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Synchronization of cell division in Astasia longa on a chemically defined medium
Experimental
Cell Reseurch
20, 401-415
(1960)
401
SYNCHRONIZATION OF CELL DIVISION ON A CHEMICALLY DEFINED G. M. Department
PADILLA’
of Zoology,
University Received
and
T.
of California, August
W.
LONGA
IN ASTASIA MEDIUM
JAMES’ Los Angeles,
Calif.,
U.S.A.
17, 1959
IN
recent years a variety of methods have been employed to achieve synchronization of cell division. They include temperature changes, biochemical techniques, and mechanical separation of cell types. The merits of the various methods have been discussed by the individual investigators and reference to these studies can be found in recent reviews, which also discuss the importance of synchronization of cell division [2, 3, 32, 353. In this work we have limited ourselves to a technique which utilizes temperature changes. The present study employs a method originally developed for the synchronization of cell division in Amoeba proteus [14], the basic rationale for which emphasizes an ecological and environrnental approach to the problem. This method consists of a repetitive temperature cycle impressed upon a mass culture of organisms. It is aimed at testing the capacity of cells not only to achieve one set of simultaneous cell divisions, but also to undergo successive divisions that remain in phase with an externally applied temperature cycle. This criterion places an added restriction on the meaning of synchronization of cell division. The success of the method depends in part on the the maintenance of a synchronized phase relationship between the externally applied temperature cycle and the cycle of cell growth and division. Rlaintenance of this condition for an indefinite number of generations would indicate that the balance between growth and cell division has not been seriously impaired [ 1:. The lack of axenic cultures of Amoeba proteus, let alone the present impossibility of growing in a chemically defined medium, prompted the use of a different organism for this study. Previous investigations [ 16, 291 indicated that the acetate flagellate, Astasia longa (Jahn strain), was well suited for synchronization studies for several reasons: It can grow and divide in a temperature range from 13 to 3O”C, it has a relatively long generation time, 1 Public Health Research 2 Supported by National
Fellow Science
of the National Cancer Institute. Foundation Grant No. 5666. Experimental
Cell
Research
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G. M. Padilla
402
and T. W. James
and it has a typical mitosis [S, 181. Astusiu of studying growth and division processes background since it can be grown in a acetate or ethanol as a carbon source and
MATERIAL
AND
longa also offers the opportunity against a well-defined chemical simple medium requiring only vitamins.
METHODS
Conditions of growth.-The organisms were grown in four liter Erlenmeyer flasks containing two liters of medium which was shaken at each sampling but was not aerated. Aeration proved to be unnecessary at the population densities used. The media were of three types: (1) a 1 per cent or 2 per cent Proteose Peptone solution (Difco), (2) Cramer-Myers medium [al], as shown in Table I, and (3) Cramer-Myers salt medium supplemented with 1.75 mg of L-methionine per liter and 100 mg L-cysteine hydrochloride per liter. The amino acids were sterilized by filtration. In those experiments in which the Cramer-Myers medium was used, sodium acetate was added at a concentration of 5 g per liter (anhydrous). No sodium acetate was added to the proteose peptone medium. Vitamins B, and B,, were added to the inorganic media at concentrations of 0.01 mg/l and 0.0005 mg/l, respectively. TABLE I. Cramer-Myers
medium
Constituents (NH,),HPO, I
MnCI, * 4H,O koS.0,. 7H,O ZnSO, * 7H,O Na,MO, * 2&O CuSO, .5H,O Sodium acetate (anhydrous) Vitamin B, Vitamin B,, pH 6.8 a From Myers, J. and Cramer, b Substituted for CaCI, 7H,O Experimental
Cell Research
20
M., Arch. Mikrobiol. used at 1.3 mg/L.
(modified).”
Concentrations mg/liter
1000 1000 200
800 20 3 1.8 1.5 0.4 0.2 0.02 5000 0.01 0.0005
17, 384 (1952).
Cell division
in Astasia longa
303
A temperature program was impressed upon the culture by means of a clock mechanism which controlled the temperature level of a water bath (American Instrument Co.). It was possible to alter the temperature of the bath from 5°C to 30°C in a variety of cycles, the only limitation being imposed by the period of transition required for the temperature shifts. This transition period was reduced to as little as 30 minutes in the final set of experiments. The culture was sampled automatically by using an automatic pipetting machine (Brewer Co.) coupled to a sample collector (Technicon Co.). The entire operation of sampling consisted of a clock-operated sequence of events in which the culture was shaken for two minutes prior to the removal of two separate &ml samples. The first sample was discarded as it essentially washed out the connecting lines. The second aliquot was delivered to a test tube containing 0.2 ml of Bouin’s fixative.Evaporative loss was minimized by the fact that the test tubes were maintained in a covered rotating table. Moreover, the samples collected through the night were examined the following day. The fixative did not cause lysis and the culture was sterile throughout the entire experiment. Cell counts were made by using a Whipple disc and a Sedgwick-Rafter counting chamber 141. While this method may seem to be laborious, it was found that optical density methods were inadequate as the relationship usually found between cell counts and optical density was disrupted by the temperature changes. Furthermore, visual examination of the samples permitted the estimation of the number of cells in cytoplasmic fission as well as the determination of any other cytological changes attendant to the phenomenon of synchrony.
OBSERVATIONS
Statistical Dispersion
treatment of the Index [7] indicated
cell
counts
by the
estimation
of the
Poisson
that the cell samples follow this distribution. of the coefficient of variability indicated that error coultl be kept below five per cent by counting at least NO cells per sampling. Any deviation in the counts beyond these limits was the result of non-random sampling by the pipetting machine. In some instances, however, there seems to be some correlation between the apparent scatter and the cytological behavior of the cells prior to cellular Moreover, routine calculation the variation due to counting
fission. Construction
of the temperature
cycle.-In the development of the temperaturc cycle used in this study, two points of departure were taken: one concerned itself with the examination of the long-term adaptation or the steadystate response of these organisms to incubation temperatures, and the other consisted of an evaluation of the efrect of temperature shifts on the growth and the mitotic activity of these cells. The first investigation [16] was designed to measure the temperature E.zperimen&al
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Research
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G. M. Padilla
404
and T. W. James
range for normal growth of Astasia and to test its capacity under a variety of incubation temperatures. Table II, which includes data from this study, demonstrates that this organism is capable of growing at temperatures ranging from 15 to 30°C. It can be seen that a reciprocal relationship exists between the incubation temperature and each of the following: the generation time, the dry weight, and the average cell volume. The respiratory activity, on the other hand, increases as the temperature is raised. TABLE
G.T.,
generation
Incubation temperature
II. Effect of incubation temperatures on cellular properties of Astasia longa. time,
“C
15 20 25 30
G.T.
V, average
hrs.
at incubation volume, Qoz, measured weight and cyto. cytoplasm.
Dry weight mg/lOB cells
1’ pL3
1.12 0.900 0.943 0.556
3320 2460 2470 1540
33 12 11 6.5
Qoz (lo6
cells)
18.0 22.7 21.6 47.2
a Calculated on a cell hour basis. (From: James, T. W. and Padilla, G. M., in Proceedings Conference. Ed. Quastler, H., Yale Univ. Press, 1959.)
mg d.w. mm3 cyto.
