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The Cell Cycle in Amoebae
CHAPTER 17
The Cell Cycle in Amoebae DAVID M. PRESCOTT
I. II. III. IV. V. VI. VII.
Introduction The Subsections of the Cell Cycle Attempts to Induce a Gi Period in Amoeba proteus How is the Cell Cycle Arrested when Amoebae Cease Proliferation? Nucleocytoplasmic Interactions in the Control of D N A Synthesis Some Observations on the G Period Concluding Remarks References 2
467 468 471 472 474 475 476 477
I. Introduction The number of published studies on the cell cycle in amoebae is still small, and most of these have dealt only with various strains of Amoeba proteus. Thus, we know a little about the cell cycle of A. proteus and almost nothing about the cell cycles of other types of amoebae. This state of affairs is due in large measure to the relatively crude and ill-defined culture methods that still must be used in order to grow any of the large, free-living amoebae. These amoebae must still be cultivated on living organisms, primarily on other protozoa and on bacteria, and this makes precise studies of the cell cycle a difficult task. In particular, the presence of living food organisms in the medium and the large amount of food vacuole material inside the amoebae put a severe limit on the kinds of experiments that can be done with radioactive tracers. Even those radioactive tracer studies that are currently feasible with amoebae often lack the degree of precision that is routinely obtainable with cell types grown on well-defined media, e.g., Tetrahymena or mammalian cells in culture. 467
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Along with the disadvantages of the large, free-living amoebae as experimental material, however, there are important advantages. These amoebae are so large that experiments with single cells are relatively easy. Particularly important, the amoebae represent the only cell type that can be maintained in culture continuously and in which nuclear transplantation can be done by a simple and straightforward technique. In addition, the few studies on the cell cycle of amoebae indicate that the arrangement of events in the cycle may be different from other cell types and may provide unique experimental opportunities to gain insight into the phenomena of cell growth and division.
II. The Subsections of the Cell Cycle The life cycle for various kinds of experimental cells is customarily divisible into four subsections defined by D N A replication and cell division, i.e., G S, G , and D . The subsections of the cell cycles for three strains of A. proteus have been studied in three different laboratories with some differences in the results. The first study was reported by Nilova in 1965 on a strain of A. proteus collected near Leningrad and designated as the " L " strain. Nilova added H-thymidine to the culture medium and first detected incorporation into D N A 7-12 hours after cell division. The period of D N A synthesis lasted between 10-14 hours. F r o m her radioautographic results she concluded that at 25°C the " L " strain of A. proteus has a G period of 7-12 hours, followed by an S period of 10-14 hours, and a G period of 4-6 hours. The generation time varied between 25 and 30 hours. I and Lauth (unpublished) have done some preliminary work on this same " L " strain of amoeba (provided by A. L. Yudin of the Cytology Institute in Leningrad) and obtained results different from those of Nilova. Dividing amoebae were individually collected and the resulting daughter cells labeled by addition of 100 ^Ci/ml of H-thymidine (10-20 Ci/mM) to the medium. In three such experiments incorporation of H-thymidine into D N A occurred during the first hour after mitosis and continued for at least 6 hours in most nuclei. The experiments were not extended far enough to define the end of the S period, but one point is clear—in contrast to Nilova's results the G period is less than 1 hour and is possibly absent altogether. In our experiments, however, the amount of labeling (detected by radioautography) varied over a wide range and occasional nuclei failed to show evidence of incorporation of H-thymidine during the first several hours after division. Whether this means that a substantial G period is occasionally present in some amoebae or that H-thymidine some times fails to enter into the thymidine triphosphate pool in a sufficient amount to label D N A , we cannot say. l 5
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The wide variation in the amount of incorporation of H-thymidine into different nuclei is possibly the result of the variation in the amount of food vacuole material from amoeba to amoeba. The food vacuole material is the normal source of thymidine for D N A synthesis in amoebae, and this food reserve greatly reduces the effectiveness of labeling with H-thymidine added to the medium (Ord, 1968). Whether a true G period is occasionally present in " L " strain amoebae will have to be decided by additional experiments, but we are inclined to believe for the present (especially in the light of the situation in other strains) that those amoeba with an apparent G period reflect the inade quacies of our isotope labeling procedures. 3
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In a strain of A. proteus of uncertain origin, cultured in this laboratory for over 10 years, 9 0 % of D N A synthesis, as indicated by H-thymidine incorpora tion, takes place in the period extending from division to about 5 hours into interphase (Ron and Prescott, 1969). Under the culture conditions used (bacteria and Tetrahymena as food), there was considerable variation in the time of termination of this period of major synthesis, some amoebae continuing until 9 hours after division. In about 10% of the amoebae a very low level of H thymidine incorporation continued for at least 13 hours after division. It is clear that in this strain a G period is absent, the S period shows some variability, and the G period occupies much of the cell cycle—in the range of 30-40 hours out of a generation time of 36-48 hours at 23°C. Goldstein and Ron (1969) have provided some additional experiments that may help to explain the large amount of variability observed in the length of the S period. They removed 3 0 - 5 0 % of the cytoplasm from dividing amoebae. The operation did not interfere with division, and two daughter cells were produced each of which was 3 0 - 5 0 % smaller than normal. In these cells, D N A synthesis continued longer than in controls (sham-operated, dividing cells). Goldstein and R o n have suggested that the prolongation of the S period might be due to a depletion of nuclear proteins. This deprivation is incurred because 9 0 % or more of the nuclear proteins in A. proteus are released to the cytoplasm during mitosis, and amputation of 3 0 - 5 0 % of the cytoplasm during mitosis removes roughly a corresponding amount of the nuclear proteins that are temporarily in the cytoplasm. These nuclear proteins normally return to the nucleus during the first 3 hours after mitosis is over (Prescott and Goldstein, 1968). But it is not really known whether or not these nuclear proteins play any role in D N A replication. As Goldstein and R o n point out, the amputation of cytoplasm necessarily means the loss of many other materials besides nuclear proteins. The small daughter cells have less cytoplasmic machinery for metabolism and less food vacuole material than a normal cell, and the prolongation of the S period may be due to a general inadequacy of the cytoplasm to support a full rate of D N A synthesis. In line with this interpretation we might then suppose that the varia tion in the length of the S period seen among normal, unoperated cells may be 3
