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Mechanism of synchrony induction
Experimental Cell Research68 (1971) 43l-436
MECHANISM
OF SYNCHRONY
INDUCTION
I. Some Features of Synchronous Rounding in Tetrahymena
pyriformis
Y. WATANABE Department
of Pathology, National Institute of Health, Shinagawa-ku, Kamiosaki 2&/O, TokJJo, Japan
SUMMARY When Tetruhymena pyriformis was kept standing in amino acid-deprived synthetic medium, it rounded periodically as a substitute for division. The abortive cell cycle was roughly estimated to be about 6 h. The synchronizing heat treatment not only prevented the cells from entering the spherical stage but it caused the cells to stop in a certain stage so that the first and second synchronous roundings occurred at 75-80 min and 190-200 min respectively after the end of the heat-treatment (EHT), the two rounding maxima well coinciding with those of synchronous divisions. The delay in the first synchronous rounding, induced by 20 min-exposures to 34’C or IO mM sodium fluoride, increased gradually when given from 0 to about 45 min after EHT and decreased sharply after that, like the response in the synchronous division system. DNA, RNA and protein amounts per cell remained nearly constant in about 70-80 % of those of logphase cells through the abortive cell cycle, which was different from that in synchronous division. From these results, it is suggested that net increase of DNA, RNA and protein contents is not necessary for the progression of the abortive cell cycle and for phasing process. It is noteworthy that the interval between the first and second synchronous rounding is shorter than the ordinary one-cell cycle. It is possible, therefore, to infer that shortening of the cycle is not due to the surplus accumulation of biomolecules, but may be attributed to the concentration of an essential factor(s) controlling the cell cycle
In 1963, we discovered a phenomenon desigrounding”. When nated as “synchronous amino acid-starved Tetrahymena cells are exposed to cyclic temperature treatment for synchronous division, they attain sphericity synchronously with no accompanying division at the time when synchronous division in ordinary proteose-peptone medium would take place [I]. Recently, Tamura et al. indicated some corresponding biochemical changes in the synchronous rounding to those in synchronous division [2]. From the studies on cortical morphogenesis in synchronous rounding cells, Frankel clearly
demonstrated that primordium development occurs after the end of the heat treatment (EHT) in regular sequence [3]. Furthermore, supplying amino acids during synchronous rounding induction deflects synchronous rounding back to synchronous division [2], which shows that the cells kept in amino acid-deprived synthetic medium are synchronized by the same mechanism of phasing as that in synchronous division by HT (heat-treatment). The present work was designed to compare some features of synchronous rounding with those of synchronous division. Exptl
Cell Res 68
432
Y. Watanabe MATERIALS
AND METHODS
Culture conditions Tetuuilytwna pyrifornh, strain W, was grown in 2 o,, proteose-peptone medium enriched with 0.5 ?; yeast extract and 0.87 9, dextrose under sterile conditions. For culturing, 50 ml flasks with 10 ml of the medium were used in the present experiments, unless otherwise specified. Culture flasks containing the cells were submerged in a water bath at 26’C and shaken horizontally.
Induction qf slwchronow rounding Exponentially growing cells in the proteose-peptone medium were quickly but thoroughly washed with an inorganic medium (2 g NaCI, 80 mg KCI, 120 mg CaCI, in I 000 ml double-distilled water), after which the cells were transferred axenically to an amino acid-deprived medium; the medium was basal medium 2A of Dewey, Parks & Kidder [4] enriched with Tween 80, dextrose and guanylic acid to make I Y,, 0.5 % and 0.015 56, respectively, minus amino acids [I]. The transferred cells were kept standing in the amino acid-deprived synthetic medium for 9-15 h at 26 C and then they were subjected to HT: 8 alternate 30 min-exposures to temperatures of 34’C and 26’C [I]. In order to elevate the maximum rounding index in the second synchronous rounding after EHT, the heat-treated ceils were washed once with the inorganic medium (26°C) after the 8th high-temperature shock and resuspended in the renewed amino acid-deprived medium and exposed to three more alternate changes of temperatures. The rounding cell in the present experiments is defined as the cell having an axial ratio (minor axis/major axis) of 0.8 or more.
Uptake of tulIpan blue during the heattreatment At appropriate stages in the inducing process of synchronous rounding, a small amount of culture was mixed with an equal volume of 4 % trypan blue solution containing 2 Y!,proteose-peptone and incubated for 20 min. A small droplet of the culture was put under a cover glass in order-to suppress cell movement and more than 400 cells were counted within 5 min in terms of presence or absence of colored food vacuoles.