0.338 0.365 0.383 0.362
of the First
temperature,
d.w.
dry
mg d.w. synthesized’ ~1 0, consumed
1.25 2.30 2.50 1.125
National
x x x x
10-3 10m3 10-z 1O-3
Biophysics
The above data were used to construct a 24-hour cycle having a w-arm period of 25°C and a cold period of 15°C which would allow the organism to grow and produce the necessary cell machinery. The last column in Table II shows the organism’s effectiveness in terms of dry weight produced per unit of oxygen consumed, indicating that at 25°C Astasia is twice as effective as it is at 15°C. If the proportion of cellular constituents is the same in both cases, the cold period would have to be approximately twice as long as the warm period to accomplish the same end. Tissue cultures [22, 301 as well as micro-organisms [36] display a reduction in the division index upon cooling. Temperature increases generally have the opposite effect. Astasia longa grown at 25°C and then exposed to 15°C do not undergo cell division for 18 to 20 hours. This length of time was taken as the upper limit for the duration of the cold period. The length of the warm period was empirically set to maintain a 1: 2 relationship with the cold period and to allow the entire cycle to be 24 hours long. Experimental
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Cell division
in Astasia longa
405
RESULTS
The term “cycle” will be used to mean one complete sequence of events of the recurrent temperature changes, i.e., from the beginning of the “cold” period through the following “warm” period up to the beginning of the period. The cycles comprising the temperature program in next “cold” each experiment are indicated in the graph by number (1, 2, 3, etc.). Fig. 1 shows an early experiment illustrating a typical temperature cycle and the growth pattern of a culture of Astasia longa in a 1 per cent proteose peptone medium. The ordinate is the logarithm (to the base 10) of the population density and the abscissa denotes the age of the culture in hours. The temperature cycles are indicated at the base of the graph and the vertical bars bracket the transitions at the level of the growth curve. In this experiment the culture was allowed to enter the logarithmic phase of growth, having attained a density of approximately 2100 cells per milliliter (3.3 on the log scale) before the temperature program was imposed. The program used on the culture in Fig. 1 consisted of a lo-hour “cold” period at 15°C followed by an &hour “warm” period at 25°C. The period of transition between the two temperatures lasted three hours. Thus the entire cycle was repeated every 24 hours. The principle of one to two ratio mentioned above, namely that the cell etl’ectiveness at 25°C is double that at lis”C, was not applied in this cycle. Fig. 1 shows the non-synchronous behavior of the population of cells on this cycle. The first two warm periods result in a doubling of the number of cells while essentially no divisions occur during the greater portion of the respective cold periods. Successive cycles, namely the third, fourth, and fifth, do not show doubling of cell number while the sixth and seventh cycles show a resumption of doubling in the warm period. Attention to the end of the division periods shows that the completion of division is gradually displaced into the following cold periods so that by the third cycle, doubling of cell numbers fails to occur, giving an irregular and asynchronous increase in the population density that continues over the fourth and fifth cycles. The resumption of the synchrony occurs when the beginning of the division period again coincides with the beginning of the warm period. The experiment was terminated at a population level of approximately 100,000 cells per milliliter (5 on the log scale). The sampling error which is implied by the scatter in Fig. 1 was reduced in subsequent experiments by adopting the improved sampling and counting technique outlined earlier. Despite this limitation an interaction between Experimentut
Cell
Research
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G. M. Padilla
and T. W. James
the temperature cycle and the cell cycle is apparent later. Fig. 2 shows the response of a culture grown on 2 per and maintained on a 24-hour cycle consisting of 15 hours at 25°C. The temperature transition periods minutes each.
and will
he discussed
cent proteose peptone hours at 15°C and 8 were reduced to 30
.Temp.
100
120
140
160
180 HOUrS
200
220
240
260
Fig. l.-Growth pattern of Astasia Zonga in a temperature cycle which is poorly matched with the cell cycle. Note the synchrony in the first and second cycles, lack of synchrony in the third, fourth, and fifth cycles, and resumption of synchrony in the sixth and seventh cycles.
In this experiment the culture was placed on the temperature program beginning with the warm period immediately after inoculation, although sampling was started only after the culture had been exposed to the cycle for 40 hours. The culture at this time had attained a population level of 2200 cells per milliliter (3.34 log units). Fig. 2 illustrates the repetitive cycle of temperature and the resulting growth curve. Consistent doubling of cell numbers was obtained within two hours after the onset of each warm period. The fission index calculated as the ratio of the number of cells in recognizable cytoplasmic fission to the total number of cells counted is also shown in Fig. 2. The fission index should not be Experimental
Cell
Research
20
Cell division
in Astasia longa
‘10’7
confused with the mitotic index since the former is obtained by scoring only cells in cytoplasmic cleavage. Since cptoplasmic cleavage occupies a time period considerably shorter than total mitosis [IS] the mitotic index would be larger than the fission index. During the warm period fission indices were determined at fifteen-minute intervals, while during the cold period the determinations Ivere made every 4 hours.
60
80
l&l
120
140
160
1
Hours
Fig. 2.--Synchronized growth and cell division of Asfctsia Zonga grown on a 2 per cent proteosc peptone medium. The temperature cycle, as indicated, consists of 15 hours at 15”C, 8 hours at 25°C and two half-hour transition periods. The fision index, expressed in per cent of cells in fission, is shown in the lower portion of the graph.
In addition to the frequent sampling, living cells removed from the synchronized culture were observed at the beginning of each burst and during the time of maximum activity in cell division. It was found that prior to cell division, cells would become non-motile, settle down, and undergo extensive metabolic movement as has been noted in other flagellates [13]. Of these cells, 30 per cent would normally be in a recognizable state of fission; at that time the cells in the parent culture also showed a maximum fission index. Discrepancy between the recorded peaks in the fission index (from 2.4 per cent to 12.1 per cent) and the higher observed value for this index, indicates Errperimenlnl
Cell Research
20
and T. W. James
G. M. Padilla
that the automatic sampling device is not removing a representative sample of dividing cells from the culture and is possibly selecting for motile, nondividing cells. In spite of this, the fission index can still be used as a measure of the improving resonance between the cell cycle and the time-temperature cycle. TABLE
III.
Analysis of the growth curve of Astasia longa in synchronous cell division. Values
Cycle
Growth constant (Ir)a for burst
1 2 3 4 5 Growth
are calculated
from
the culture
shown
in Fig.
Duration of burst (hrs)
% Cell divisions in warm period
1.3 3.5 1.5 1.1 1.0
90 93 96 100 100
0.12 0.10 0.22 0.24 0.12
2. Maximum index
fission (%)
4.1 7.5 9.6 12.1 6.3
constant (k)b for log phase: Culture at 25°C 0.024 Culture at 15°C 0.005
a Calculated
from
b Calculated
from
the
growth
cultures
grown
equation:
log N/N k = d.
t
in 2 per cent proteose
peptone
as for culture
shown
in Fig.
2.
Table III is an analysis of the synchronous pattern of cell division for the culture shown in Fig. 2. The first column indicates the cycles of the temperature program used in this experiment as they are numbered in Fig. 2 (i.e., 1, 2, 3, etc.). Column two gives the values of the instantaneous growth constant (k) calculated for the log-linear portion of the burst of division occurring in each warm period. For purposes of comparison Table III also includes the growth constants for the logarithmic phase of growth of cells in the same medium (2 per cent proteose peptone) grown at 25 and 15°C. Column three indicates the duration of each burst in hours. Column four gives the proportion of cells that have completed cell division by the end of each warm period. Column live shows the maximum fission index achieved in each of the cycles. Comparison of the minimum fission time (12 minutes) and the minimum duration of the synchronous burst (1 hour) indicates that about 20 per cent of the cells should be seen in fission during the burst. Experimental
Cell Research
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Cell diuision
in Astasia longa
409
Various attempts to bring about synchronization of cell division in Astasia grown in the chemically defined Cramer-Myers medium resulted only in poorly defined synchronous growth. Alterations of the relative lengths of the warm and cold periods, together with a decrease in the temperature shifts, failed to improve the synchrony with this medium. TABLE
IV.
of generation
Comparison
time, fission time, and fission in various media at 15” and 25°C.
Astasia longa grown The
generation
time (G.T.), the logarithmic
fission index (F.I.) phase of growth. Incubation temperature OC
Medium 2 :b Proteose peptone Cramer-Myers only Cramer-Myers, cysteine and methionine a Calculated is expressed
from
Crick’s
as a decimal
Generation time (hrs.)
