocol: after the cell division, one cell was fixed immediately and the value of its protein content was denoted as the initial [9,25] protein level value. The second cell from each pair was grown for one more full cell cycle and its generation time was measured. After the division of this cell, its daughter cellswere immediately fixed and the protein contents of both cells were measured. The terminology and notation used in the paper are as follows: we distinguish between the true mother cell (with unobservable protein contents denoted m-) and the adopted mother cell called, for short, a mother cell (with observable protein contents denoted m). The daughters' protein contents, registered in random order, are denoted d l and d 2 • The unobservable increment is (d, + d 2)-mT, its observable counterpart is (d l + d 2)-m. Since differences between mT and m happen to be substantial, we investigated only the global and not the individual effects of substituting observable m for unobservable Hl-p, The fixation and staining protocols for total protein with 2,4-dinitrofluorobenzene (DNFB) were as previously described [20, 25, 28]. The measurements were made with a SPM-05 scanning cytophotometer (Zeiss, Oberkochen, Germany) at 438 nm wave-length. Standard statistical methods including the Wilcoxon's and Spearman's statistic were used for the analysis of the suitably transformed data [3].
Table 1. Parameters describing protein contents of mother and daughter cells (Gl ) in au (arbitrary units): total and quartile distinctions (I-IV) Variable
Mothers
Daughters
N x sd
37 2217.62 421.26 990.1 7 3266.50 19
57 2050.21 349. 76 1417.35 2953.38 17
8 1651 326 990 1944
13 1769 207 1482 2191
10 2057 68 1959 2128
14 2022 315 1417 2410
11 2285 133 2135 2497
17 2187 309 1602 2953 14 2176 385 1521 2801
rrun,
max. VC% N x sd min.
max. II
N
x sd min. max. III
N
x sd min.
max. IV
N
x sd Results
mm.
max.
8 2851 235 2575 3266
1. The Total Protein Content in G 1 Phase - the Relationship Between Mother (Adopted Mother) and Daughter Cells We have analysed the total protein content of adopted mother(m) and daughter (d, and d 2 ) cells. In the first experiment (Table 1) we analysed 94 cells. The means, standard deviations, and ranges of the total protein contents were similar in both the mother and its daughter cells . The total protein content of mother cells (G 1 ) ranged from 990 au to 3266 au, with the mean value equal to 2217 au . The total protein content in daughter cells (G 1 ) ranged from 1417 au to 2953 au with the mean value equal to 2050 au. The values of the total protein contents in individual lines of mother - daughter cells were variable. We hypothesised that the protein level in daughter cells may depend on the protein level of their mother cells . Thus we carried out the analysis of such dependence for mother cells with extreme values (low or high) of protein levels. We noticed that when the protein content value of the mother cell was very low the daughter cell 's protein contents tended to be higher, whereas when the total protein level of the mother cell was high, the daughter cells tended to have lower protein contents. To investigate this phenomenon more closely we performed the quartile analysis of the data. We grouped the mother cells into quartiles (I, II, III, IV) whereby each contained 25 % of the total number of cells from the low-
est to the highest protein content. For each quartile we calculated the mean value and standard deviation of the total protein content for mothers and their daughter cells. The results are presented in the second part of Table 1 (see also Fig. 1). The analysis of these data shows that in quartile I the mean content of total protein in mother cells is lower than that of the daughters by 118 au (7.1 %), whereas in quartiles II, III and IV daughter cells have lower total protein contents than their mothers by 35 au (1. 7 %), 98 au (4 %) and 675 au (23.7%), respectively. This descriptive analysis of the first experiment was followed by a more formal analysis of the next experiments (A, B, C) which disproved the hypothesis of constant increment. The results of experiments A, B, and C are presented in Table 2 A, B, and C. For each experiment, we registered n triples (m, d., d 2 ) for the adopted mother and both daughters separately withn =20, 17,and24inA,B,andC,respectively. Then we calculated the differences (d, + d 2)-m. These differences are analysed in Table 2 for quartiles I-IV and for the whole experiment. Further we used the observed data (m, d h d 2 ) to verify the hypothesis H, that, under stable experiment conditions, the increment h =d, + drmT is constant. We remind that m- denotes the unobservable true mother protein content.
Distribution and Regulation of Total Protein Content· 169 Table 2. The quartile analysis of the mothers' and daughters' total protein contents and the increment value in experiments A, B, and C; m =mother cells, d., d 2 =sister cells (daughters), (d, + d 2)-m = increment value
total proteins (au)
3000
A
m
N x sd
2000
5
5
5
5
1691 95
1743 174
1720 209
1772 176
5
5
5
5
2071 95
1923 284
2168 454 5 2121 260
2019 698
N II
x
sd N 1000
III
x
IV
N x sd
sd
~
II
III
N x sd
IV
5
5
2442 171
2267 320
5
1946 499
5
5
5
5
2926 225 20 2282 522
2200 388 20 2033 291
2225 238 20 2058 347
1499 637 20 1809 538
quartiles
Fig. 1. The quartile analysis of the total protein content values (au) of mother and daughter cells. _ = mothers, o = daughters.
B
m
x
sd
Consider a single experiment with n triples m, db d 2 • We take for granted that in the population which they represent the absolute difference of protein contents in sister cells has the same distribution in the generation of mothers (where it is denoted by Im-mTI) and the generation of daughters (where it is denoted by Idl-d 2 1). Let symbol>- denote the equality of distributions. So we assume that
5
5
5
sd
2082 579
2284 408
2326 699
N x sd
4
4
4
4
III
2507 46
2710 252
2402 488
2605 688
IV
N x sd
~
N x sd
C
4
4
2024 364 17 2161 701
1340 1060 17 2104 542
6
6
6
6
x
880 255
1018 494
1078 437
1217 979
6
6
6
6
1431
sd
1228 67
1413 898
6
III
N x sd
477 6
1210 573
1620 181
1823 450
IV
N x sd
x
N
(d l + drmT) =O.