Setback in the process of synchronous rounding The heat-treated cells in the amino acid-deprived medium were exoosed. at various sees after EHT. to 34’C for 20 min and’then returned 70 the optimum temperature (26°C) or IO mM sodium fluoride for 20 Ain and washed twice in the inorganic medium. The delays in synchronous rounding were plotted against the age of the cells at the initial time of the setback treatment. Exptl Cell Rrs 68
Measurements of DNA, RNA and protein amounts Usually, 20 to 50 ml samples were taken at appropriate stages in the inducing process of synchronous rounding. The sampled cells were washed 3 times with the cold inorganic medium and suspended in cold 5 ‘111 trichloroacetic acid. Fractionation of DNA, RNA and protein was followed by the method of Schmidt & Thannhauser [5]. DNA and RNA amounts were measured calorimetrically by use of the diphenylamine reaction [6] and the orcinol reaction [7], respectively. Protein contents were determined by the method of Lowry et al. [8]. The cell number was counted by the use of a Fuchs-Rosenthal chamber as described in the previous paper [I].
RESULTS Abortil;e cell cycle in amino acid-deprived medium When log-phase cells in proteose-peptone medium are transferred to amino aciddeprived medium, only about 30 % of the total cells can divide once within the first 5 h and afterwards cell numbers remain constant for a long time [l]. Under these non-dividing conditions, spherical cells appeared with nearly constant rate, about 7 “b of the total cells. The rounding was not an irreversible change and the interval of sphericity was observed to be about 25 min. The facts suggest that each cell in amino acid-deprived medium may repeat an abortive cell cycle (from sphericity to sphericity) asynchronously. If that be the case, the one cycle would be estimated to be about 6 h by a simple proportion from the percentage of spherical cells and the interval of rounding. The time well coincides with one generation time of the cell kept in complete synthetic medium
[Il. Effect of heat-treatment on the amino acidstarted cells As shown in fig. I, when the amino acidstarved cells are subjected to HT, the percentage of the spherical cells decreases rapidly and all acquire pyriform shape.
Mechanism
Afterwards, the cell shape did not change up to the onset of the first synchronous rounding. The disappearance of rounding cells at the early phase of HT corresponds well to the disappearance of dividing cells in the synchronous division-induction system [9]. Next, trypan blue test were performed, since oral replacement by HT is understandable from the experiment of the uptake of the dye (fig. 1). Before the synchronizing HT, about 20 96 of the cells fail to take up trypan blue. The percentages of the non-feeding cells in terms of trypan blue uptake remain nearly constant as long as the rate of cell rounding is fixed. However, the population of the non-feeding cells begins to increase gradually after the commencement of HT and reaches about 80~; at EHT. The fact suggests that a cell in prerounding stage is incapable of taking up trypan blue and that HT involves the accumulation of such cells. Incapability of trypan blue uptake is not due to an irreversible damage of the cell. As shown in fig. 2, most cells after one synchronous rounding can again take up trypan blue. From EHT to the end of 2nd synchronous rounding, percentages of the non-feeding cell fluctuate in synchronous fashion, for instance, two maxima of the curve nearly coincide with those of synchronous roundings. Tile first
and second synchronous
of slxchronmv induction.
0 12
3
4
5
6
I
433
-,
c
7
Fig. 1. Abscissa: hours: ordinate: (left) cl, % nonfeeding cells; (right) l , 9: rate of rounding cell. Effect of HT on amino acid-starved cells. Log-phase cells were kept in the amino acid-deprived synthetic medium at 26’C for 12 h before HT. l , “b of rounding cells; il, ‘%Aof non-feeding cells as determined by 2 % trypan blue for 20 min, each point representing the middle of 20 min-incubation. Top: system of temperature treatment for synchronization.