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due to the varying amounts of food vacuole material present in the amoebae entering the S phase and to the varying amount of metabolic machinery present in one cell to another. Cytokinesis in amoebae rarely produces two exactly equal-sized daughter cells and not infrequently divisions are severely unequal, creating extensive variation in daughter cell sizes that might lead to variations in the length of the S phase. This suggestion that such variation in size gives rise to variation in the length of the S period remains to be tested. The most detailed study of the timing of D N A synthesis has been done by Ord (1968) on another strain of A. proteus. She found no measurable G period. D N A synthesis begins with the completion of cell division and continues for 12-14 hours. Within the S period two peak intervals of D N A synthesis occur: the first extends from about 30 minutes to 4 hours after division and accounts for 75 % of D N A synthesis and the second extends from about 9-13 hours after division and accounts for 1 5 % of D N A synthesis. Whether the two peaks are separated by an interval of low synthesis or an interval of no synthesis at all is not clear. A small amount of H-thymidine incorporation (about 1 0 % of the total) into D N A occurs at other times in the cycle, including incorporation in some cases during early prophase. This latter finding is unusual and deserves further study. In confirmation of Ord's report, Goldstein and R o n (1969) also noted a second peak of D N A synthesis at about 12 hours after cytokinesis in the amoebae maintained in our laboratory. F r o m the several foregoing studies the following conclusions may be drawn (see Fig. 1). In A. proteus D N A synthesis normally begins immediately at the end of cell division, although the possibility of a period sometimes (under different culture conditions?) being present cannot be excluded with certainty (Nilova, 1965). The length of the S period is variable, ending somewhere between x
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Fig. 1. The diagram gives a summary of the main events that compose the cell cycle in A. proteus. D N A synthesis (S period) begins at the end of mitosis (no Gi period). Most of the cell cycle is occupied by the G period. Two hours before mitosis (in G ) the amoeba passes T-l (transition point one). At this point the amoeba becomes insensitive to actinomycin D in the sense that mitosis occurs and D N A synthesis is initiated when mitosis is completed. 2
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5-14 hours after division, possibly depending upon the nutritional state of the individual amoebae. The S period may consist of two subphases of D N A synthesis, a major phase followed by a minor one. A small amount of nuclear D N A synthesis may occur at other times in the cell cycle. Obviously, more work is needed to clear up some of the uncertainties that appear in the published data, particularly, to give us a sharper definition of the timing of D N A synthesis in A. proteus and to explain the variability and anomalous timing of D N A synthesis within and between strains of amoebae.
III. Attempts to Induce a
Period in A. proteus
As a general rule for most cell types (mammalian cells, ciliated protozoa, bacteria, etc.), the cells come to rest in the G j period when proliferation stops. For those few nuclear types that normally lack a G period (Physarum, the micronucleus in some ciliates, A. proteus), there are two alternative possibilities. Either a G period must be induced when proliferation ceases or the cells must come to rest in some subsection of the cycle other than the G period. We have tried in several ways to induce a G period in our strain of A. proteus. During the several days following removal of all food organisms (Tetrahymena) from a mass population of amoebae, the frequency of cell division in the popula tion gradually falls to zero. The last amoebae to divide in this situation are relatively well-starved as judged by the nearly complete absence of any remnants of food vacuoles. All such starving amoebae that manage to go through one final mitosis also incorporate H-thymidine (25 μΟ/ιηΙ added to the inorganic medium) into D N A as soon as mitosis is completed (Prescott and R a o , un published). Thus, any amoeba that has sufficient resources to reach mitosis will also enter D N A replication as soon as mitosis is finished, i.e., a G period cannot be induced by starvation. (It is also possible that the S period in such starving amoebae is greatly extended over the normal length, but this point has not been studied.) Second, as already mentioned, it is possible to remove 50 % of the cytoplasm from a dividing amoeba without affecting mitosis and often not affecting cyto kinesis. The two daughter cells contain only half of the normal amount of cyto plasm, yet such cells begin D N A synthesis without delay as soon as mitosis is completed (Goldstein and Prescott, 1967). D N A synthesis starts whether or not the daughter cells are fed. Goldstein and R o n (1969) have shown that the S period is lengthened in such experimentally treated amoebae. Third, cytokinesis can be prevented in amoebae without affecting mitosis by immersing dividing amoebae in a 1 % solution of bovine albumin. The daughter nuclei in the resulting binucleated amoebae all begin D N A synthesis without a l
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G period. Thus, not unexpectedly, the initiation of the S period is not influenced by the absence of cytokinesis. The fourth observation is perhaps the most striking. When a proliferating population of amoebae is exposed to actinomycin D ( A M D ) (100 /ig/ml or 1 mg/ml), cells continue to divide for 2 hours. Since essentially all R N A synthesis in amoeba is shut down within 15 minutes after addition of 1 mg/ml of A M D to the medium (Rao and Prescott, 1970), we conclude that the synthesis of any R N A that is essential for mitosis and cytokinesis is completed 2 hours before mitosis. Even the very last amoebae that are able to divide in the presence of A M D nevertheless begin D N A synthesis without delay at the end of mitosis. (We do not know whether such cells complete the S period or whether the S period is otherwise changed, e.g., lengthened.) This experiment with A M D shows that mitosis and D N A synthesis in amoeba are indeed tightly coupled. We have concluded (Rao and Prescott, 1970) that the cell becomes committed to enter D N