15 25 15 25 15 25 equation:
in this
and fission time (F.T.) were measured One standard deviation is given.
52.4 t 11.5k2.7 42.7k4.5 12.8+ 61.9 + 13.0t F.T.
=Iog,
Fission
index
2.3 7.1 2.6
$$I$$
0.75f0.16 0.94 * 0.10 2.59+ 1.17 3.79f 1.52 1.58?0.20 1.62 & 0.49 x G.TL. log,
2
(Kate:
in
for cells in
Fission time’ (min.)
6) 7.3
index
45.0 t 10 9.9F 2.3 133.0*53 64.5/20 96.Oi 35 19.2f 7.8 the fission
index
(F. I.)
equation.)
=Zcomparison of the generation time and fission index between cells grown in 2 per cent proteose peptone and Cramer-Myers showed that while the difference in generation times was slight, the fission index was much higher in cells grown in the defined medium. This increased fission index was reflected in a prolonged fission time as calculated by the equation attributed to Crick [‘3] and as used by Scherbaum [28]. The implication was that the cells grown in the defined medium lacked some constituent involved in the division process. It had been previously shown that Astasia does not utilize amino acids as sources of nitrogen [.?I but the ef’fect of such compounds on the division process had not been examined. A series of experiments were performed in which cells were grown in Cramer-Myers supplemented with cysteine and methionine in concentrations comparable to those used in the growth of other microorganisms. Table IV summarizes the results of these experiments. Column one shows the medium used (i.e., 2 per cent proteose peptone, Cramer-Myers and Cramer-hlyers supplemented with cysteine and methionine). Column two gives the incubation temperature. Column three gives the average generation Experimental
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G. M. Padilla
410
and T. W. James
time ((;.‘I’.) in hours for cells in the logarithmic four shows the average fission index (I:.I.) as a gives the average fission time (F.T.) in minutes equation. One standard deviation is given for all
phase of growth. Column per cent, and column five as calculated from Crick’s determinations.
5.0 4.8 * 4.6 * 4.4 -
4.2 4.0 -
Fig. S.--Synchronous growth and cell division of dsfasia longa grown on a chemically defined medium (modified Cramer-Myers). The 24-hour temperature cycle consists of 15 hours at 15”C, 8 hours at 25”C, and two 30 minute transition periods. The fission index is expressed in per cent of cells in fission (O-O).
Table IV demonstrates that for cells grown at 2,5”C there is a marked reduction in the fission time upon addition of cysteine and methionine to CramerMyers medium without any apparent decrease in the generation time. The fission time, however, is still considerably longer than found for cells grown on proteose peptone. At 15°C the addition of these amino acids results in some reduction in the fission time, but the wide variation in these values renders them rather inconclusive. Other experiments indicate that either cysteine or methionine alone do not reduce the fission time to the same degree as when they are combined. Further experiments are being conducted to determine the optimum concentration for the greatest reduction of the fission time. Experimental
Cell Research
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Cell division
in Astnsia
longa
411
Having determined the effect of cysteine and methionine on the fission time of Astasia longa, the supplemented medium was used in subsequent synchronization experiments. Fig. 3 shows the results of such an experiment. The temperature cycle is exactly the same as that used in the experiment shown in Fig. 2. The culture quickly establishes a close relationship with pattern of growth and cell the time-temperature cycle. The synchronized division can be seen to continue until the medium becomes exhausted; this occurs at a population density of approximately 400,000 cells per ml (5.6 in the log scale). It was also interesting to note that the calculated fission times for cells at 25°C generally agree with the observed fission times which were 12.6 i 4.03 minutes for cells grown in 2 per cent proteose peptone and 13.0 i 2.20 minutes for cells grown in the supplemented Cramer-Myers medium. DISCUSSION
The growth and development of a cell from cell division to cell division has been classically subdivided into an interkinetic or growth phase and a kinetic or mitotic phase. This at once introduces the idea that in its broadest sense cellular processes are sequential. If one now considers a sequence of cell generations, or a cell lineage, these phases recurring in time give form to what may be called a cell cycle; the generation time being one period. From this point of view a mass culture of cells in the logarithmic phase of growth becomes an assemblage of these cell cycles randomly distributed in time. A further extension of this argument would allow one to define synchronization of cell division as the alignment of cell cycles so that each growth and division phase occurs simultaneously throughout the culture. The question now arises as to whether one can achieve synchronization of cell division by means of a repetitive temperature cycle. The success encountered in the early investigations of this problem in bacteria [ 10, 11, 15 ] and protozoa [la], as well as in recent experiments with tissue cultures [22I, demonstrates that this question is answerable in the affirmative. These studies strongly suggest that an interaction can be established between the cell cycle and the applied temperature cycle. One may also examine the outstanding synchronization experiments on Tetrahymena [25] in this light. In this case the bursts of cell division following release from the heat treatment can be likened to self-dampened oscillations. The entire question of the cyclical nature of a cell’s activity has been re-examined in recent reviews [3, 32, X5]. The primary aim of the present investigation has been to devise a tempera27 - 0017R?5”
Experimental
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G. M. Padilla
and T. W. James
ture cycle for Astasia longa, grown in a chemically defined medium, that would result in the type of synchrony outlined above. It is obvious that the temperature cycle which will best interlock with the cell cycle can take a variety of forms, particularly since the cell cycle is a function of both time and temperature. Moreover, there is generally a reciprocal relationship between time and temperature for the physico-chemical processes of a cell. Consideration of this time-temperature relationship led to the investigation of the adaptation of Astasia to incubation temperature (cf. Table II). As pointed out earlier, these data, which express the oxygen consumption, the generation time, the cell volume, and the synthetic activity of Astasia as a function of temperature, give a measure of this cell’s capacities at temperatures ranging from 15 to 30°C. This information, together with the observations on the effects of acute temperature changes on the mitotic activity of Astasia, was basic to the construction of the temperature cycle as shown in Fig. 1. The results indicate that the success of this or any other repetitive temperature cycle is largely dependent on the establishment of resonance between the temperature cycle and the cell cycle. This resonance phenomenon is illustrated by Fig. 1 as the interplay between poorly matched temperature and cell cycles. Such behavior is typical of a culture in which the cell cycle is completed in a period different from the period of the impressed temperature cycle. It results in phases of synchrony and lack of synchrony. This is analogous to the interference of waves of slightly different periods. Figs. 2 and 3, on the other hand, show the effects of resonance between the two cycles. This improved interlocking of the cycles was the result of different time intervals and reduced temperature transition periods. The results indicate that the establishment of resonance depends on at least three factors: (1) the energy relationships established between the various phases of the cell cycle; (2) segregation of cell types through differential effect of temperature on different phases of the cell cycle; and (3) the nature of the medium, particularly with respect to the processes involved in cell division. These factors are separated solely for purposes of discussion since the interrelationships existing between them are probably also important in the evaluation of these experiments. The proposition that a temperature cycle brings about synchronization of cell division by altering the time course and direction of the energy-yielding processes involved in cell division, can be examined in terms of the “energy reservoir” idea proposed by Swarm !32]; a somewhat similar approach was used in the kinetic considerations of synchronization in Tetrahymena [26]. Experimental