the degree of synchrony (rounding index. about 25 Y,) was considerably lower than that of the first rounding. However. the second rounding occurred at much the same time as that of second synchronous division (190-200 min after EHT). In the experiments, the heat-treated cells were resuspended in new amino acid-deprived medium at the end
roundings
In fig. 3, photographs of synchronous rounding are shown in comparison with those of synchronous division. A glance at the photographs clearly shows that the degree of synchrony and the time sequence of synchronous rounding bear a strong resemblance to those of synchronous division. At the maximum (75-80 min after EHT), the rounding index reached nearly 90’&, the completely spherical cells (axial ratio, 1.0) being more than 50:;, of the total. As to the second synchronous rounding,
0
33
60
90
120
150
'80
210 240
270
300
Fig. 2. Abscissa: min after EHT; ordinate: “:, nonfeeding cell. Uptake of trypan blue at various stages after EHT to the end of the second synchronous rounding. The incubation condition wi:h trypan blue is the same as that of fig. I. Top: periods of the first and second synchronous rounding are shown with black bars. Exptl Cell Res 68
434
Y. Watanabe
Fig. S. Tetrahymena cells in six matching stages of temperature-induced synchronous rounding (A-F)
and
synchronous division (G-L). Photographs (A) and (G) were taken at 0 min; (B) and (If), at 30 min; (C) and (I), at 65 min; (D) and (J), at 75 min; (E) and (K), at 80 min; (F) and (L), at 120 min after EHT. Note rounding cells in (C, D, E) and division (I, J, K).
of the 8th temperature-shock and exposed to 3 more temperature cycles, since a small part of the total cells tends to enter division at the second synchronous rounding unless the medium is renewed.
50
1
,'
,'
,’
Setback found in the process of synckronous rounding Fig. 4 shows the delays in the first synchronous rounding induced by 20 min exposures to 34°C or to 10 mM sodium fluoride at various stages after EHT. In both curves, the delays increase gradually from EHT to approx. 45 min after EHT, followed by a very sharp decrease. The physiological transition point is present about 45 min after EHT. After the transition point, the cells show little or no delay by the exposure to 34°C or 10 mM sodium fluoride. The setback curves in synchronous rounding as shown in fig. 4 are essentially the same as those in synchronous division [l, 10, 111. DNA, RNA and protein amounts in the induction process of synchronous rounding
Fig. 4. Abscissa: min after EHT; ordina~ec excess delay (min), o-3, heat shock at 34°C; O--O, 10 mM sodium fluoride; m, transition point. Effect of 20 min-exposure to 34°C or IOmM sodium fluoride on synchronous rounding. The plot shows the delay of the first synchronous rounding as function of cell age. The rounding period was shaded. Note the transition point about 45 min after EHT. Exptl Cell Res 68
Fig. 5 represents DNA, RNA and protein amounts of the amino acid-starved cells during HT and in the period between EHT and the end of the first synchronous rounding. When the amounts of DNA, RNA and protein per 1O6cells in log-phase are taken as 100, those of the cells kept in amino acid-deprived
Mechanism of synchrony induction. I
435
events in the induction process of synchrony and the changes underlying in the process of cell division. We have endeavored to divide independent biological phenomena, two namely “induction of synchrony” and “cell 47 division”, from the complicated synchronous 31) zc division. The synchronous rounding we found appears to provide a simpler experimental system for analysing a cause involved in the induction of synchrony. Fig. 5. Abscissa:-min after EHT; ordinate: % amount. It is certain that HT induces synchronizdC~C, DNA; y -- i, RNA; O-.-O, protein. DNA, RNA and protein amounts in the inducing tion of the cell. When amino acids are added processof synchronous rounding. DNA, RNA and protein contents per cell in log phase are taken as to the amino acid-deprived medium, starved 100, and those of amino acid-starvedcells at various cells without HT divide only asynchronously stagesare shown in percentages.Each point represents a meanvalue of six separateexperiments.Bottom: The but cells which have undergone HT show system of heat-treatment and minutes after EHT. synchronous division [2]. Disappearance of SR, the period of synchronousrounding. spherical cells at the beginning of HT suggests a phasing effect of such treatment, medium for 5 h were about 77 96. The value since dividing cells also disappear at the reflects the fact that those cells which had corresponding period in the system of synreached proximity to division under the chronous division [9]. Furthermore, the previous log-phase culture (about 30 %) must number of non-feeding cells in terms of have