A synthesis at about the same moment that it becomes committed to undergo mitosis. This situation is fundamentally different from what is observed in plant and animal cells generally. In the latter the decision to enter D N A synthesis would appear to be made in the G period, perhaps several hours before the beginning of the S phase. In amoeba, the available evidence shows that the decision to enter D N A synthesis has been made 2 hours before mitosis. Thus, because the decision to begin the S period is made before mitosis in amoeba and after mitosis in most plants and animals, one can conclude that the decision to enter mitosis and the decision to enter D N A synthesis are not obligatorily tied to each other in the same temporal relationship in all cell types. In fact, the decision to enter mitosis and the decision to begin D N A synthesis may not always show the same temporal relationship even in a given, single type of cell. In mammalian cells, for example, a G period is absent altogether under some circumstances; it is conceivable therefore that the decision to enter the next D N A replication may be made before mitosis in some circumstances (no and after mitosis in other circumstances ( a G j of several hours). 1
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IV. How is the Cell Cycle Arrested when Amoebae Cease Proliferation? As already described, the arrest of proliferation in amoebae by starvation does not induce the appearance of a G period. This suggests that amoebae come to rest in the G period of the cell cycle. This would reflect another major difference between amoebae and most other cell types since most cell types come to rest in the cell cycle in the G period (no information is available on the arrest point in the cycle for the two other cases of G ^ l e s s nuclei, i.e., the micronucleus in some ciliates and the nuclei in Physarum). 1
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Since amoebae appear to come to arrest in the G period when cell prolifera tion ceases in a population subjected to starvation, we expected that starving amoebae, upon refeeding, would again take up progress through the G period and subsequently divide. It turned out not to be so simple. When dividing amoebae are selected from a well-fed culture and starved, they finish the S period but do not divide (presumed G arrest). If such amoebae are kept without food for 24 hours after cell division and then refed, they will divide again between 24-48 hours after the refeeding. Within 2 hours after refeeding has begun, however, all amoebae again begin to make D N A . This can be demonstrated by using Tetrahymena labeled with H-thymidine as food or by adding ^ - t h y m i dine (100 /xCi/ml) to the medium in the presence of nonlabeled food organisms. 2
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This period of D N A synthesis that follows the refeeding of starved amoebae lasts for about 6 hours. Whether this synthesis represents a full redoubling of the G amount of D N A has not yet been determined, but it is clear from radioautographs that the rate of synthesis during the 6-hour period is comparable to the rate observed in the normal S period following mitosis. If amoebae that have made extra D N A are again starved for at least 24 hours by withdrawing food as soon as the extra D N A synthesis is over, most of these amoebae will respond to a second refeeding by again synthesizing D N A . Thus by an alternation of long periods of starvation and short periods of refeeding, amoebae are induced to go through several periods of D N A synthesis without the occurrence of mitosis. As yet we do not know the fate of the extra D N A produced following starva tion, except that it is retained by the nucleus and distributed equally between the two daughter nuclei at the subsequent mitosis. In some manner, amoebae that have synthesized extra D N A after starvation must have a means of subse quently reducing their D N A content. If this were not so, we would expect to find a steadily increasing amount of D N A per nucleus in stock cultures of amoebae, since stock cultures are constantly subjected to periods of starvation alternating with periods of feeding. The amount of D N A per nucleus obviously does not undergo a progressive, permanent increase in amoeba cultures. The two strains of amoebae studied so far, our strain and the Leningrad strain, behave in about the same way with respect to the extra synthesis of D N A follow ing a period of starvation. H o w the amoebae regulate their nuclear content of D N A in this situation remains to be determined. Either the amoeba is able to destroy the extra amount of D N A in such a regulated manner that the genetic intactness of the nucleus is not jeopardized, or else an amoeba is able to undergo a reduction division at a mitosis following the buildup of the extra D N A . Finally, this induction of extra D N A synthesis by refeeding of starved amoebae demonstrates that the tight coupling between mitosis and the initiation of D N A synthesis can be uncoupled at least to the extent of the initiation of D N A syn thesis independently of mitosis. 2
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V. Nucleocytoplasmic Interactions in the Control of DNA Synthesis The readiness with which nuclei can be transplanted in amoebae has been used to advantage in the study of the possible role of nucleocytoplasmic inter actions in progress of amoebae through the cell cycle. The first experiments concern the influence of G cytoplasm on the synthesis of D N A in S phase nuclei and the possibility that S phase cytoplasm may induce D N A synthesis in a G nucleus. We reported in 1967 (Prescott and Goldstein) that the incorporation of H-thymidine into D N A in an S phase nucleus was reduced when that nucleus was implanted into a G phase amoeba. We have recently confirmed this result (unpublished). In control experiments an S phase nucleus was transferred into another S phase cell. In such cases the rate of incorporation of H-thymidine was not detectably diminished, indicating that the operation in itself was not disrupting D N A synthesis. In the reciprocal experiment, G nuclei were transferred into S phase cells. Many but not all of such transferred G nuclei again incorporated H-thymidine. In the control experiment involving the transfer of a G nucleus into another G cell, there was no stimulation of incorporation of H-thymidine into the transferred nucleus. We concluded from these observations (7) that the cytoplasm of a G phase cell lacks those properties necessary for the support of D N A synthesis, and (2) that S phase cytoplasm has properties that can induce D N A synthesis in a G nucleus. Ord (1969) has done the same kinds of experiments on other strains of A . proteus with quite different results. In agreement with our experiments her data do show a decline in H-thymidine incorporation in an S phase nucleus after implantation of that nucleus into a G cell, but Ord believes that this reduction is due to a greater dilution of the H-thymidine by endogenous unlabeled thymidine in G cells in comparison to S phase cells. Whether this is the correct explanation for reduced labeling or whether a real decline in the rate of D N A synthesis occurs in S phase nuclei implanted into G cells, could be determined by feeding the recipient G cells on Tetrahymena labeled with H-thymidine for several hours prior to implantation of the S nuclei. This would avoid changes in the rate of H-thymidine incorporation due to unlabeled thymidine derived from the food organisms. Contrary to our results Ord did not detect any stimulation of H-thymidine incorporation in a G nucleus implanted into an S phase cell. The reason for the difference between her results and ours remains to be explained. As Ord points out the explanation is unlikely to be found in differences between strains of amoebae. One should recall, however, that the G nucleus in A. proteus allowed to starve for 24 hours or more does revert into a state in which D N A synthesis is, indeed, reinitiated within 2 hours of refeeding with Tetrahymena. It is not 2