4
2315 920 17 2139 806
N
II
(2)
h-h =d, + drmT -
4
2997 78 17 2195 671 m
sd
Since the mean sample increment h is also equal to c, hypothesis H, implies that
4
2090 1623
5
x
N
Clearly, SI is observable but S is not. Under H, (constant increment hypothesis), for each experiment there exists a number c such that:
4
1902 898
2040 288
II
S = m-rn- - (ill-I!!T), SI = d l-d 2 - (d cd 2 )· So (1) is equivalent to the assumption:
4
1465 1010
N
(1) Let m, m-, db and d 2 denote the sample means of ill, mT, db and d 2 • In the experiment we calculate m, db d 2 from the data, mTis not observable. Obviously, the differences in (1) can be replaced by centered differences:
4
1277 370
N
~
x sd
6
6
1441 473
1645 565
6
6
6
6
2518 732 24 1562 727
1582 645 24 1464 571
1673 548 24 1350 530
737 959 24 1253 877
170 . S. Radzikowski et al.
Let us define
IS 2 '
S2 = S + (h-ll) = d, + drm -
1000
(d1 + d2- m).
*
Evidently, S2 is observable and equal to S whenever hypothesis H o is true. Therefore, by (2), H , implies that the distributions of Sand S2 are equal: H,': IS21"-'ISd .
(3)
It follows that rejecting H, ' implies rejecting H o • Our task now is to test (3). Let us start with examining the scatter plots of (lSd, IS21) in the three experiments (Fig. 2, experiments A, B, C). We see from these plots that the sample marginal distributions of IS11 and IS2\ are distinctly different in each case: IS21 tends to assume larger values than IS11. Therefore we should test H,' against the hypothesis that IS21 is stochastically larger than ISli. Let us check first whether it is legitimate to use a test suitable for independent samples. Clearly, IS11and IS21 are the functions of the same n triples (m, db d 2) and are not independent. However, their scatter plots indicate that their dependence is very weak and should not influence the result of testing. Indeed, the related values of the Spearman' s correlation coefficient Ps are: - 0.122 in expo A, - 0.336 in expo B, and - O. 220 in expo C. This suggests a weak tendency to negative dependence, far from the possibility of rejecting the hypothesis of independence between IS11 and IS21 (the corresponding critical values in a one-sided test of inde pendence at the significance level ex = 0.05 are - 0.45 for experiment A, - 0.49 for expo B, and - 0.40 for expo C). Thus, we used the Wilcoxon's statistic W to test H , ' against the hypothesis that IS21 is stochastically larger than ISd. The values ofW are: 313,233, and 466 in experiments A, B, and C, respectively. It follows that hypothesis Ho': IS11"-'IS21 is rejected in each experiment at the significance level ex = 0.02 (the respective critical values of W at this significance level are 337, 240, and 492, respectively). We supplemented the analysis of (1St!, IS21) by the resuits of the Wilcoxon matched-pairs signed rank test . Now, the null hypothesis tested is that the differences IS11- IS21and IS21-I S11 have the same distribution, and the alternative one-sided hypothesis states that IS211St! is stochastically larger than ISII-IS21. Under the null hypothesis, the probabilities that the Wilcoxon signed rank statistic takes on values smaller than its observed value are: 0.005 in experiment A, 0.081 in experiment B, and 0.0013 in experiment C. Then the null hypothesis is strongly rejected in experiments A and C. The result of experiment B is not significant at the level 0.05, but is close to the corresponding critical value. Moreover, the three results for A, B, and C considered jointly by
* *
500
* *
Fig. 2. Scatter plots of (\S21, IStll.in th...ree independentexperj- ~
=d 1-d2-(d 1-d2); S1 =d l +d 2-m-(d1
+ d2 - Iii) (see Results).
* *
*
*
*
* *
.",
0
*
*
*
* IS11
A
100
IS 2 ' 2500~
*
300
1000
* *
*
*
* *
* 500
*
*
*
*
*
* *
0
* 1S11
B
200
IS 21
400
600
800
1000 1200
*
17oo~ 1000
500
*
* *
* **
*
* *
*
*
• * *
500
* * **
ments A, B, and C. S2
*
*
* *
*
* *
0
* 1S11
C
200
400
600
800
1000 1200 1400
Distribution and Regulation of Total Protein Content . 171
means of the "test of tests" procedure reject the null hypothesis at the level 0.005!
2. The Relation Between the Initial (G 1) Total Protein Content and the Length of the Cell Cycle In order to study the relation between total protein content and the GT we performed another experiment. We adopted the total protein content values of the sisters of mother cells as the initial G I content values, and analyzed the G I total protein levels and the length of the cell cycle of the respective sister cells. The values of the total protein content in G I were divided according to the quartiles which represent the values of the total protein content reaching 25%, 50%, and 75%. For each quartile we calculated the mean value of the total protein content and the mean value of the length of the cell cycle. The analysis of the results presented in Fig. 3 indicates that the cells with the low value of the total protein content (quartile I, the mean value equal to 1726.9 au) have the mean generation time of 11 h 23 min. Since the cells in quartiles II and III have similar means of the total protein content, we combined them into one group with the mean value of proteins equal to 2177.4 au. Their mean generation time was shorter than in quartile I, equaling 10 h 45 min. The mean generation time of the cells in quartile IV (the mean value of the total protein content equal to 2814 au) was equal to 10 h 24 min. These results indicate that the cells with low protein contents have a rather long generation time, and when the protein content is higher, the mean generation time is shorter (Fig. 3).
proteins (au)
time (h)
3500
13.00
3250
12.30
3000
12.00
2750
11.30
2500
11.00
2250
10.30
2000
10.00
1750
9.30
1500
9.00
0
00.00 II, III
IV
quarter
Fig. 3. The quartile analysis of the initial total protein content (G I ) and the length of the cell cycle. _ = mothers' protein content (au), 0 = generation time.