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Cell division
in Asfasia
longa
,113
Hriefly, this concept includes the notion that there is an energy reservoir which is filled through the interkinetic phase and is expended during cell division. A cyclical sequence of events is thus established relating the filling and emptying of this energy reservoir to the phases of growth and cell division. With a temperature cycle in which the duration of the warm and cold periods match the cell’s capacities at these temperatures, the cellular processes are channelled into the filling and emptying of this energy reservoir. In terms of the temperature program used in these studies, the cells are forced into an increased production of metabolic respiratory machinery in the cold portion of the cycle (15°C). This is reflected in the increased dry weight and cell size found for cells adapted to this temperature (cf. Table II). This is analogous to an increase in a capacity factor for the cellular system. Toward the end of the cold period the cells approach a size and metabolic capacity of cells grown at 15°C. Upon exposure to the warm period of the cycle, this increase in the metabolic respiratory machinery would in essence operate at a greater rate resulting in the rapid filling of the energy reservoir which then saturates the division machinery. The temperature-dependent rate of metabolic activity c,an be considered as an intensity factor of the cellular system. To bring about cell division, some minimum value of the product of the capacity and intensity factors and time would be required. Evidence for the interplay between these capacity and intensity factors through temperature changes can be gained from direct measurements of the oxygen consumption of cells. For Astnsirr longa we have found that 25°C cells upon cooling to 15°C undergo changes in respiratory activity indicating a gradual increase in respiratory machinery. Upon returning to 25°C these same cells demonstrate a greater respiratory capacity [23]. It is clear that for these energy relationships to contribute to the synchronization process there must also be some mechanism that at one time or another brings the cells to the same level of development jl4, 221. In previous investigations [ 10, 12, 17, 27 j attention has been directed to the processes related to the synthesis of nucleic acids. In general, it is felt that at a lower temperature there is a blockage or even a reversal in the synthesis of nuclear components. In bacterial systems evidence has been presented to show that low temperature exerts drastic changes in the nucleus that can be observed cytologically [ll]. Recent investigations on the synchronization of Amoehrr j13-, using an interference microscope, show that there is loss of materials from the nucleus following cold or warm treatment; a process which may reset the cells to the same stage of nuclear synthesis. This would result in the segregation of large numbers of cells into categories with nearly equivalent nuclei. Experimental
Cell
Reseurrh 20
G. M. Padilla
and T. W. James
A similar situation concerning the equivalence of nuclei has been suggested for cells in the stationary phase of growth [31]. This segregation of cell types can be related to the improvement of synchrony illustrated in Fig. 2. As the data presented in Table III show, this improvement can be appraised in several ways: (1) there is an increase in the growth constant (k) for each successive burst of cell division; (2) there is an increase in the percentage of cells completing division during each warm period; and (3) there is a progressive increase in the maximum division index during each burst. Yet, each burst of cell division occurs at the same time: approximately 2 hours after the cells are exposed to the warm temperature. This suggests that the synchronized cells are at the same initial stage of mitosis and cell cleavage, processes which last approximately 3; hours in some species of Astasia [ 19 j. The results shown in Fig. 3, while essentially the same as those of Fig. 2, point to the importance of conducting synchronization experiments on a chemically defined medium. This is particularly true if one wishes to investigate the role played by certain compounds in the division process. In the synchronization of Astasia, cysteine and methionine have proved to be key factors in the reduction of the fission time, an effect similar to that found in Amoeba [33]. Such an effect can be due to several things: (1) both cysteine and methionine are precursors to compounds such as coenzyme A, glutathione, etc. [34]; (2) as protein sulfhydryl residues they may be directly involved in the make-up of the mitotic apparatus 120, 241, and lastly (3) their primary role may be one dealing with the oxidation-reduction level of the cellular environment [6]. The fact that the generation time is unaffected by their presence, implies that these amino acids are primarily involved in the division process. This points to the possibility of an insight into the biochemical pathways leading to cell division. We feel that in order to perpetuate the synchronized pattern of growth within a culture, chemostatic conditions of growth will have to be adopted. SUMMARY
1. A 24-hour repeating temperature cycle, consisting of 15 hours at 15”C, 8 hours at 25”C, and two 30-minute transition periods, has been used for the synchronization of cell division of Astasia longa grown in 2 per cent proteose peptone and in a chemically defined medium. This results in a burst of cell division every 24 hours for as many as seven generations. 2. Cysteine and methionine were found to be important in the mechanism Experimental
Cell Research
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Cell division
in Astasia longa
415
of synchronization of cells grown in Cramer-Myers medium. ‘Their addition appears to reduce the fission time of these cells without affecting the generation time. 3. The synchronization process is evaluated in terms of a cell cycle. The possible manner in which synchronization is achieved 1-1~means of a repcaling temperature cycle is discussed. REFERENCES 1. BSRXER, 2. BRUCE, 3. 4. 5. 6. 7. 8. 9 1;: 11. 12. 13. 11. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. ‘-0 _I>. 29. 30. 31. 32. 33. 34. 35. 36.
H. D. and COHEX, S. S., J. Bmteriol. 72, 115 (1956). V. B., in Influence of Temperature on Biological Systems. Ed. Johnson, I’. I-I., Am. Physiological Sot., Washington, 1957. CAMPBELL, A., Bucleriol. 12~s. 21. 263 (1957). HALL, R. P., JOHNSON, D. I?. and LOEFER, J. B., Truns. ,4m. Microscop. Sot. 54, 218 (1935). HASSOS, R. W., Ph.D. Thesis, Univ. Calif., Los Angeles, Calif., 1952. Oxidation-Reduction Potentials in Bacteriology and Biochemistry. E. & S. HEWITT, L. F., Livingstone Ltd., Edinburgh, 1950. HOEL, P. G., Introduction to Mathematical Statistics. John Wiley & Sons, New York, 1954. HOLLANDE, A., Arch. Zool. exptt gCn. 83, 73 (1942). HUGHES, A., The Mitotic Cycle. Academic Press, New York, 1952. HOTCHKISS, R. D., Proc. Xatl. Acad. Sci. 40, 49 (1954). HUSTER-SZYBALSKA, AI., SZYBALSKI, W. and DELAMATER, E., -7. Bucleriol. 71, 17 (1956). IVERSON, R. M. and GIESE, A. C., Exptl. Cell Research 13, 213 (1957). JAIIN, T. L., and MCKIBB&N, R. W., Trans. Am. Microscop. Sot. 56, 48 (1937). JAILIES, T. W., Ph.D. Thesis Univ. Calif., Berkclcy, Calif. (1954). __ Ann. S. I’. Acad. Sci. 78, 501 (1959). J.~~xEs, T. W. and PADILLA, G. M., in Proceedings of the First National Biophysics Conference. Ed. Quastler, H., Yale IJniv. Press, 1959. LARK, K. G. and MA.~LBF,, O., Biochim. ef Biophys. Acta 21, 448 (1956). LEEDALE, G. F., Arch. Mikrobiot. 32, 32 (1958). __ Ibid. 32, 352 (1959). RI~ix.4, D., in Glutathione. Ed. Colowick, S. P. and Kaplan, iX. G. Academic Press, Sew York, 1954. MYERS, J. and CRAMER, M., Arch. Mikrobiol. 17, 384 (1952). NEWTON, A. A. and WILDY, P., Exptl. Cell Research 16, 624 (1959). PADILLA. G. M. and JAMES, T. W. (In preparation). RAPKIN~, L., Ann. Physiol: Phgsiochem. Viol. 7, 382 (1931). SCHERBAUI\I. 0. and ZEurnEN, E., Exptl. Cell Research 6, 221 (1954). Ibid. i3, 11 (1957). __ Ibid. 13, 24 (1957). J. Protozool. 4, 257 (1957). SCHOEXBORN, H. W., .I. Exptl. Zool. 105, 269 (1947). SPEAR, F. G., Arch. exptl. Zellforsch. 7, 484 (1928). SUMMERS, L. G., BERNSTEIK, E. and JAMES, T. W., Expfl. Cell Research 13, 436 (1957). SWANN, M. M., Cancer Research 17, 727 (1957). VOEGTLIN, C. and CIIALKLEY, H. W., U.S. Publ. Health Repts. 45, 3051 (1930). YOUSG, L. and MAW, G. A., The Metabolism of Sulphur Compounds. Methuen and Co. Ltd., London, 1958. ZEUTHEN, E., Advances in Biol. and Med. Phys. VI, 37 (1958). ZEUTHEN, E. and SCHERBAUM, O., in Cell Physiology. Ed. Kitching, J. A., Butterworth, London, 1954.