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clear, however, whether this latter stimulation of D N A synthesis in a G nucleus is related to the D N A synthesis we observed in a G nucleus transferred into an S phase cell. Ord also transferred nuclei from late S phase cells into early S phase cells to see whether the early S phase cytoplasm could prevent the switch off of D N A synthesis in late S phase nuclei. Her results show that the late S phase nucleus apparently cannot be stimulated to synthesize extra D N A by implantation into an early S phase cell. Ord concludes that the switching off of D N A synthesis is therefore not prevented by the properties of the early S phase cytoplasm. This again implies the lack of any role of the cytoplasm in the regulation of D N A synthesis in A. proteus. The contradiction between Ord's results and ours should be investigated by further experiments. Particularly, it will be necessary to settle the question of whether the cytoplasm or nucleocytoplasmic interaction plays any role in initiat ing, supporting, or switching off D N A synthesis in A. proteus. 2
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We (Goldstein and Prescott, 1967) have also studied nucleocytoplasmic interactions at other parts of the cell cycle with the objective of determining factors that underlie progress of the cell through its cycle. We asked whether the long G period in amoeba might be due only to a progression of nuclear events. If this were so we might expect that a cell constructed by implanting a late G nucleus into an S phase cell (with the S phase nucleus removed) would divide relatively quickly and without a requirement for cytoplasmic growth. Such cells, however, must feed and grow an average of 90 hours before division can take place. This suggests that " m a t u r a t i o n " of the cytoplasm occurs during the normal G period. A cell constructed of late G cytoplasm and an S nucleus requires on the average about 70 hours to reach division. Thus, the cell containing a mature nucleus (late G ) and an immature cytoplasm (early S) requires more time to progress through the rest of the cycle than does a cell containing an immature nucleus and a mature cytoplasm. We may conclude that the possession of mature cytoplasm permits the cell to reach division sooner. This suggests, indirectly at least, that the maturation of the cytoplasm is important for progress of the cell through the cell cycle. Since most of the cell cycle involved in these experiments is G we are led to believe that cytoplasmic maturation may form the major basis of the G period. We (Goldstein and Prescott, 1967) have obtained other indications that progress through G is dependent upon cytoplasmic maturation, but at present, we do not have any clues regarding the specific nature of the events that 2
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constitute cytoplasmic maturation in the G phase. Certainly the fact that most of the cell cycle in amoeba is occupied by a G period points to a much greater complexity of the G period in amoebae compared to the cells of metaphyta and metazoa. We must consider that the G period in amoeba may contain many cell cycle events and functions that may occur partly or completely in other subsections of the cycle in cells of multicellular organisms. One of the few specific clues about the G period in amoeba is the transition point that is passed about 2 hours before mitosis. Once a G amoeba has passed this point, it is no longer dependent upon R N A synthesis in order to proceed to mitosis, divide, and initiate D N A synthesis in the new, postmitotic nucleus. There are a number of possible experiments of this general type, using inhibitors, that might tell us more about the G period in amoeba. And by no means have the possibilities for using nuclear transplantation to study the cell cycle in amoeba been exhausted. 2
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VII. Concluding Remarks It must be admitted that we still know relatively little about the cell life cycle in the amoeba (summarized in Fig. 1). We do know that mitosis and D N A synthesis are normally tightly coupled, and there is no G period. Second, we know that the long G period is complex, and its completion requires some form of maturation of the cytoplasm. Third, if an amoeba is arrested in the G period by withholding nutrients, the state of the cell changes such that it synthesizes more D N A upon refeeding, instead of simply proceeding to mitosis. It appears that in spite of the starvation, the amoeba progresses to a state in which it is now prepared to initiate D N A synthesis. It would be interesting to determine whether the initiation of D N A synthesis in the starved amoeba is dependent on R N A synthesis. This could be determined in a manner analogous to the demonstration that amoebae progressing normally through the cycle do not require R N A synthesis during the last two hours of G in order to divide and initiate D N A synthesis. Thus, there are meaningful experiments on the amoeba cell cycle that could be done in spite of the inadequacies of present culture methods for this cell. There is no doubt, however, that the task would be far easier if amoeba could be grown axenically on a defined medium. 1
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Acknowledgment The work of the author described in this chapter was supported by a grant from the National Science Foundation.
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References Goldstein, L., and Prescott, D. M. (1967). In "The Control of Nuclear Activity" (L. Goldstein, ed.), pp. 3-17. Prentice-Hall, Englewood-Cliffs, New Jersey. Goldstein, L., and Ron, A. (1969). Exp. Cell Res. 5 5 , 144. Nilova, V. K. (1965). Tsitologiya 7, 633. Ord, M. J. (1968). / . Cell Sci. 3 , 483. Ord, M. J. (1969). Nature (London) 2 2 1 , 964. Prescott, D. M., and Goldstein, L. (1967). Science 1 5 5 , 469. Prescott, D. M., and Goldstein, L. (1968). / . Cell Biol. 3 9 , 404. Rao, M. V. N., and Prescott, D. M. (1970). Exp. Cell Res. 6 2 , 286. Ron, Α., and Prescott, D. M. (1969). Exp. Cell Res. 5 6 , 430.