divided once in the starvation period trypan blue uptake increases gradually during before the heat-treatment. After that, the HT. Studies of oral morphogenesis also amounts of DNA, RNA and protein per reveal the phasing effect of HT. According cell remained constant. Especially, DNA and to Frankel’s observations [3], about IO ‘“b of RNA contents do not change at all during the cells kept standing in amino acidthe heat-treatment and in the period from deprived medium are performing so called EHT to the end of the synchronous rounding, oral replacement and the percentage increases while protein amounts per cell decreased gradually up to nearly 100~; during HT. slightly at the latter half of HT (fig. 5). Oral replacement leading to synchronous rounding corresponds to the accumulation of cells possessing “anarchic fields” (the DISCUSSION early stage of primordium) in the inducing The discovery of the method inducing synprocess of synchronous division. The result chronous division by a controlled temperature concerning oral replacement well agrees with cycle [12] provided many interesting problems that of our present experiment using trypan and developments in the solution of the blue. mechanism of cell division. However, the The role that HT plays in synchronization artificially induced synchrony of cell division may also be deduced from the setback curve is initiated by a phase of recovery from speciin synchronous rounding (fig. 4). It has been fied imposed stress, that is to say, serial accepted that cells at various stages between biochemical events toward the synchronous 0 and 45 min after EHT are returned quickly division are undoubtedly composed of the to their initial physiological condition (0 min E,q~tl Cell Rex 68
436
Y. Watanabe
after EHT) by the heat shock [13]. Whether the cells are kept in amino acid-deprived synthetic medium or in proteose-peptone medium, the setback curves induced by 34°C or sodium fluoride treatment are essentially the same. It is, therefore, concluded that both systems (synchronous rounding and synchronous division) lie on common ground. The present experimental results show that DNA, RNA and protein amounts per cell remain constant through most process of synchronization. This is one of the marked differences between synchronous rounding and synchronous division; in the latter system, the cells synthesize DNA, RNA and protein in larger quantities with no division during HT [9, 141. It is of utmost importance that the progress of the abortive cell life cycle and synchrony induction by HT are possible without doubling of DNA, RNA and protein. Moreover, it must be emphasized here that the interval between the first and second synchronous rounding after EHT is clearly shorter than the usual one cell cycle. It has sometimes been assumed that the reduction of generation time found between the first and second synchronous division is attributed to the intracellular surplus accumulation of various biomolecules produced during HT. This seems to be logical if one takes into account only the results obtained in the system of synchronous division. However, the idea is not always tenable, since the net amounts of DNA, RNA and protein per
Exprl Cell Res 68
cell do not increase in the system of synchronous rounding. Nevertheless, even in the latter circumstances, one cannot exclude the possibility of accumulation of a particular factor(s) closely related to the reduction of one cell cycle. Further scrutiny of the factor will be discussed in succeeding papers. I am grateful to Professor K. Dan, President of Tokyo Metropolitan University, for the critical reading of the manuscript. Thanks are also due to Dr S. Tamura and Miss A. Mochizuki for their assistance in carrying out some of the experiments.
REFERENCES I. Watanabe, Y, Japan j med sci biol I6 (1963) 107. 2. Tamura, S, Toyoshima, Y & Watanabe, Y, Japan j med sci biol I9 (1966) 85. 3. Frankel, J, J exptl zoo1 173 (1970) 79. 4. Dewey, V C, Parks, R E Jr & Kidder, G W, Arch biochem 29 (1950) 281. 5. Schmidt, G & Thannhauser, S J, J biol them 161 (1945) 83. 6. Dische, Z, Mikrochem 8 (1930) 4. I. Majbaum, W, Z physiol them 25X (1939) 117. 8. Lowry, 0 H, Rosebrough, N J, Farr. A L & Randall. R J. J biol them 193 (1951) 265. 9. Zeuthen; E & Scherbaum, 0, Colston papers 7 (1954) 141. 10. Hamburger, K, Compt rend trav lab Carlsberg 32 (1962) 359. I I. Frankel, J, Compt rend trav lab Carlsberg 33 (1962) I. 12. Scherbaum, 0 & Zeuthen, E, Exptl cell res 6 (1954) 221. 13. Zeutgen, E, Synchrony in cell division and growth, p. 99. Interscience, New York, London Sydney, 1964. 14. Scherbaum, 0, Exptl cell res I3 ( 1957) 24. Received January 11, 1971 Revised version received May 24, 1971
MECHANISM
OF SYNCHRONY
INDUCTION
I. Some Features of Synchronous Rounding in Tetrahymena
pyriformis
Y. WATANABE Department