Experimental
Cell Research
20
Cell Reseurch
20, 401-415
(1960)
401
SYNCHRONIZATION OF CELL DIVISION ON A CHEMICALLY DEFINED G. M. Department
PADILLA’
of Zoology,
University Received
and
T.
of California, August
W.
LONGA
IN ASTASIA MEDIUM
JAMES’ Los Angeles,
Calif.,
U.S.A.
17, 1959
IN
recent years a variety of methods have been employed to achieve synchronization of cell division. They include temperature changes, biochemical techniques, and mechanical separation of cell types. The merits of the various methods have been discussed by the individual investigators and reference to these studies can be found in recent reviews, which also discuss the importance of synchronization of cell division [2, 3, 32, 353. In this work we have limited ourselves to a technique which utilizes temperature changes. The present study employs a method originally developed for the synchronization of cell division in Amoeba proteus [14], the basic rationale for which emphasizes an ecological and environrnental approach to the problem. This method consists of a repetitive temperature cycle impressed upon a mass culture of organisms. It is aimed at testing the capacity of cells not only to achieve one set of simultaneous cell divisions, but also to undergo successive divisions that remain in phase with an externally applied temperature cycle. This criterion places an added restriction on the meaning of synchronization of cell division. The success of the method depends in part on the the maintenance of a synchronized phase relationship between the externally applied temperature cycle and the cycle of cell growth and division. Rlaintenance of this condition for an indefinite number of generations would indicate that the balance between growth and cell division has not been seriously impaired [ 1:. The lack of axenic cultures of Amoeba proteus, let alone the present impossibility of growing in a chemically defined medium, prompted the use of a different organism for this study. Previous investigations [ 16, 291 indicated that the acetate flagellate, Astasia longa (Jahn strain), was well suited for synchronization studies for several reasons: It can grow and divide in a temperature range from 13 to 3O”C, it has a relatively long generation time, 1 Public Health Research 2 Supported by National
Fellow Science
of the National Cancer Institute. Foundation Grant No. 5666. Experimental
Cell
Research
20
G. M. Padilla
402
and T. W. James
and it has a typical mitosis [S, 181. Astusiu of studying growth and division processes background since it can be grown in a acetate or ethanol as a carbon source and
MATERIAL
AND
longa also offers the opportunity against a well-defined chemical simple medium requiring only vitamins.
METHODS
Conditions of growth.-The organisms were grown in four liter Erlenmeyer flasks containing two liters of medium which was shaken at each sampling but was not aerated. Aeration proved to be unnecessary at the population densities used. The media were of three types: (1) a 1 per cent or 2 per cent Proteose Peptone solution (Difco), (2) Cramer-Myers medium [al], as shown in Table I, and (3) Cramer-Myers salt medium supplemented with 1.75 mg of L-methionine per liter and 100 mg L-cysteine hydrochloride per liter. The amino acids were sterilized by filtration. In those experiments in which the Cramer-Myers medium was used, sodium acetate was added at a concentration of 5 g per liter (anhydrous). No sodium acetate was added to the proteose peptone medium. Vitamins B, and B,, were added to the inorganic media at concentrations of 0.01 mg/l and 0.0005 mg/l, respectively. TABLE I. Cramer-Myers
medium
Constituents (NH,),HPO, I
MnCI, * 4H,O koS.0,. 7H,O ZnSO, * 7H,O Na,MO, * 2&O CuSO, .5H,O Sodium acetate (anhydrous) Vitamin B, Vitamin B,, pH 6.8 a From Myers, J. and Cramer, b Substituted for CaCI, 7H,O Experimental
Cell Research
20
M., Arch. Mikrobiol. used at 1.3 mg/L.
(modified).”
Concentrations mg/liter
1000 1000 200
800 20 3 1.8 1.5 0.4 0.2 0.02 5000 0.01 0.0005
17, 384 (1952).
Cell division
in Astasia longa
303
A temperature program was impressed upon the culture by means of a clock mechanism which controlled the temperature level of a water bath (American Instrument Co.). It was possible to alter the temperature of the bath from 5°C to 30°C in a variety of cycles, the only limitation being imposed by the period of transition required for the temperature shifts. This transition period was reduced to as little as 30 minutes in the final set of experiments. The culture was sampled automatically by using an automatic pipetting machine (Brewer Co.) coupled to a sample collector (Technicon Co.). The entire operation of sampling consisted of a clock-operated sequence of events in which the culture was shaken for two minutes prior to the removal of two separate &ml samples. The first sample was discarded as it essentially washed out the connecting lines. The second aliquot was delivered to a test tube containing 0.2 ml of Bouin’s fixative.Evaporative loss was minimized by the fact that the test tubes were maintained in a covered rotating table. Moreover, the samples collected through the night were examined the following day. The fixative did not cause lysis and the culture was sterile throughout the entire experiment. Cell counts were made by using a Whipple disc and a Sedgwick-Rafter counting chamber 141. While this method may seem to be laborious, it was found that optical density methods were inadequate as the relationship usually found between cell counts and optical density was disrupted by the temperature changes. Furthermore, visual examination of the samples permitted the estimation of the number of cells in cytoplasmic fission as well as the determination of any other cytological changes attendant to the phenomenon of synchrony.
OBSERVATIONS
Statistical Dispersion
treatment of the Index [7] indicated
cell
counts
by the
estimation
of the
Poisson
that the cell samples follow this distribution. of the coefficient of variability indicated that error coultl be kept below five per cent by counting at least NO cells per sampling. Any deviation in the counts beyond these limits was the result of non-random sampling by the pipetting machine. In some instances, however, there seems to be some correlation between the apparent scatter and the cytological behavior of the cells prior to cellular Moreover, routine calculation the variation due to counting
fission. Construction
of the temperature
cycle.-In the development of the temperaturc cycle used in this study, two points of departure were taken: one concerned itself with the examination of the long-term adaptation or the steadystate response of these organisms to incubation temperatures, and the other consisted of an evaluation of the efrect of temperature shifts on the growth and the mitotic activity of these cells. The first investigation [16] was designed to measure the temperature E.zperimen&al
Cell
Research
20
G. M. Padilla
404
and T. W. James
range for normal growth of Astasia and to test its capacity under a variety of incubation temperatures. Table II, which includes data from this study, demonstrates that this organism is capable of growing at temperatures ranging from 15 to 30°C. It can be seen that a reciprocal relationship exists between the incubation temperature and each of the following: the generation time, the dry weight, and the average cell volume. The respiratory activity, on the other hand, increases as the temperature is raised. TABLE
G.T.,
generation
Incubation temperature
II. Effect of incubation temperatures on cellular properties of Astasia longa. time,
“C
15 20 25 30
G.T.
V, average
hrs.
at incubation volume, Qoz, measured weight and cyto. cytoplasm.
Dry weight mg/lOB cells
1’ pL3
1.12 0.900 0.943 0.556
3320 2460 2470 1540
33 12 11 6.5
Qoz (lo6
cells)
18.0 22.7 21.6 47.2
a Calculated on a cell hour basis. (From: James, T. W. and Padilla, G. M., in Proceedings Conference. Ed. Quastler, H., Yale Univ. Press, 1959.)
mg d.w. mm3 cyto.