The Cell Cycle in Amoebae DAVID M. PRESCOTT
I. II. III. IV. V. VI. VII.
Introduction The Subsections of the Cell Cycle Attempts to Induce a Gi Period in Amoeba proteus How is the Cell Cycle Arrested when Amoebae Cease Proliferation? Nucleocytoplasmic Interactions in the Control of D N A Synthesis Some Observations on the G Period Concluding Remarks References 2
467 468 471 472 474 475 476 477
I. Introduction The number of published studies on the cell cycle in amoebae is still small, and most of these have dealt only with various strains of Amoeba proteus. Thus, we know a little about the cell cycle of A. proteus and almost nothing about the cell cycles of other types of amoebae. This state of affairs is due in large measure to the relatively crude and ill-defined culture methods that still must be used in order to grow any of the large, free-living amoebae. These amoebae must still be cultivated on living organisms, primarily on other protozoa and on bacteria, and this makes precise studies of the cell cycle a difficult task. In particular, the presence of living food organisms in the medium and the large amount of food vacuole material inside the amoebae put a severe limit on the kinds of experiments that can be done with radioactive tracers. Even those radioactive tracer studies that are currently feasible with amoebae often lack the degree of precision that is routinely obtainable with cell types grown on well-defined media, e.g., Tetrahymena or mammalian cells in culture. 467
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Along with the disadvantages of the large, free-living amoebae as experimental material, however, there are important advantages. These amoebae are so large that experiments with single cells are relatively easy. Particularly important, the amoebae represent the only cell type that can be maintained in culture continuously and in which nuclear transplantation can be done by a simple and straightforward technique. In addition, the few studies on the cell cycle of amoebae indicate that the arrangement of events in the cycle may be different from other cell types and may provide unique experimental opportunities to gain insight into the phenomena of cell growth and division.
II. The Subsections of the Cell Cycle The life cycle for various kinds of experimental cells is customarily divisible into four subsections defined by D N A replication and cell division, i.e., G S, G , and D . The subsections of the cell cycles for three strains of A. proteus have been studied in three different laboratories with some differences in the results. The first study was reported by Nilova in 1965 on a strain of A. proteus collected near Leningrad and designated as the " L " strain. Nilova added H-thymidine to the culture medium and first detected incorporation into D N A 7-12 hours after cell division. The period of D N A synthesis lasted between 10-14 hours. F r o m her radioautographic results she concluded that at 25°C the " L " strain of A. proteus has a G period of 7-12 hours, followed by an S period of 10-14 hours, and a G period of 4-6 hours. The generation time varied between 25 and 30 hours. I and Lauth (unpublished) have done some preliminary work on this same " L " strain of amoeba (provided by A. L. Yudin of the Cytology Institute in Leningrad) and obtained results different from those of Nilova. Dividing amoebae were individually collected and the resulting daughter cells labeled by addition of 100 ^Ci/ml of H-thymidine (10-20 Ci/mM) to the medium. In three such experiments incorporation of H-thymidine into D N A occurred during the first hour after mitosis and continued for at least 6 hours in most nuclei. The experiments were not extended far enough to define the end of the S period, but one point is clear—in contrast to Nilova's results the G period is less than 1 hour and is possibly absent altogether. In our experiments, however, the amount of labeling (detected by radioautography) varied over a wide range and occasional nuclei failed to show evidence of incorporation of H-thymidine during the first several hours after division. Whether this means that a substantial G period is occasionally present in some amoebae or that H-thymidine some times fails to enter into the thymidine triphosphate pool in a sufficient amount to label D N A , we cannot say. l 5
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The wide variation in the amount of incorporation of H-thymidine into different nuclei is possibly the result of the variation in the amount of food vacuole material from amoeba to amoeba. The food vacuole material is the normal source of thymidine for D N A synthesis in amoebae, and this food reserve greatly reduces the effectiveness of labeling with H-thymidine added to the medium (Ord, 1968). Whether a true G period is occasionally present in " L " strain amoebae will have to be decided by additional experiments, but we are inclined to believe for the present (especially in the light of the situation in other strains) that those amoeba with an apparent G period reflect the inade quacies of our isotope labeling procedures. 3
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In a strain of A. proteus of uncertain origin, cultured in this laboratory for over 10 years, 9 0 % of D N A synthesis, as indicated by H-thymidine incorpora tion, takes place in the period extending from division to about 5 hours into interphase (Ron and Prescott, 1969). Under the culture conditions used (bacteria and Tetrahymena as food), there was considerable variation in the time of termination of this period of major synthesis, some amoebae continuing until 9 hours after division. In about 10% of the amoebae a very low level of H thymidine incorporation continued for at least 13 hours after division. It is clear that in this strain a G period is absent, the S period shows some variability, and the G period occupies much of the cell cycle—in the range of 30-40 hours out of a generation time of 36-48 hours at 23°C. Goldstein and Ron (1969) have provided some additional experiments that may help to explain the large amount of variability observed in the length of the S period. They removed 3 0 - 5 0 % of the cytoplasm from dividing amoebae. The operation did not interfere with division, and two daughter cells were produced each of which was 3 0 - 5 0 % smaller than normal. In these cells, D N A synthesis continued longer than in controls (sham-operated, dividing cells). Goldstein and R o n have suggested that the prolongation of the S period might be due to a depletion of nuclear proteins. This deprivation is incurred because 9 0 % or more of the nuclear proteins in A. proteus are released to the cytoplasm during mitosis, and amputation of 3 0 - 5 0 % of the cytoplasm during mitosis removes roughly a corresponding amount of the nuclear proteins that are temporarily in the cytoplasm. These nuclear proteins normally return to the nucleus during the first 3 hours after mitosis is over (Prescott and Goldstein, 1968). But it is not really known whether or not these nuclear proteins play any role in D N A replication. As Goldstein and R o n point out, the amputation of cytoplasm necessarily means the loss of many other materials besides nuclear proteins. The small daughter cells have less cytoplasmic machinery for metabolism and less food vacuole material than a normal cell, and the prolongation of the S period may be due to a general inadequacy of the cytoplasm to support a full rate of D N A synthesis. In line with this interpretation we might then suppose that the varia tion in the length of the S period seen among normal, unoperated cells may be 3