of Pathology, National Institute of Health, Shinagawa-ku, Kamiosaki 2&/O, TokJJo, Japan
SUMMARY When Tetruhymena pyriformis was kept standing in amino acid-deprived synthetic medium, it rounded periodically as a substitute for division. The abortive cell cycle was roughly estimated to be about 6 h. The synchronizing heat treatment not only prevented the cells from entering the spherical stage but it caused the cells to stop in a certain stage so that the first and second synchronous roundings occurred at 75-80 min and 190-200 min respectively after the end of the heat-treatment (EHT), the two rounding maxima well coinciding with those of synchronous divisions. The delay in the first synchronous rounding, induced by 20 min-exposures to 34’C or IO mM sodium fluoride, increased gradually when given from 0 to about 45 min after EHT and decreased sharply after that, like the response in the synchronous division system. DNA, RNA and protein amounts per cell remained nearly constant in about 70-80 % of those of logphase cells through the abortive cell cycle, which was different from that in synchronous division. From these results, it is suggested that net increase of DNA, RNA and protein contents is not necessary for the progression of the abortive cell cycle and for phasing process. It is noteworthy that the interval between the first and second synchronous rounding is shorter than the ordinary one-cell cycle. It is possible, therefore, to infer that shortening of the cycle is not due to the surplus accumulation of biomolecules, but may be attributed to the concentration of an essential factor(s) controlling the cell cycle
In 1963, we discovered a phenomenon desigrounding”. When nated as “synchronous amino acid-starved Tetrahymena cells are exposed to cyclic temperature treatment for synchronous division, they attain sphericity synchronously with no accompanying division at the time when synchronous division in ordinary proteose-peptone medium would take place [I]. Recently, Tamura et al. indicated some corresponding biochemical changes in the synchronous rounding to those in synchronous division [2]. From the studies on cortical morphogenesis in synchronous rounding cells, Frankel clearly
demonstrated that primordium development occurs after the end of the heat treatment (EHT) in regular sequence [3]. Furthermore, supplying amino acids during synchronous rounding induction deflects synchronous rounding back to synchronous division [2], which shows that the cells kept in amino acid-deprived synthetic medium are synchronized by the same mechanism of phasing as that in synchronous division by HT (heat-treatment). The present work was designed to compare some features of synchronous rounding with those of synchronous division. Exptl
Cell Res 68
432
Y. Watanabe MATERIALS
AND METHODS
Culture conditions Tetuuilytwna pyrifornh, strain W, was grown in 2 o,, proteose-peptone medium enriched with 0.5 ?; yeast extract and 0.87 9, dextrose under sterile conditions. For culturing, 50 ml flasks with 10 ml of the medium were used in the present experiments, unless otherwise specified. Culture flasks containing the cells were submerged in a water bath at 26’C and shaken horizontally.
Induction qf slwchronow rounding Exponentially growing cells in the proteose-peptone medium were quickly but thoroughly washed with an inorganic medium (2 g NaCI, 80 mg KCI, 120 mg CaCI, in I 000 ml double-distilled water), after which the cells were transferred axenically to an amino acid-deprived medium; the medium was basal medium 2A of Dewey, Parks & Kidder [4] enriched with Tween 80, dextrose and guanylic acid to make I Y,, 0.5 % and 0.015 56, respectively, minus amino acids [I]. The transferred cells were kept standing in the amino acid-deprived synthetic medium for 9-15 h at 26 C and then they were subjected to HT: 8 alternate 30 min-exposures to temperatures of 34’C and 26’C [I]. In order to elevate the maximum rounding index in the second synchronous rounding after EHT, the heat-treated ceils were washed once with the inorganic medium (26°C) after the 8th high-temperature shock and resuspended in the renewed amino acid-deprived medium and exposed to three more alternate changes of temperatures. The rounding cell in the present experiments is defined as the cell having an axial ratio (minor axis/major axis) of 0.8 or more.
Uptake of tulIpan blue during the heattreatment At appropriate stages in the inducing process of synchronous rounding, a small amount of culture was mixed with an equal volume of 4 % trypan blue solution containing 2 Y!,proteose-peptone and incubated for 20 min. A small droplet of the culture was put under a cover glass in order-to suppress cell movement and more than 400 cells were counted within 5 min in terms of presence or absence of colored food vacuoles.