0.338 0.365 0.383 0.362
of the First
temperature,
d.w.
dry
mg d.w. synthesized’ ~1 0, consumed
1.25 2.30 2.50 1.125
National
x x x x
10-3 10m3 10-z 1O-3
Biophysics
The above data were used to construct a 24-hour cycle having a w-arm period of 25°C and a cold period of 15°C which would allow the organism to grow and produce the necessary cell machinery. The last column in Table II shows the organism’s effectiveness in terms of dry weight produced per unit of oxygen consumed, indicating that at 25°C Astasia is twice as effective as it is at 15°C. If the proportion of cellular constituents is the same in both cases, the cold period would have to be approximately twice as long as the warm period to accomplish the same end. Tissue cultures [22, 301 as well as micro-organisms [36] display a reduction in the division index upon cooling. Temperature increases generally have the opposite effect. Astasia longa grown at 25°C and then exposed to 15°C do not undergo cell division for 18 to 20 hours. This length of time was taken as the upper limit for the duration of the cold period. The length of the warm period was empirically set to maintain a 1: 2 relationship with the cold period and to allow the entire cycle to be 24 hours long. Experimental
Cell
Research
20
Cell division
in Astasia longa
405
RESULTS
The term “cycle” will be used to mean one complete sequence of events of the recurrent temperature changes, i.e., from the beginning of the “cold” period through the following “warm” period up to the beginning of the period. The cycles comprising the temperature program in next “cold” each experiment are indicated in the graph by number (1, 2, 3, etc.). Fig. 1 shows an early experiment illustrating a typical temperature cycle and the growth pattern of a culture of Astasia longa in a 1 per cent proteose peptone medium. The ordinate is the logarithm (to the base 10) of the population density and the abscissa denotes the age of the culture in hours. The temperature cycles are indicated at the base of the graph and the vertical bars bracket the transitions at the level of the growth curve. In this experiment the culture was allowed to enter the logarithmic phase of growth, having attained a density of approximately 2100 cells per milliliter (3.3 on the log scale) before the temperature program was imposed. The program used on the culture in Fig. 1 consisted of a lo-hour “cold” period at 15°C followed by an &hour “warm” period at 25°C. The period of transition between the two temperatures lasted three hours. Thus the entire cycle was repeated every 24 hours. The principle of one to two ratio mentioned above, namely that the cell etl’ectiveness at 25°C is double that at lis”C, was not applied in this cycle. Fig. 1 shows the non-synchronous behavior of the population of cells on this cycle. The first two warm periods result in a doubling of the number of cells while essentially no divisions occur during the greater portion of the respective cold periods. Successive cycles, namely the third, fourth, and fifth, do not show doubling of cell number while the sixth and seventh cycles show a resumption of doubling in the warm period. Attention to the end of the division periods shows that the completion of division is gradually displaced into the following cold periods so that by the third cycle, doubling of cell numbers fails to occur, giving an irregular and asynchronous increase in the population density that continues over the fourth and fifth cycles. The resumption of the synchrony occurs when the beginning of the division period again coincides with the beginning of the warm period. The experiment was terminated at a population level of approximately 100,000 cells per milliliter (5 on the log scale). The sampling error which is implied by the scatter in Fig. 1 was reduced in subsequent experiments by adopting the improved sampling and counting technique outlined earlier. Despite this limitation an interaction between Experimentut
Cell
Research
20
406
G. M. Padilla
and T. W. James
the temperature cycle and the cell cycle is apparent later. Fig. 2 shows the response of a culture grown on 2 per and maintained on a 24-hour cycle consisting of 15 hours at 25°C. The temperature transition periods minutes each.
and will
he discussed
cent proteose peptone hours at 15°C and 8 were reduced to 30
.Temp.
100
120
140
160
180 HOUrS
200
220
240
260
Fig. l.-Growth pattern of Astasia Zonga in a temperature cycle which is poorly matched with the cell cycle. Note the synchrony in the first and second cycles, lack of synchrony in the third, fourth, and fifth cycles, and resumption of synchrony in the sixth and seventh cycles.
In this experiment the culture was placed on the temperature program beginning with the warm period immediately after inoculation, although sampling was started only after the culture had been exposed to the cycle for 40 hours. The culture at this time had attained a population level of 2200 cells per milliliter (3.34 log units). Fig. 2 illustrates the repetitive cycle of temperature and the resulting growth curve. Consistent doubling of cell numbers was obtained within two hours after the onset of each warm period. The fission index calculated as the ratio of the number of cells in recognizable cytoplasmic fission to the total number of cells counted is also shown in Fig. 2. The fission index should not be Experimental
Cell
Research
20
Cell division
in Astasia longa
‘10’7
confused with the mitotic index since the former is obtained by scoring only cells in cytoplasmic cleavage. Since cptoplasmic cleavage occupies a time period considerably shorter than total mitosis [IS] the mitotic index would be larger than the fission index. During the warm period fission indices were determined at fifteen-minute intervals, while during the cold period the determinations Ivere made every 4 hours.
60
80
l&l
120
140
160
1
Hours
Fig. 2.--Synchronized growth and cell division of Asfctsia Zonga grown on a 2 per cent proteosc peptone medium. The temperature cycle, as indicated, consists of 15 hours at 15”C, 8 hours at 25°C and two half-hour transition periods. The fision index, expressed in per cent of cells in fission, is shown in the lower portion of the graph.
In addition to the frequent sampling, living cells removed from the synchronized culture were observed at the beginning of each burst and during the time of maximum activity in cell division. It was found that prior to cell division, cells would become non-motile, settle down, and undergo extensive metabolic movement as has been noted in other flagellates [13]. Of these cells, 30 per cent would normally be in a recognizable state of fission; at that time the cells in the parent culture also showed a maximum fission index. Discrepancy between the recorded peaks in the fission index (from 2.4 per cent to 12.1 per cent) and the higher observed value for this index, indicates Errperimenlnl
Cell Research
20
and T. W. James
G. M. Padilla
that the automatic sampling device is not removing a representative sample of dividing cells from the culture and is possibly selecting for motile, nondividing cells. In spite of this, the fission index can still be used as a measure of the improving resonance between the cell cycle and the time-temperature cycle. TABLE
III.
Analysis of the growth curve of Astasia longa in synchronous cell division. Values
Cycle
Growth constant (Ir)a for burst
1 2 3 4 5 Growth
are calculated
from
the culture
shown
in Fig.
Duration of burst (hrs)
% Cell divisions in warm period
1.3 3.5 1.5 1.1 1.0
90 93 96 100 100
0.12 0.10 0.22 0.24 0.12
2. Maximum index
fission (%)
4.1 7.5 9.6 12.1 6.3
constant (k)b for log phase: Culture at 25°C 0.024 Culture at 15°C 0.005
a Calculated
from
b Calculated
from
the
growth
cultures
grown
equation:
log N/N k = d.
t
in 2 per cent proteose
peptone
as for culture
shown
in Fig.
2.
Table III is an analysis of the synchronous pattern of cell division for the culture shown in Fig. 2. The first column indicates the cycles of the temperature program used in this experiment as they are numbered in Fig. 2 (i.e., 1, 2, 3, etc.). Column two gives the values of the instantaneous growth constant (k) calculated for the log-linear portion of the burst of division occurring in each warm period. For purposes of comparison Table III also includes the growth constants for the logarithmic phase of growth of cells in the same medium (2 per cent proteose peptone) grown at 25 and 15°C. Column three indicates the duration of each burst in hours. Column four gives the proportion of cells that have completed cell division by the end of each warm period. Column live shows the maximum fission index achieved in each of the cycles. Comparison of the minimum fission time (12 minutes) and the minimum duration of the synchronous burst (1 hour) indicates that about 20 per cent of the cells should be seen in fission during the burst. Experimental
Cell Research
20
Cell diuision
in Astasia longa
409
Various attempts to bring about synchronization of cell division in Astasia grown in the chemically defined Cramer-Myers medium resulted only in poorly defined synchronous growth. Alterations of the relative lengths of the warm and cold periods, together with a decrease in the temperature shifts, failed to improve the synchrony with this medium. TABLE
IV.
of generation
Comparison
time, fission time, and fission in various media at 15” and 25°C.
Astasia longa grown The
generation
time (G.T.), the logarithmic
fission index (F.I.) phase of growth. Incubation temperature OC
Medium 2 :b Proteose peptone Cramer-Myers only Cramer-Myers, cysteine and methionine a Calculated is expressed
from
Crick’s
as a decimal
Generation time (hrs.)
15 25 15 25 15 25 equation:
in this
and fission time (F.T.) were measured One standard deviation is given.