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due to the varying amounts of food vacuole material present in the amoebae entering the S phase and to the varying amount of metabolic machinery present in one cell to another. Cytokinesis in amoebae rarely produces two exactly equal-sized daughter cells and not infrequently divisions are severely unequal, creating extensive variation in daughter cell sizes that might lead to variations in the length of the S phase. This suggestion that such variation in size gives rise to variation in the length of the S period remains to be tested. The most detailed study of the timing of D N A synthesis has been done by Ord (1968) on another strain of A. proteus. She found no measurable G period. D N A synthesis begins with the completion of cell division and continues for 12-14 hours. Within the S period two peak intervals of D N A synthesis occur: the first extends from about 30 minutes to 4 hours after division and accounts for 75 % of D N A synthesis and the second extends from about 9-13 hours after division and accounts for 1 5 % of D N A synthesis. Whether the two peaks are separated by an interval of low synthesis or an interval of no synthesis at all is not clear. A small amount of H-thymidine incorporation (about 1 0 % of the total) into D N A occurs at other times in the cycle, including incorporation in some cases during early prophase. This latter finding is unusual and deserves further study. In confirmation of Ord's report, Goldstein and R o n (1969) also noted a second peak of D N A synthesis at about 12 hours after cytokinesis in the amoebae maintained in our laboratory. F r o m the several foregoing studies the following conclusions may be drawn (see Fig. 1). In A. proteus D N A synthesis normally begins immediately at the end of cell division, although the possibility of a period sometimes (under different culture conditions?) being present cannot be excluded with certainty (Nilova, 1965). The length of the S period is variable, ending somewhere between x
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Fig. 1. The diagram gives a summary of the main events that compose the cell cycle in A. proteus. D N A synthesis (S period) begins at the end of mitosis (no Gi period). Most of the cell cycle is occupied by the G period. Two hours before mitosis (in G ) the amoeba passes T-l (transition point one). At this point the amoeba becomes insensitive to actinomycin D in the sense that mitosis occurs and D N A synthesis is initiated when mitosis is completed. 2
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5-14 hours after division, possibly depending upon the nutritional state of the individual amoebae. The S period may consist of two subphases of D N A synthesis, a major phase followed by a minor one. A small amount of nuclear D N A synthesis may occur at other times in the cell cycle. Obviously, more work is needed to clear up some of the uncertainties that appear in the published data, particularly, to give us a sharper definition of the timing of D N A synthesis in A. proteus and to explain the variability and anomalous timing of D N A synthesis within and between strains of amoebae.
III. Attempts to Induce a
Period in A. proteus
As a general rule for most cell types (mammalian cells, ciliated protozoa, bacteria, etc.), the cells come to rest in the G j period when proliferation stops. For those few nuclear types that normally lack a G period (Physarum, the micronucleus in some ciliates, A. proteus), there are two alternative possibilities. Either a G period must be induced when proliferation ceases or the cells must come to rest in some subsection of the cycle other than the G period. We have tried in several ways to induce a G period in our strain of A. proteus. During the several days following removal of all food organisms (Tetrahymena) from a mass population of amoebae, the frequency of cell division in the popula tion gradually falls to zero. The last amoebae to divide in this situation are relatively well-starved as judged by the nearly complete absence of any remnants of food vacuoles. All such starving amoebae that manage to go through one final mitosis also incorporate H-thymidine (25 μΟ/ιηΙ added to the inorganic medium) into D N A as soon as mitosis is completed (Prescott and R a o , un published). Thus, any amoeba that has sufficient resources to reach mitosis will also enter D N A replication as soon as mitosis is finished, i.e., a G period cannot be induced by starvation. (It is also possible that the S period in such starving amoebae is greatly extended over the normal length, but this point has not been studied.) Second, as already mentioned, it is possible to remove 50 % of the cytoplasm from a dividing amoeba without affecting mitosis and often not affecting cyto kinesis. The two daughter cells contain only half of the normal amount of cyto plasm, yet such cells begin D N A synthesis without delay as soon as mitosis is completed (Goldstein and Prescott, 1967). D N A synthesis starts whether or not the daughter cells are fed. Goldstein and R o n (1969) have shown that the S period is lengthened in such experimentally treated amoebae. Third, cytokinesis can be prevented in amoebae without affecting mitosis by immersing dividing amoebae in a 1 % solution of bovine albumin. The daughter nuclei in the resulting binucleated amoebae all begin D N A synthesis without a l
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G period. Thus, not unexpectedly, the initiation of the S period is not influenced by the absence of cytokinesis. The fourth observation is perhaps the most striking. When a proliferating population of amoebae is exposed to actinomycin D ( A M D ) (100 /ig/ml or 1 mg/ml), cells continue to divide for 2 hours. Since essentially all R N A synthesis in amoeba is shut down within 15 minutes after addition of 1 mg/ml of A M D to the medium (Rao and Prescott, 1970), we conclude that the synthesis of any R N A that is essential for mitosis and cytokinesis is completed 2 hours before mitosis. Even the very last amoebae that are able to divide in the presence of A M D nevertheless begin D N A synthesis without delay at the end of mitosis. (We do not know whether such cells complete the S period or whether the S period is otherwise changed, e.g., lengthened.) This experiment with A M D shows that mitosis and D N A synthesis in amoeba are indeed tightly coupled. We have concluded (Rao and Prescott, 1970) that the cell becomes committed to enter D N A synthesis at about the same moment that it becomes committed