We were in fact interested in the dependence of mon GT, where mT was not observable. However, our starting point was the hypothesis of the lack of correlation between the two pairs of results: a pair (m, mT)concerning protein contents and a pair of GT's for both cells, and so we could take advantage of the known positive dependence within these pairs. Then, we could use the observable values m for one cell and GT for the other cell, and test their independence as a substitute null hypothesis. The observed value of the Spearman's rho in the experiment of size n = 37 is - 0.28 while the critical value of the one-sided test is - 0.275 at the significance level 0.05. So we find that GT is negatively dependent on m. Since rri- is positively dependent on m, the obtained result suggests negative dependence of mT on GT. Discussion It is known that cytokinesis is never a fully precise bipartition, and in consequence, the masses of the sibling cells usually differ to some extent [17]. Thus, if there were no regulatory mechanisms to eliminate the differences in cell masses arising during each cell division, the variation of the cell size within a certain population would increase without limits. However, this does not occur, and the cell masses of the individual cells show only limited variations [16,17,18,19,29,30]. This suggests the existence of a mechanism which regulates the mass of the cells in a population. This conjecture finds strong support in our present results regarding the division of Trithigmostoma. It was suggested that regulation of cell mass in Paramecium [2, 10] and Tetrahymena [27] occurs through an incremental model (in such regulative system all cells synthesize a standard amount of increment in cell mass during each cell cycle, regardless of the initial values). In the present paper we analysed the congruence between the data on the regulation of total protein in Trithigmostoma steini and the proposed model of regulation (increment). Although the direct measurements of protein levels in true mother was not possible, the statistical analysis of the results allowed to reject decisively a hypothesis of a constant (standard in experimental conditions) protein increment in the cells (see Results). In all our experiments the initial (adopted mother) total protein content influences the increment value, giving a slight deviation from theoretical values. This is in sharp disagreement with a model of a constant increment in all cells, and justifies the rejection of the assumption of the proposed model in the case of Trithigmostoma steini. The results of Rasmussen and Berger [26] in studies of Paramecium are also incongruent with the model of a constant increment. Berger [2] (p, 113) states that downward regulation of cell mass in Paramecium does "occur more rapidly than would be expected from a purely incremental mechanism alone". It is widely accepted that the "yeast" mechanism of cell cycle regulation is universal for all eucaryotic organisms and involves similar sequences of events. The crucial points in cell cycle regulation are considered the
172 . S. Radzikowski et aI.
beginning of S phase and M phase with the participation of genes from the groups cdc and wee, active in regulation of these phases. The products of these genes take part in the molecular re~ulatory processes of the cell cycle, e.g. the protein p34 c c2 [6,21]. The discovery of yeast gene cdc2 homologues in other organisms showed that the proteins produced are similar but not identical. In man, protein 34 kDa has a sequence homologous only in 63 % with the yeast product. Another example is a phosphorylation of p34 cdc2 in yeast at Tyr-15, whereas in murine cells both Tyr-15 and Thr-14 are phosphorylated. Homologues of the gene cdc2, found in Paramecium, code for a protein homologous in 50-60% with the mammalian sequences [31]. These similarities of the molecular mechanisms might suggest that the observed patterns regulating individual cell components are similar, although the details may differ in various eukaryotes. We observed that the length of the cell cycle in Trithigmostoma depends on the initial total protein content in G] phase of the cell cycle, i.e. the cells with a low total protein content tend to have a longer generation time than the cells with high protein levels. This may indicate that the cells entering the cell cycle with a low total protein content require a longer generation time to complete synthesis and to increase their protein content. This is in agreement with observations [2,28] made in Paramecium and Tetrahymena. A possible interpretation of the observed interdependence is that the duration of the cell cycle constitutes the main factor limiting the total protein level, provided that the rate of protein accumulation is constant throughout the cell cycle. This was shown earlier in studies of a conical mutant of Tetrahymena pyriformis by Schaffer and Cleffmann [28], who suggested that differences between sisters in cell size introduced at division are eliminated exclusively by cell cycle duration and not by growth rate. Thus, faced with a constant rate of protein accumulation , the cells do not have enough time for synthesis leading to the doubling of the initial mass. Consequently, the protein levels in daughter cells are lowered. Higher levels of proteins in true mothers leads to shorter generation time, and in consequence a greater decrease of protein levels is observed in daughter cells. There are studies [9, 13, 28] showing that the cells whose mass is larger than the average mass in a population have a reduced G] phase of cell cycle. Kimball et al. [19] and Berger [1] postulate that most if not all of the variation in the cell mass is eliminated within a single generation. Our data do not allow to decide the question whether one cell cycle is sufficient or not to eliminate deviation from the average protein content. Among more interesting problems is a monitoring in total protein content G] (initial) or G 2 from the preceding generation in Trithigmostoma and transmission of this "information" which influences the duration of the remaining part of the cell cycle. Nurse and Fantes [11,23] state that if the cells are perturbed so that they divide at a different size than normal, then this is corrected in the