52.4 t 11.5k2.7 42.7k4.5 12.8+ 61.9 + 13.0t F.T.
=Iog,
Fission
index
2.3 7.1 2.6
$$I$$
0.75f0.16 0.94 * 0.10 2.59+ 1.17 3.79f 1.52 1.58?0.20 1.62 & 0.49 x G.TL. log,
2
(Kate:
in
for cells in
Fission time’ (min.)
6) 7.3
index
45.0 t 10 9.9F 2.3 133.0*53 64.5/20 96.Oi 35 19.2f 7.8 the fission
index
(F. I.)
equation.)
=Zcomparison of the generation time and fission index between cells grown in 2 per cent proteose peptone and Cramer-Myers showed that while the difference in generation times was slight, the fission index was much higher in cells grown in the defined medium. This increased fission index was reflected in a prolonged fission time as calculated by the equation attributed to Crick [‘3] and as used by Scherbaum [28]. The implication was that the cells grown in the defined medium lacked some constituent involved in the division process. It had been previously shown that Astasia does not utilize amino acids as sources of nitrogen [.?I but the ef’fect of such compounds on the division process had not been examined. A series of experiments were performed in which cells were grown in Cramer-Myers supplemented with cysteine and methionine in concentrations comparable to those used in the growth of other microorganisms. Table IV summarizes the results of these experiments. Column one shows the medium used (i.e., 2 per cent proteose peptone, Cramer-Myers and Cramer-hlyers supplemented with cysteine and methionine). Column two gives the incubation temperature. Column three gives the average generation Experimental
Cell Research
20
G. M. Padilla
410
and T. W. James
time ((;.‘I’.) in hours for cells in the logarithmic four shows the average fission index (I:.I.) as a gives the average fission time (F.T.) in minutes equation. One standard deviation is given for all
phase of growth. Column per cent, and column five as calculated from Crick’s determinations.
5.0 4.8 * 4.6 * 4.4 -
4.2 4.0 -
Fig. S.--Synchronous growth and cell division of dsfasia longa grown on a chemically defined medium (modified Cramer-Myers). The 24-hour temperature cycle consists of 15 hours at 15”C, 8 hours at 25”C, and two 30 minute transition periods. The fission index is expressed in per cent of cells in fission (O-O).
Table IV demonstrates that for cells grown at 2,5”C there is a marked reduction in the fission time upon addition of cysteine and methionine to CramerMyers medium without any apparent decrease in the generation time. The fission time, however, is still considerably longer than found for cells grown on proteose peptone. At 15°C the addition of these amino acids results in some reduction in the fission time, but the wide variation in these values renders them rather inconclusive. Other experiments indicate that either cysteine or methionine alone do not reduce the fission time to the same degree as when they are combined. Further experiments are being conducted to determine the optimum concentration for the greatest reduction of the fission time. Experimental
Cell Research
20
Cell division
in Astnsia
longa
411
Having determined the effect of cysteine and methionine on the fission time of Astasia longa, the supplemented medium was used in subsequent synchronization experiments. Fig. 3 shows the results of such an experiment. The temperature cycle is exactly the same as that used in the experiment shown in Fig. 2. The culture quickly establishes a close relationship with pattern of growth and cell the time-temperature cycle. The synchronized division can be seen to continue until the medium becomes exhausted; this occurs at a population density of approximately 400,000 cells per ml (5.6 in the log scale). It was also interesting to note that the calculated fission times for cells at 25°C generally agree with the observed fission times which were 12.6 i 4.03 minutes for cells grown in 2 per cent proteose peptone and 13.0 i 2.20 minutes for cells grown in the supplemented Cramer-Myers medium. DISCUSSION
The growth and development of a cell from cell division to cell division has been classically subdivided into an interkinetic or growth phase and a kinetic or mitotic phase. This at once introduces the idea that in its broadest sense cellular processes are sequential. If one now considers a sequence of cell generations, or a cell lineage, these phases recurring in time give form to what may be called a cell cycle; the generation time being one period. From this point of view a mass culture of cells in the logarithmic phase of growth becomes an assemblage of these cell cycles randomly distributed in time. A further extension of this argument would allow one to define synchronization of cell division as the alignment of cell cycles so that each growth and division phase occurs simultaneously throughout the culture. The question now arises as to whether one can achieve synchronization of cell division by means of a repetitive temperature cycle. The success encountered in the early investigations of this problem in bacteria [ 10, 11, 15 ] and protozoa [la], as well as in recent experiments with tissue cultures [22I, demonstrates that this question is answerable in the affirmative. These studies strongly suggest that an interaction can be established between the cell cycle and the applied temperature cycle. One may also examine the outstanding synchronization experiments on Tetrahymena [25] in this light. In this case the bursts of cell division following release from the heat treatment can be likened to self-dampened oscillations. The entire question of the cyclical nature of a cell’s activity has been re-examined in recent reviews [3, 32, X5]. The primary aim of the present investigation has been to devise a tempera27 - 0017R?5”
Experimental
Cell Research
20
412
G. M. Padilla
and T. W. James
ture cycle for Astasia longa, grown in a chemically defined medium, that would result in the type of synchrony outlined above. It is obvious that the temperature cycle which will best interlock with the cell cycle can take a variety of forms, particularly since the cell cycle is a function of both time and temperature. Moreover, there is generally a reciprocal relationship between time and temperature for the physico-chemical processes of a cell. Consideration of this time-temperature relationship led to the investigation of the adaptation of Astasia to incubation temperature (cf. Table II). As pointed out earlier, these data, which express the oxygen consumption, the generation time, the cell volume, and the synthetic activity of Astasia as a function of temperature, give a measure of this cell’s capacities at temperatures ranging from 15 to 30°C. This information, together with the observations on the effects of acute temperature changes on the mitotic activity of Astasia, was basic to the construction of the temperature cycle as shown in Fig. 1. The results indicate that the success of this or any other repetitive temperature cycle is largely dependent on the establishment of resonance between the temperature cycle and the cell cycle. This resonance phenomenon is illustrated by Fig. 1 as the interplay between poorly matched temperature and cell cycles. Such behavior is typical of a culture in which the cell cycle is completed in a period different from the period of the impressed temperature cycle. It results in phases of synchrony and lack of synchrony. This is analogous to the interference of waves of slightly different periods. Figs. 2 and 3, on the other hand, show the effects of resonance between the two cycles. This improved interlocking of the cycles was the result of different time intervals and reduced temperature transition periods. The results indicate that the establishment of resonance depends on at least three factors: (1) the energy relationships established between the various phases of the cell cycle; (2) segregation of cell types through differential effect of temperature on different phases of the cell cycle; and (3) the nature of the medium, particularly with respect to the processes involved in cell division. These factors are separated solely for purposes of discussion since the interrelationships existing between them are probably also important in the evaluation of these experiments. The proposition that a temperature cycle brings about synchronization of cell division by altering the time course and direction of the energy-yielding processes involved in cell division, can be examined in terms of the “energy reservoir” idea proposed by Swarm !32]; a somewhat similar approach was used in the kinetic considerations of synchronization in Tetrahymena [26]. Experimental
Cell Research
20
Cell division
in Asfasia
longa
,113