to undergo mitosis. This situation is fundamentally different from what is observed in plant and animal cells generally. In the latter the decision to enter D N A synthesis would appear to be made in the G period, perhaps several hours before the beginning of the S phase. In amoeba, the available evidence shows that the decision to enter D N A synthesis has been made 2 hours before mitosis. Thus, because the decision to begin the S period is made before mitosis in amoeba and after mitosis in most plants and animals, one can conclude that the decision to enter mitosis and the decision to enter D N A synthesis are not obligatorily tied to each other in the same temporal relationship in all cell types. In fact, the decision to enter mitosis and the decision to begin D N A synthesis may not always show the same temporal relationship even in a given, single type of cell. In mammalian cells, for example, a G period is absent altogether under some circumstances; it is conceivable therefore that the decision to enter the next D N A replication may be made before mitosis in some circumstances (no and after mitosis in other circumstances ( a G j of several hours). 1
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IV. How is the Cell Cycle Arrested when Amoebae Cease Proliferation? As already described, the arrest of proliferation in amoebae by starvation does not induce the appearance of a G period. This suggests that amoebae come to rest in the G period of the cell cycle. This would reflect another major difference between amoebae and most other cell types since most cell types come to rest in the cell cycle in the G period (no information is available on the arrest point in the cycle for the two other cases of G ^ l e s s nuclei, i.e., the micronucleus in some ciliates and the nuclei in Physarum). 1
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Since amoebae appear to come to arrest in the G period when cell prolifera tion ceases in a population subjected to starvation, we expected that starving amoebae, upon refeeding, would again take up progress through the G period and subsequently divide. It turned out not to be so simple. When dividing amoebae are selected from a well-fed culture and starved, they finish the S period but do not divide (presumed G arrest). If such amoebae are kept without food for 24 hours after cell division and then refed, they will divide again between 24-48 hours after the refeeding. Within 2 hours after refeeding has begun, however, all amoebae again begin to make D N A . This can be demonstrated by using Tetrahymena labeled with H-thymidine as food or by adding ^ - t h y m i dine (100 /xCi/ml) to the medium in the presence of nonlabeled food organisms. 2
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This period of D N A synthesis that follows the refeeding of starved amoebae lasts for about 6 hours. Whether this synthesis represents a full redoubling of the G amount of D N A has not yet been determined, but it is clear from radioautographs that the rate of synthesis during the 6-hour period is comparable to the rate observed in the normal S period following mitosis. If amoebae that have made extra D N A are again starved for at least 24 hours by withdrawing food as soon as the extra D N A synthesis is over, most of these amoebae will respond to a second refeeding by again synthesizing D N A . Thus by an alternation of long periods of starvation and short periods of refeeding, amoebae are induced to go through several periods of D N A synthesis without the occurrence of mitosis. As yet we do not know the fate of the extra D N A produced following starva tion, except that it is retained by the nucleus and distributed equally between the two daughter nuclei at the subsequent mitosis. In some manner, amoebae that have synthesized extra D N A after starvation must have a means of subse quently reducing their D N A content. If this were not so, we would expect to find a steadily increasing amount of D N A per nucleus in stock cultures of amoebae, since stock cultures are constantly subjected to periods of starvation alternating with periods of feeding. The amount of D N A per nucleus obviously does not undergo a progressive, permanent increase in amoeba cultures. The two strains of amoebae studied so far, our strain and the Leningrad strain, behave in about the same way with respect to the extra synthesis of D N A follow ing a period of starvation. H o w the amoebae regulate their nuclear content of D N A in this situation remains to be determined. Either the amoeba is able to destroy the extra amount of D N A in such a regulated manner that the genetic intactness of the nucleus is not jeopardized, or else an amoeba is able to undergo a reduction division at a mitosis following the buildup of the extra D N A . Finally, this induction of extra D N A synthesis by refeeding of starved amoebae demonstrates that the tight coupling between mitosis and the initiation of D N A synthesis can be uncoupled at least to the extent of the initiation of D N A syn thesis independently of mitosis. 2
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V. Nucleocytoplasmic Interactions in the Control of DNA Synthesis The readiness with which nuclei can be transplanted in amoebae has been used to advantage in the study of the possible role of nucleocytoplasmic inter actions in progress of amoebae through the cell cycle. The first experiments concern the influence of G cytoplasm on the synthesis of D N A in S phase nuclei and the possibility that S phase cytoplasm may induce D N A synthesis in a G nucleus. We reported in 1967 (Prescott and Goldstein) that the incorporation of H-thymidine into D N A in an S phase nucleus was reduced when that nucleus was implanted into a G phase amoeba. We have recently confirmed this result (unpublished). In control experiments an S phase nucleus was transferred into another S phase cell. In such cases the rate of incorporation of H-thymidine was not detectably diminished, indicating that the operation in itself was not disrupting D N A synthesis. In the reciprocal experiment, G nuclei were transferred into S phase cells. Many but not all of such transferred G nuclei again incorporated H-thymidine. In the control experiment involving the transfer of a G nucleus into another G cell, there was no stimulation of incorporation of H-thymidine into the transferred nucleus. We concluded from these observations (7) that the cytoplasm of a G phase cell lacks those properties necessary for the support of D N A synthesis, and (2) that S phase cytoplasm has properties that can induce D N A synthesis in a G nucleus. Ord (1969) has done the same kinds of experiments on other strains of A . proteus with quite different results. In agreement with our experiments her data do show a decline in H-thymidine incorporation in an S phase nucleus after implantation of that nucleus into a G cell, but Ord believes that this reduction is due to a greater dilution of the H-thymidine by endogenous unlabeled thymidine in G cells in comparison to S phase cells. Whether this is the correct explanation for reduced labeling or whether a real decline in the rate of D N A synthesis occurs in S phase nuclei implanted into G cells, could be determined by feeding the recipient G cells on Tetrahymena labeled with H-thymidine for several hours prior to implantation of the S nuclei. This would avoid changes in the rate of H-thymidine incorporation due to unlabeled thymidine derived from the food organisms. Contrary to our results Ord did not detect any stimulation of H-thymidine incorporation in a G nucleus implanted into an S phase cell. The reason for the difference between her results and ours remains to be explained. As Ord points out the explanation is unlikely to be found in differences between strains of amoebae. One should recall, however, that the G nucleus in A. proteus allowed to starve for 24 hours or more does revert into a state in which D N A synthesis is, indeed, reinitiated within 2 hours of refeeding with Tetrahymena. It is not 2