next cell cycle. The cells that are larger after division grow less before the next division so that division takes place in cells closer to the normal size. It implies the ability of cells to discriminate among the phases of the cell cycle. Nurse [22] suggests that it is p34 cdc2 which provides a memory for the cell of whether it is in G] or in G 2 • It has been suggested that in Paramecium and Tetrahymena the rate of protein synthesis in the cell is a function of its macronuclear DNA content [2, 28]. Berger [1, 2] postulates that in ciliates the mechanism which controls the rate of protein synthesis isan essential element of the cell cycle control circuit. Studies on CHO [10] suggested that the duration of G] and S phases depends on the amount of RNA in the cell. The cells with a high level of RNA have clearly shorter G] and S phases than the cells with the low RNA content. Authors of these studies postulate that the rate of transition of G] into Sphase depends on the number of ribosomes in the cell. The results of the studies on Tetrahymena support this conclusion [4, 27]. Cleffmann and Miyake [8] and Cleffmann er al. [7] demonstrated that the length of the cell cycle in Tetrahymena reflects the amounts of RNA in the cell - the lower the level of RNA the longer the cell cycle and vice versa. Studies on undifferentiated PCC3 (EC) cells showed that the postmitotic differences in the protein contents between sister cells have a minor impact on the cell mass variability in the population but they affect the length of cell interphases [32]. Rasmussen and Berger [26, 27] suggest among others that the regulation of cell mass in Paramecium involves a shortening of the cell cycle in the cells with an increased cell mass: as the initial cell mass increases there is a progressive decrease in the length of the cell cycle up to 75 % of its usual time. This is probably achieved by the shortening of the G1/S interval. Studies on budding yeast (S. cerevisiae) [14, 15, 21] demonstrated that after the mitosis the mother cell is large enough to enter a new cell cycle immediately, but the much smaller daughter bud has to pass through a period of intense growth which significantly delays its entry into the new cell cycle. Hola and Riley [13] have compared the cell volume, growth rate, and interdivision times of mammalian epithelial cells in culture, and concluded that the unequal distribution of cytoplasm at cytokinesis may be one of the causes of heterogeneity ofthe length of cell generation time in the population of these cells. In conclusion we would like to point out that the mechanism responsible for the observed interdependence between an initial protein content and the amount of proteins synthesized in a cell cycle are likely to be found among the molecular processes of cell cycle regulation' which therefore have been shown to be universal for all eukaryotes.
Acknowledgements This work was supported by grant 14-501/v GR-45 Poland to Dr. S. Radzikowski.
Distribution and Regulation of Total Protein Content . 173
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mass and initiation of DNA synthesis in mouse fibroblast in vitro. Exptl, Cell Res., 40, 12-20. Kimball R. E (1967): Persistent intraclonal variation in cell dry mass and DNA content in Paramecium aurelia. Expt\. Cell Res., 48, 378-394. Kimball R. E, Caspers son T. 0., Svensson G., and Carlson L. (1959): Quantitative cytochemical studies of Paramecium aurelia. 1. Growth in total dry weight measured by the scanning interference microscope and X-ray absorption methods. Exptl. Cell Res., 17, 160 -172. Kimball R. E, Perdue S. w., Chu E. H. Y., and Oritz J. R. (1971): Microphotometric and autographic studies on the cell cycle size during growth and decline of Chinese hamster cell cultures. Exptl, Cell Res., 66, 17-94. Lew D. J., Marini N. ]., and Reed S. 1. (1992): Different G j cyclins control the timing of cell cycle commitment in mother and daughter cells of the budding yeast S. cerevisiae. Cell, 69, 317-327. Nurse P. (1992): Eucariotic cell-cycle contro\. Bioch. Soc. Trans., 20, 239-242. Nurse P. and Fantes A. P. (1981): Cell cycle controls in fission yeast: a genetic analysis. In: John P. C. L. (ed.): The cell cycle., pp. 85-105. Univ. Press, Cambridge. Radzikowski S. and Golembiewska M. (1977): Morphology and culture method of Chilodonella steini. Protistologica, 13, 381-389. Radzikowski S. and Golembiewska-Skoczylas M. (1985): Relation between macronuclear DNA and total protein content and generation time in the Chilodonella steini (Ciliata) sister cells. Protoplasma, 129, 44-51. Rasmussen C. D. and Berger J. D. (1982): Downward regulation of cell size in Paramecium tetraurelia: effects of increased cell size, with or without increased DNA content, on the cell cycle. J. Cell Sci., 57, 315 -329. Rasmussen C. D., Berger J. D., and Ada S.-L. Ching (1986): Effects of increased cell mass and altered gene dosage on the timing of initiation of macronuclear DNA synthesis in Paramecium tetraurelia. Exptl, Cell Res., 165,53-62. Schafer E. and Cleffmann G. (1982): Division and growth kinetics of the division mutant of Tetrahymena. Exptl, Cell Res., 137,277-284. Sennerstam R. (1988): Partition of protein (mass) to sister cell pairs at mitosis: are-evaluation. J. Cell Sci., 90, 301306. Sennerstam R. and Stromberg J. O. (1984): A comparative study of the cell cycles of nullipotent and multipotent embryonal carcinoma cell lines during exponential growth. Dev. BioI., 103, 221-229. Tang L., Pelech S. R., and Berger J. D. (1993): Isolation and characterization of cdc2 genes from Paramecium tetraurelia. Abstracts, IX Intern. Congr. Protozoo\. Berlin, Germany, p. 126. Yen A., Fried]., Kitahara T. A., Strife A., and Clarkson B. D. (1975): The kinetic significance of cell size. 1. Variation of cell cycle parameters with size measured at mitosis. Exptl, Cell Res., 95, 205 -302.