Hriefly, this concept includes the notion that there is an energy reservoir which is filled through the interkinetic phase and is expended during cell division. A cyclical sequence of events is thus established relating the filling and emptying of this energy reservoir to the phases of growth and cell division. With a temperature cycle in which the duration of the warm and cold periods match the cell’s capacities at these temperatures, the cellular processes are channelled into the filling and emptying of this energy reservoir. In terms of the temperature program used in these studies, the cells are forced into an increased production of metabolic respiratory machinery in the cold portion of the cycle (15°C). This is reflected in the increased dry weight and cell size found for cells adapted to this temperature (cf. Table II). This is analogous to an increase in a capacity factor for the cellular system. Toward the end of the cold period the cells approach a size and metabolic capacity of cells grown at 15°C. Upon exposure to the warm period of the cycle, this increase in the metabolic respiratory machinery would in essence operate at a greater rate resulting in the rapid filling of the energy reservoir which then saturates the division machinery. The temperature-dependent rate of metabolic activity c,an be considered as an intensity factor of the cellular system. To bring about cell division, some minimum value of the product of the capacity and intensity factors and time would be required. Evidence for the interplay between these capacity and intensity factors through temperature changes can be gained from direct measurements of the oxygen consumption of cells. For Astnsirr longa we have found that 25°C cells upon cooling to 15°C undergo changes in respiratory activity indicating a gradual increase in respiratory machinery. Upon returning to 25°C these same cells demonstrate a greater respiratory capacity [23]. It is clear that for these energy relationships to contribute to the synchronization process there must also be some mechanism that at one time or another brings the cells to the same level of development jl4, 221. In previous investigations [ 10, 12, 17, 27 j attention has been directed to the processes related to the synthesis of nucleic acids. In general, it is felt that at a lower temperature there is a blockage or even a reversal in the synthesis of nuclear components. In bacterial systems evidence has been presented to show that low temperature exerts drastic changes in the nucleus that can be observed cytologically [ll]. Recent investigations on the synchronization of Amoehrr j13-, using an interference microscope, show that there is loss of materials from the nucleus following cold or warm treatment; a process which may reset the cells to the same stage of nuclear synthesis. This would result in the segregation of large numbers of cells into categories with nearly equivalent nuclei. Experimental
Cell
Reseurrh 20
G. M. Padilla
and T. W. James
A similar situation concerning the equivalence of nuclei has been suggested for cells in the stationary phase of growth [31]. This segregation of cell types can be related to the improvement of synchrony illustrated in Fig. 2. As the data presented in Table III show, this improvement can be appraised in several ways: (1) there is an increase in the growth constant (k) for each successive burst of cell division; (2) there is an increase in the percentage of cells completing division during each warm period; and (3) there is a progressive increase in the maximum division index during each burst. Yet, each burst of cell division occurs at the same time: approximately 2 hours after the cells are exposed to the warm temperature. This suggests that the synchronized cells are at the same initial stage of mitosis and cell cleavage, processes which last approximately 3; hours in some species of Astasia [ 19 j. The results shown in Fig. 3, while essentially the same as those of Fig. 2, point to the importance of conducting synchronization experiments on a chemically defined medium. This is particularly true if one wishes to investigate the role played by certain compounds in the division process. In the synchronization of Astasia, cysteine and methionine have proved to be key factors in the reduction of the fission time, an effect similar to that found in Amoeba [33]. Such an effect can be due to several things: (1) both cysteine and methionine are precursors to compounds such as coenzyme A, glutathione, etc. [34]; (2) as protein sulfhydryl residues they may be directly involved in the make-up of the mitotic apparatus 120, 241, and lastly (3) their primary role may be one dealing with the oxidation-reduction level of the cellular environment [6]. The fact that the generation time is unaffected by their presence, implies that these amino acids are primarily involved in the division process. This points to the possibility of an insight into the biochemical pathways leading to cell division. We feel that in order to perpetuate the synchronized pattern of growth within a culture, chemostatic conditions of growth will have to be adopted. SUMMARY
1. A 24-hour repeating temperature cycle, consisting of 15 hours at 15”C, 8 hours at 25”C, and two 30-minute transition periods, has been used for the synchronization of cell division of Astasia longa grown in 2 per cent proteose peptone and in a chemically defined medium. This results in a burst of cell division every 24 hours for as many as seven generations. 2. Cysteine and methionine were found to be important in the mechanism Experimental
Cell Research
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Cell division
in Astasia longa
415
of synchronization of cells grown in Cramer-Myers medium. ‘Their addition appears to reduce the fission time of these cells without affecting the generation time. 3. The synchronization process is evaluated in terms of a cell cycle. The possible manner in which synchronization is achieved 1-1~means of a repcaling temperature cycle is discussed. REFERENCES 1. BSRXER, 2. BRUCE, 3. 4. 5. 6. 7. 8. 9 1;: 11. 12. 13. 11. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. ‘-0 _I>. 29. 30. 31. 32. 33. 34. 35. 36.
H. D. and COHEX, S. S., J. Bmteriol. 72, 115 (1956). V. B., in Influence of Temperature on Biological Systems. Ed. Johnson, I’. I-I., Am. Physiological Sot., Washington, 1957. CAMPBELL, A., Bucleriol. 12~s. 21. 263 (1957). HALL, R. P., JOHNSON, D. I?. and LOEFER, J. B., Truns. ,4m. Microscop. Sot. 54, 218 (1935). HASSOS, R. W., Ph.D. Thesis, Univ. Calif., Los Angeles, Calif., 1952. Oxidation-Reduction Potentials in Bacteriology and Biochemistry. E. & S. HEWITT, L. F., Livingstone Ltd., Edinburgh, 1950. HOEL, P. G., Introduction to Mathematical Statistics. John Wiley & Sons, New York, 1954. HOLLANDE, A., Arch. Zool. exptt gCn. 83, 73 (1942). HUGHES, A., The Mitotic Cycle. Academic Press, New York, 1952. HOTCHKISS, R. D., Proc. Xatl. Acad. Sci. 40, 49 (1954). HUSTER-SZYBALSKA, AI., SZYBALSKI, W. and DELAMATER, E., -7. Bucleriol. 71, 17 (1956). IVERSON, R. M. and GIESE, A. C., Exptl. Cell Research 13, 213 (1957). JAIIN, T. L., and MCKIBB&N, R. W., Trans. Am. Microscop. Sot. 56, 48 (1937). JAILIES, T. W., Ph.D. Thesis Univ. Calif., Berkclcy, Calif. (1954). __ Ann. S. I’. Acad. Sci. 78, 501 (1959). J.~~xEs, T. W. and PADILLA, G. M., in Proceedings of the First National Biophysics Conference. Ed. Quastler, H., Yale IJniv. Press, 1959. LARK, K. G. and MA.~LBF,, O., Biochim. ef Biophys. Acta 21, 448 (1956). LEEDALE, G. F., Arch. Mikrobiot. 32, 32 (1958). __ Ibid. 32, 352 (1959). RI~ix.4, D., in Glutathione. Ed. Colowick, S. P. and Kaplan, iX. G. Academic Press, Sew York, 1954. MYERS, J. and CRAMER, M., Arch. Mikrobiol. 17, 384 (1952). NEWTON, A. A. and WILDY, P., Exptl. Cell Research 16, 624 (1959). PADILLA. G. M. and JAMES, T. W. (In preparation). RAPKIN~, L., Ann. Physiol: Phgsiochem. Viol. 7, 382 (1931). SCHERBAUI\I. 0. and ZEurnEN, E., Exptl. Cell Research 6, 221 (1954). Ibid. i3, 11 (1957). __ Ibid. 13, 24 (1957). J. Protozool. 4, 257 (1957). SCHOEXBORN, H. W., .I. Exptl. Zool. 105, 269 (1947). SPEAR, F. G., Arch. exptl. Zellforsch. 7, 484 (1928). SUMMERS, L. G., BERNSTEIK, E. and JAMES, T. W., Expfl. Cell Research 13, 436 (1957). SWANN, M. M., Cancer Research 17, 727 (1957). VOEGTLIN, C. and CIIALKLEY, H. W., U.S. Publ. Health Repts. 45, 3051 (1930). YOUSG, L. and MAW, G. A., The Metabolism of Sulphur Compounds. Methuen and Co. Ltd., London, 1958. ZEUTHEN, E., Advances in Biol. and Med. Phys. VI, 37 (1958). ZEUTHEN, E. and SCHERBAUM, O., in Cell Physiology. Ed. Kitching, J. A., Butterworth, London, 1954.
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