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clear, however, whether this latter stimulation of D N A synthesis in a G nucleus is related to the D N A synthesis we observed in a G nucleus transferred into an S phase cell. Ord also transferred nuclei from late S phase cells into early S phase cells to see whether the early S phase cytoplasm could prevent the switch off of D N A synthesis in late S phase nuclei. Her results show that the late S phase nucleus apparently cannot be stimulated to synthesize extra D N A by implantation into an early S phase cell. Ord concludes that the switching off of D N A synthesis is therefore not prevented by the properties of the early S phase cytoplasm. This again implies the lack of any role of the cytoplasm in the regulation of D N A synthesis in A. proteus. The contradiction between Ord's results and ours should be investigated by further experiments. Particularly, it will be necessary to settle the question of whether the cytoplasm or nucleocytoplasmic interaction plays any role in initiat ing, supporting, or switching off D N A synthesis in A. proteus. 2
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VI. Some Observations on the G Period 2
We (Goldstein and Prescott, 1967) have also studied nucleocytoplasmic interactions at other parts of the cell cycle with the objective of determining factors that underlie progress of the cell through its cycle. We asked whether the long G period in amoeba might be due only to a progression of nuclear events. If this were so we might expect that a cell constructed by implanting a late G nucleus into an S phase cell (with the S phase nucleus removed) would divide relatively quickly and without a requirement for cytoplasmic growth. Such cells, however, must feed and grow an average of 90 hours before division can take place. This suggests that " m a t u r a t i o n " of the cytoplasm occurs during the normal G period. A cell constructed of late G cytoplasm and an S nucleus requires on the average about 70 hours to reach division. Thus, the cell containing a mature nucleus (late G ) and an immature cytoplasm (early S) requires more time to progress through the rest of the cycle than does a cell containing an immature nucleus and a mature cytoplasm. We may conclude that the possession of mature cytoplasm permits the cell to reach division sooner. This suggests, indirectly at least, that the maturation of the cytoplasm is important for progress of the cell through the cell cycle. Since most of the cell cycle involved in these experiments is G we are led to believe that cytoplasmic maturation may form the major basis of the G period. We (Goldstein and Prescott, 1967) have obtained other indications that progress through G is dependent upon cytoplasmic maturation, but at present, we do not have any clues regarding the specific nature of the events that 2
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constitute cytoplasmic maturation in the G phase. Certainly the fact that most of the cell cycle in amoeba is occupied by a G period points to a much greater complexity of the G period in amoebae compared to the cells of metaphyta and metazoa. We must consider that the G period in amoeba may contain many cell cycle events and functions that may occur partly or completely in other subsections of the cycle in cells of multicellular organisms. One of the few specific clues about the G period in amoeba is the transition point that is passed about 2 hours before mitosis. Once a G amoeba has passed this point, it is no longer dependent upon R N A synthesis in order to proceed to mitosis, divide, and initiate D N A synthesis in the new, postmitotic nucleus. There are a number of possible experiments of this general type, using inhibitors, that might tell us more about the G period in amoeba. And by no means have the possibilities for using nuclear transplantation to study the cell cycle in amoeba been exhausted. 2
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VII. Concluding Remarks It must be admitted that we still know relatively little about the cell life cycle in the amoeba (summarized in Fig. 1). We do know that mitosis and D N A synthesis are normally tightly coupled, and there is no G period. Second, we know that the long G period is complex, and its completion requires some form of maturation of the cytoplasm. Third, if an amoeba is arrested in the G period by withholding nutrients, the state of the cell changes such that it synthesizes more D N A upon refeeding, instead of simply proceeding to mitosis. It appears that in spite of the starvation, the amoeba progresses to a state in which it is now prepared to initiate D N A synthesis. It would be interesting to determine whether the initiation of D N A synthesis in the starved amoeba is dependent on R N A synthesis. This could be determined in a manner analogous to the demonstration that amoebae progressing normally through the cycle do not require R N A synthesis during the last two hours of G in order to divide and initiate D N A synthesis. Thus, there are meaningful experiments on the amoeba cell cycle that could be done in spite of the inadequacies of present culture methods for this cell. There is no doubt, however, that the task would be far easier if amoeba could be grown axenically on a defined medium. 1
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Acknowledgment The work of the author described in this chapter was supported by a grant from the National Science Foundation.
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References Goldstein, L., and Prescott, D. M. (1967). In "The Control of Nuclear Activity" (L. Goldstein, ed.), pp. 3-17. Prentice-Hall, Englewood-Cliffs, New Jersey. Goldstein, L., and Ron, A. (1969). Exp. Cell Res. 5 5 , 144. Nilova, V. K. (1965). Tsitologiya 7, 633. Ord, M. J. (1968). / . Cell Sci. 3 , 483. Ord, M. J. (1969). Nature (London) 2 2 1 , 964. Prescott, D. M., and Goldstein, L. (1967). Science 1 5 5 , 469. Prescott, D. M., and Goldstein, L. (1968). / . Cell Biol. 3 9 , 404. Rao, M. V. N., and Prescott, D. M. (1970). Exp. Cell Res. 6 2 , 286. Ron, Α., and Prescott, D. M. (1969). Exp. Cell Res. 5 6 , 430.