Key words: Trithigmostoma steini - Sister cells - Protein content - Generation time - Size control Stefan Radzikowski, University of Warsaw, Institute of Zoology, Department of Zoology, Krakowskie Przedrniescie 26/28 00-927/1 Warszawa, Poland '
European Journal of
PROTISTOLOGY
Pattern of Total Protein Content Regulation in Trithigmostoma steini (Chilodonella steinij Stefan Radzikowski, "Elzbleta Pleszczynska, Matgorzata Gotembiewska-Skoczylas, and Beata Sapetto-Rebow University of Warsaw, Institute of Zoology, Warsaw, Poland and *Institute of Computer Science of the Polish Academy of Science, Warsaw, Poland
SUMMARY A series of experiments on the ciliate Trithigmostoma steini was conducted in order to analyze interrelation of total protein contents between two daughter cells and their "adopted mother" (mother's sister) cell. We discuss the pattern of total proteins regulation in respect to the incremental model [1, 2,4]. The results allow to reject the hypothesis of constant protein increment in Trithigmostoma steini. Instead we suggest that the increment tends to diminish when the true mother's content of protein is large. We also noticed a negative correlation between the length of the cell cycle and the initial protein content of the cell. However, one cell cycle is not a sufficient period to investigate this effect. The regulation processes observed in Trithigmostoma steini seem to be similar to those of other ciliates and we believethat they are based on molecular control mechanisms known for other eukaryotes. Introduction In ciliates, the partition of the cell mass during division is not precise, and the newly formed sister cells differ in their protein content. This phenomenon is probably responsible for the striking individual variability in the cell total protein contents in the population. However, in Trithigmostoma, in spite of the great variability in the total protein contents, the mean values of this parameter remain constant for many generations at stable culture conditions. Moreover, the total protein contents of sister cells are always positively, strongly, and linearly codependent [25]. These findings suggest the existence of (a) special mechanism(s) which either regulate(s) the total protein level in the course ofthe cell cycle or operate(s) during cytokinesis and maintain(s) the differences between protein content of sister cells within certain limits
[2,25]. In the present study we analysed: - the interdependence of total protein contents between two daughter cells and their "adopted mother" (mother's sister) cell, and © 1995 by Gustav Fischer Verlag, Stuttgart
- the correlation between initial G 1 levels of total protein and the length of the cell cycle.
Material and Methods The study was based on a clone of Trithigmostoma steini that is nonmature for conjugation. In this paper we present experiments carried out on more than 450 cells. The methods of cultivation were as described earlier [24]. All experiments were conducted at 19 DC in constant light of 500 lux. We have used the "sister model" of experiment [5,9, 12,25]. The "sister model" was thoroughly checked with respect to different parameters. We accepted that the total protein levels of sister cellswere always positively and linearly codependent in all experiments. This relationship does not depend on the kind of the cell (prater or opisthe). In all experiments, the values of mean absolute differences, expressed as percentage of the mean value, between total protein content of sister cells never exceeded the values ofVC% of the total population. On the basis of the data described in Table 1, the mean absolute difference between sister cells is 10.6% of the mean value while VC% is equal to 17%. The mean absolute difference 0932-4739-95-0031-0167$3.50-0
168 . S. Radzikowski et aI. obtained for the same cells after accidental formation of sister pairs was 23.6%. To investigate the amount of total protein in the mother cell and its progeny we employed the following experimental protocol: after the cell division, one cell was fixed immediately and the value of its protein content was denoted as the initial [9,25] protein level value. The second cell from each pair was grown for one more full cell cycle and its generation time was measured. After the division of this cell, its daughter cellswere immediately fixed and the protein contents of both cells were measured. The terminology and notation used in the paper are as follows: we distinguish between the true mother cell (with unobservable protein contents denoted m-) and the adopted mother cell called, for short, a mother cell (with observable protein contents denoted m). The daughters' protein contents, registered in random order, are denoted d l and d 2 • The unobservable increment is (d, + d 2)-mT, its observable counterpart is (d l + d 2)-m. Since differences between mT and m happen to be substantial, we investigated only the global and not the individual effects of substituting observable m for unobservable Hl-p, The fixation and staining protocols for total protein with 2,4-dinitrofluorobenzene (DNFB) were as previously described [20, 25, 28]. The measurements were made with a SPM-05 scanning cytophotometer (Zeiss, Oberkochen, Germany) at 438 nm wave-length. Standard statistical methods including the Wilcoxon's and Spearman's statistic were used for the analysis of the suitably transformed data [3].
Table 1. Parameters describing protein contents of mother and daughter cells (Gl ) in au (arbitrary units): total and quartile distinctions (I-IV) Variable
Mothers
Daughters
N x sd
37 2217.62 421.26 990.1 7 3266.50 19
57 2050.21 349. 76 1417.35 2953.38 17
8 1651 326 990 1944
13 1769 207 1482 2191
10 2057 68 1959 2128
14 2022 315 1417 2410
11 2285 133 2135 2497
17 2187 309 1602 2953 14 2176 385 1521 2801
rrun,
max. VC% N x sd min.
max. II
N
x sd min. max. III
N
x sd min.
max. IV
N
x sd Results
mm.
max.
8 2851 235 2575 3266
1. The Total Protein Content in G 1 Phase - the Relationship Between Mother (Adopted Mother) and Daughter Cells We have analysed the total protein content of adopted mother(m) and daughter (d, and d 2 ) cells. In the first experiment (Table 1) we analysed 94 cells. The means, standard deviations, and ranges of the total protein contents were similar in both the mother and its daughter cells . The total protein content of mother cells (G 1 ) ranged from 990 au to 3266 au, with the mean value equal to 2217 au . The total protein content in daughter cells (G 1 ) ranged from 1417 au to 2953 au with the mean value equal to 2050 au. The values of the total protein contents in individual lines of mother - daughter cells were variable. We hypothesised that the protein level in daughter cells may depend on the protein level of their mother cells . Thus we carried out the analysis of such dependence for mother cells with extreme values (low or high) of protein levels. We noticed that when the protein content value of the mother cell was very low the daughter cell 's protein contents tended to be higher, whereas when the total protein level of the mother cell was high, the daughter cells tended to have lower protein contents. To investigate this phenomenon more closely we performed the quartile analysis of the data. We grouped the mother cells into quartiles (I, II, III, IV) whereby each contained 25 % of the total number of cells from the low-
est to the highest protein content. For each quartile we calculated the mean value and standard deviation of the total protein content for mothers and their daughter cells. The results are presented in the second part of Table 1 (see also Fig. 1). The analysis of these data shows that in quartile I the mean content of total protein in mother cells is lower than that of the daughters by 118 au (7.1 %), whereas in quartiles II, III and IV daughter cells have lower total protein contents than their mothers by 35 au (1. 7 %), 98 au (4 %) and 675 au (23.7%), respectively. This descriptive analysis of the first experiment was followed by a more formal analysis of the next experiments (A, B, C) which disproved the hypothesis of constant increment. The results of experiments A, B, and C are presented in Table 2 A, B, and C. For each experiment, we registered n triples (m, d., d 2 ) for the adopted mother and both daughters separately withn =20, 17,and24inA,B,andC,respectively. Then we calculated the differences (d, + d 2)-m. These differences are analysed in Table 2 for quartiles I-IV and for the whole experiment. Further we used the observed data (m, d h d 2 ) to verify the hypothesis H, that, under stable experiment conditions, the increment h =d, + drmT is constant. We remind that m- denotes the unobservable true mother protein content.
Distribution and Regulation of Total Protein Content· 169 Table 2. The quartile analysis of the mothers' and daughters' total protein contents and the increment value in experiments A, B, and C; m =mother cells, d., d 2 =sister cells (daughters), (d, + d 2)-m = increment value
total proteins (au)
3000
A
m
N x sd
2000
5
5
5
5
1691 95
1743 174
1720 209
1772 176
5
5
5
5
2071 95
1923 284
2168 454 5 2121 260
2019 698
N II
x
sd N 1000
III
x
IV
N x sd
sd
~
II
III
N x sd
IV
5
5
2442 171
2267 320
5
1946 499
5
5
5
5
2926 225 20 2282 522
2200 388 20 2033 291
2225 238 20 2058 347
1499 637 20 1809 538
quartiles
Fig. 1. The quartile analysis of the total protein content values (au) of mother and daughter cells. _ = mothers, o = daughters.
B
m
x
sd
Consider a single experiment with n triples m, db d 2 • We take for granted that in the population which they represent the absolute difference of protein contents in sister cells has the same distribution in the generation of mothers (where it is denoted by Im-mTI) and the generation of daughters (where it is denoted by Idl-d 2 1). Let symbol>- denote the equality of distributions. So we assume that
5
5
5
sd
2082 579
2284 408
2326 699
N x sd
4
4
4
4
III
2507 46
2710 252
2402 488
2605 688
IV
N x sd
~
N x sd
C
4
4
2024 364 17 2161 701
1340 1060 17 2104 542
6
6
6
6
x
880 255
1018 494
1078 437
1217 979
6
6
6
6
1431
sd
1228 67
1413 898
6
III
N x sd
477 6
1210 573
1620 181
1823 450
IV
N x sd
x
N
(d l + drmT) =O.
4
2315 920 17 2139 806
N
II
(2)
h-h =d, + drmT -
4
2997 78 17 2195 671 m
sd
Since the mean sample increment h is also equal to c, hypothesis H, implies that
4
2090 1623
5
x
N
Clearly, SI is observable but S is not. Under H, (constant increment hypothesis), for each experiment there exists a number c such that:
4
1902 898
2040 288
II
S = m-rn- - (ill-I!!T), SI = d l-d 2 - (d cd 2 )· So (1) is equivalent to the assumption:
4
1465 1010
N
(1) Let m, m-, db and d 2 denote the sample means of ill, mT, db and d 2 • In the experiment we calculate m, db d 2 from the data, mTis not observable. Obviously, the differences in (1) can be replaced by centered differences:
4
1277 370
N
~
x sd
6
6
1441 473
1645 565
6
6
6
6
2518 732 24 1562 727
1582 645 24 1464 571
1673 548 24 1350 530
737 959 24 1253 877
170 . S. Radzikowski et al.
Let us define
IS 2 '
S2 = S + (h-ll) = d, + drm -
1000
(d1 + d2- m).
*
Evidently, S2 is observable and equal to S whenever hypothesis H o is true. Therefore, by (2), H , implies that the distributions of Sand S2 are equal: H,': IS21"-'ISd .
(3)
It follows that rejecting H, ' implies rejecting H o • Our task now is to test (3). Let us start with examining the scatter plots of (lSd, IS21) in the three experiments (Fig. 2, experiments A, B, C). We see from these plots that the sample marginal distributions of IS11 and IS2\ are distinctly different in each case: IS21 tends to assume larger values than IS11. Therefore we should test H,' against the hypothesis that IS21 is stochastically larger than ISli. Let us check first whether it is legitimate to use a test suitable for independent samples. Clearly, IS11and IS21 are the functions of the same n triples (m, db d 2) and are not independent. However, their scatter plots indicate that their dependence is very weak and should not influence the result of testing. Indeed, the related values of the Spearman' s correlation coefficient Ps are: - 0.122 in expo A, - 0.336 in expo B, and - O. 220 in expo C. This suggests a weak tendency to negative dependence, far from the possibility of rejecting the hypothesis of independence between IS11 and IS21 (the corresponding critical values in a one-sided test of inde pendence at the significance level ex = 0.05 are - 0.45 for experiment A, - 0.49 for expo B, and - 0.40 for expo C). Thus, we used the Wilcoxon's statistic W to test H , ' against the hypothesis that IS21 is stochastically larger than ISd. The values ofW are: 313,233, and 466 in experiments A, B, and C, respectively. It follows that hypothesis Ho': IS11"-'IS21 is rejected in each experiment at the significance level ex = 0.02 (the respective critical values of W at this significance level are 337, 240, and 492, respectively). We supplemented the analysis of (1St!, IS21) by the resuits of the Wilcoxon matched-pairs signed rank test . Now, the null hypothesis tested is that the differences IS11- IS21and IS21-I S11 have the same distribution, and the alternative one-sided hypothesis states that IS211St! is stochastically larger than ISII-IS21. Under the null hypothesis, the probabilities that the Wilcoxon signed rank statistic takes on values smaller than its observed value are: 0.005 in experiment A, 0.081 in experiment B, and 0.0013 in experiment C. Then the null hypothesis is strongly rejected in experiments A and C. The result of experiment B is not significant at the level 0.05, but is close to the corresponding critical value. Moreover, the three results for A, B, and C considered jointly by
* *
500
* *
Fig. 2. Scatter plots of (\S21, IStll.in th...ree independentexperj- ~
=d 1-d2-(d 1-d2); S1 =d l +d 2-m-(d1
+ d2 - Iii) (see Results).
* *
*
*
*
* *
.",
0
*
*
*
* IS11
A
100
IS 2 ' 2500~
*
300
1000
* *
*
*
* *
* 500
*
*
*
*
*
* *
0
* 1S11
B
200
IS 21
400
600
800
1000 1200
*
17oo~ 1000
500
*
* *
* **
*
* *
*
*
• * *
500
* * **
ments A, B, and C. S2
*
*
* *
*
* *
0
* 1S11
C
200
400
600
800
1000 1200 1400
Distribution and Regulation of Total Protein Content . 171
means of the "test of tests" procedure reject the null hypothesis at the level 0.005!
2. The Relation Between the Initial (G 1) Total Protein Content and the Length of the Cell Cycle In order to study the relation between total protein content and the GT we performed another experiment. We adopted the total protein content values of the sisters of mother cells as the initial G I content values, and analyzed the G I total protein levels and the length of the cell cycle of the respective sister cells. The values of the total protein content in G I were divided according to the quartiles which represent the values of the total protein content reaching 25%, 50%, and 75%. For each quartile we calculated the mean value of the total protein content and the mean value of the length of the cell cycle. The analysis of the results presented in Fig. 3 indicates that the cells with the low value of the total protein content (quartile I, the mean value equal to 1726.9 au) have the mean generation time of 11 h 23 min. Since the cells in quartiles II and III have similar means of the total protein content, we combined them into one group with the mean value of proteins equal to 2177.4 au. Their mean generation time was shorter than in quartile I, equaling 10 h 45 min. The mean generation time of the cells in quartile IV (the mean value of the total protein content equal to 2814 au) was equal to 10 h 24 min. These results indicate that the cells with low protein contents have a rather long generation time, and when the protein content is higher, the mean generation time is shorter (Fig. 3).
proteins (au)
time (h)
3500
13.00
3250
12.30
3000
12.00
2750
11.30
2500
11.00
2250
10.30
2000
10.00
1750
9.30
1500
9.00
0
00.00 II, III
IV
quarter
Fig. 3. The quartile analysis of the initial total protein content (G I ) and the length of the cell cycle. _ = mothers' protein content (au), 0 = generation time.
We were in fact interested in the dependence of mon GT, where mT was not observable. However, our starting point was the hypothesis of the lack of correlation between the two pairs of results: a pair (m, mT)concerning protein contents and a pair of GT's for both cells, and so we could take advantage of the known positive dependence within these pairs. Then, we could use the observable values m for one cell and GT for the other cell, and test their independence as a substitute null hypothesis. The observed value of the Spearman's rho in the experiment of size n = 37 is - 0.28 while the critical value of the one-sided test is - 0.275 at the significance level 0.05. So we find that GT is negatively dependent on m. Since rri- is positively dependent on m, the obtained result suggests negative dependence of mT on GT. Discussion It is known that cytokinesis is never a fully precise bipartition, and in consequence, the masses of the sibling cells usually differ to some extent [17]. Thus, if there were no regulatory mechanisms to eliminate the differences in cell masses arising during each cell division, the variation of the cell size within a certain population would increase without limits. However, this does not occur, and the cell masses of the individual cells show only limited variations [16,17,18,19,29,30]. This suggests the existence of a mechanism which regulates the mass of the cells in a population. This conjecture finds strong support in our present results regarding the division of Trithigmostoma. It was suggested that regulation of cell mass in Paramecium [2, 10] and Tetrahymena [27] occurs through an incremental model (in such regulative system all cells synthesize a standard amount of increment in cell mass during each cell cycle, regardless of the initial values). In the present paper we analysed the congruence between the data on the regulation of total protein in Trithigmostoma steini and the proposed model of regulation (increment). Although the direct measurements of protein levels in true mother was not possible, the statistical analysis of the results allowed to reject decisively a hypothesis of a constant (standard in experimental conditions) protein increment in the cells (see Results). In all our experiments the initial (adopted mother) total protein content influences the increment value, giving a slight deviation from theoretical values. This is in sharp disagreement with a model of a constant increment in all cells, and justifies the rejection of the assumption of the proposed model in the case of Trithigmostoma steini. The results of Rasmussen and Berger [26] in studies of Paramecium are also incongruent with the model of a constant increment. Berger [2] (p, 113) states that downward regulation of cell mass in Paramecium does "occur more rapidly than would be expected from a purely incremental mechanism alone". It is widely accepted that the "yeast" mechanism of cell cycle regulation is universal for all eucaryotic organisms and involves similar sequences of events. The crucial points in cell cycle regulation are considered the
172 . S. Radzikowski et aI.
beginning of S phase and M phase with the participation of genes from the groups cdc and wee, active in regulation of these phases. The products of these genes take part in the molecular re~ulatory processes of the cell cycle, e.g. the protein p34 c c2 [6,21]. The discovery of yeast gene cdc2 homologues in other organisms showed that the proteins produced are similar but not identical. In man, protein 34 kDa has a sequence homologous only in 63 % with the yeast product. Another example is a phosphorylation of p34 cdc2 in yeast at Tyr-15, whereas in murine cells both Tyr-15 and Thr-14 are phosphorylated. Homologues of the gene cdc2, found in Paramecium, code for a protein homologous in 50-60% with the mammalian sequences [31]. These similarities of the molecular mechanisms might suggest that the observed patterns regulating individual cell components are similar, although the details may differ in various eukaryotes. We observed that the length of the cell cycle in Trithigmostoma depends on the initial total protein content in G] phase of the cell cycle, i.e. the cells with a low total protein content tend to have a longer generation time than the cells with high protein levels. This may indicate that the cells entering the cell cycle with a low total protein content require a longer generation time to complete synthesis and to increase their protein content. This is in agreement with observations [2,28] made in Paramecium and Tetrahymena. A possible interpretation of the observed interdependence is that the duration of the cell cycle constitutes the main factor limiting the total protein level, provided that the rate of protein accumulation is constant throughout the cell cycle. This was shown earlier in studies of a conical mutant of Tetrahymena pyriformis by Schaffer and Cleffmann [28], who suggested that differences between sisters in cell size introduced at division are eliminated exclusively by cell cycle duration and not by growth rate. Thus, faced with a constant rate of protein accumulation , the cells do not have enough time for synthesis leading to the doubling of the initial mass. Consequently, the protein levels in daughter cells are lowered. Higher levels of proteins in true mothers leads to shorter generation time, and in consequence a greater decrease of protein levels is observed in daughter cells. There are studies [9, 13, 28] showing that the cells whose mass is larger than the average mass in a population have a reduced G] phase of cell cycle. Kimball et al. [19] and Berger [1] postulate that most if not all of the variation in the cell mass is eliminated within a single generation. Our data do not allow to decide the question whether one cell cycle is sufficient or not to eliminate deviation from the average protein content. Among more interesting problems is a monitoring in total protein content G] (initial) or G 2 from the preceding generation in Trithigmostoma and transmission of this "information" which influences the duration of the remaining part of the cell cycle. Nurse and Fantes [11,23] state that if the cells are perturbed so that they divide at a different size than normal, then this is corrected in the
next cell cycle. The cells that are larger after division grow less before the next division so that division takes place in cells closer to the normal size. It implies the ability of cells to discriminate among the phases of the cell cycle. Nurse [22] suggests that it is p34 cdc2 which provides a memory for the cell of whether it is in G] or in G 2 • It has been suggested that in Paramecium and Tetrahymena the rate of protein synthesis in the cell is a function of its macronuclear DNA content [2, 28]. Berger [1, 2] postulates that in ciliates the mechanism which controls the rate of protein synthesis isan essential element of the cell cycle control circuit. Studies on CHO [10] suggested that the duration of G] and S phases depends on the amount of RNA in the cell. The cells with a high level of RNA have clearly shorter G] and S phases than the cells with the low RNA content. Authors of these studies postulate that the rate of transition of G] into Sphase depends on the number of ribosomes in the cell. The results of the studies on Tetrahymena support this conclusion [4, 27]. Cleffmann and Miyake [8] and Cleffmann er al. [7] demonstrated that the length of the cell cycle in Tetrahymena reflects the amounts of RNA in the cell - the lower the level of RNA the longer the cell cycle and vice versa. Studies on undifferentiated PCC3 (EC) cells showed that the postmitotic differences in the protein contents between sister cells have a minor impact on the cell mass variability in the population but they affect the length of cell interphases [32]. Rasmussen and Berger [26, 27] suggest among others that the regulation of cell mass in Paramecium involves a shortening of the cell cycle in the cells with an increased cell mass: as the initial cell mass increases there is a progressive decrease in the length of the cell cycle up to 75 % of its usual time. This is probably achieved by the shortening of the G1/S interval. Studies on budding yeast (S. cerevisiae) [14, 15, 21] demonstrated that after the mitosis the mother cell is large enough to enter a new cell cycle immediately, but the much smaller daughter bud has to pass through a period of intense growth which significantly delays its entry into the new cell cycle. Hola and Riley [13] have compared the cell volume, growth rate, and interdivision times of mammalian epithelial cells in culture, and concluded that the unequal distribution of cytoplasm at cytokinesis may be one of the causes of heterogeneity ofthe length of cell generation time in the population of these cells. In conclusion we would like to point out that the mechanism responsible for the observed interdependence between an initial protein content and the amount of proteins synthesized in a cell cycle are likely to be found among the molecular processes of cell cycle regulation' which therefore have been shown to be universal for all eukaryotes.
Acknowledgements This work was supported by grant 14-501/v GR-45 Poland to Dr. S. Radzikowski.
Distribution and Regulation of Total Protein Content . 173
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18 19
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22 23 24 25
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Key words: Trithigmostoma steini - Sister cells - Protein content - Generation time - Size control Stefan Radzikowski, University of Warsaw, Institute of Zoology, Department of Zoology, Krakowskie Przedrniescie 26/28 00-927/1 Warszawa, Poland '