Setback in the process of synchronous rounding The heat-treated cells in the amino acid-deprived medium were exoosed. at various sees after EHT. to 34’C for 20 min and’then returned 70 the optimum temperature (26°C) or IO mM sodium fluoride for 20 Ain and washed twice in the inorganic medium. The delays in synchronous rounding were plotted against the age of the cells at the initial time of the setback treatment. Exptl Cell Rrs 68
Measurements of DNA, RNA and protein amounts Usually, 20 to 50 ml samples were taken at appropriate stages in the inducing process of synchronous rounding. The sampled cells were washed 3 times with the cold inorganic medium and suspended in cold 5 ‘111 trichloroacetic acid. Fractionation of DNA, RNA and protein was followed by the method of Schmidt & Thannhauser [5]. DNA and RNA amounts were measured calorimetrically by use of the diphenylamine reaction [6] and the orcinol reaction [7], respectively. Protein contents were determined by the method of Lowry et al. [8]. The cell number was counted by the use of a Fuchs-Rosenthal chamber as described in the previous paper [I].
RESULTS Abortil;e cell cycle in amino acid-deprived medium When log-phase cells in proteose-peptone medium are transferred to amino aciddeprived medium, only about 30 % of the total cells can divide once within the first 5 h and afterwards cell numbers remain constant for a long time [l]. Under these non-dividing conditions, spherical cells appeared with nearly constant rate, about 7 “b of the total cells. The rounding was not an irreversible change and the interval of sphericity was observed to be about 25 min. The facts suggest that each cell in amino acid-deprived medium may repeat an abortive cell cycle (from sphericity to sphericity) asynchronously. If that be the case, the one cycle would be estimated to be about 6 h by a simple proportion from the percentage of spherical cells and the interval of rounding. The time well coincides with one generation time of the cell kept in complete synthetic medium
[Il. Effect of heat-treatment on the amino acidstarted cells As shown in fig. I, when the amino acidstarved cells are subjected to HT, the percentage of the spherical cells decreases rapidly and all acquire pyriform shape.
Mechanism
Afterwards, the cell shape did not change up to the onset of the first synchronous rounding. The disappearance of rounding cells at the early phase of HT corresponds well to the disappearance of dividing cells in the synchronous division-induction system [9]. Next, trypan blue test were performed, since oral replacement by HT is understandable from the experiment of the uptake of the dye (fig. 1). Before the synchronizing HT, about 20 96 of the cells fail to take up trypan blue. The percentages of the non-feeding cells in terms of trypan blue uptake remain nearly constant as long as the rate of cell rounding is fixed. However, the population of the non-feeding cells begins to increase gradually after the commencement of HT and reaches about 80~; at EHT. The fact suggests that a cell in prerounding stage is incapable of taking up trypan blue and that HT involves the accumulation of such cells. Incapability of trypan blue uptake is not due to an irreversible damage of the cell. As shown in fig. 2, most cells after one synchronous rounding can again take up trypan blue. From EHT to the end of 2nd synchronous rounding, percentages of the non-feeding cell fluctuate in synchronous fashion, for instance, two maxima of the curve nearly coincide with those of synchronous roundings. Tile first
and second synchronous
of slxchronmv induction.
0 12
3
4
5
6
I
433
-,
c
7
Fig. 1. Abscissa: hours: ordinate: (left) cl, % nonfeeding cells; (right) l , 9: rate of rounding cell. Effect of HT on amino acid-starved cells. Log-phase cells were kept in the amino acid-deprived synthetic medium at 26’C for 12 h before HT. l , “b of rounding cells; il, ‘%Aof non-feeding cells as determined by 2 % trypan blue for 20 min, each point representing the middle of 20 min-incubation. Top: system of temperature treatment for synchronization.
the degree of synchrony (rounding index. about 25 Y,) was considerably lower than that of the first rounding. However. the second rounding occurred at much the same time as that of second synchronous division (190-200 min after EHT). In the experiments, the heat-treated cells were resuspended in new amino acid-deprived medium at the end
roundings
In fig. 3, photographs of synchronous rounding are shown in comparison with those of synchronous division. A glance at the photographs clearly shows that the degree of synchrony and the time sequence of synchronous rounding bear a strong resemblance to those of synchronous division. At the maximum (75-80 min after EHT), the rounding index reached nearly 90’&, the completely spherical cells (axial ratio, 1.0) being more than 50:;, of the total. As to the second synchronous rounding,
0
33
60
90
120
150
'80
210 240
270
300
Fig. 2. Abscissa: min after EHT; ordinate: “:, nonfeeding cell. Uptake of trypan blue at various stages after EHT to the end of the second synchronous rounding. The incubation condition wi:h trypan blue is the same as that of fig. I. Top: periods of the first and second synchronous rounding are shown with black bars. Exptl Cell Res 68
434
Y. Watanabe
Fig. S. Tetrahymena cells in six matching stages of temperature-induced synchronous rounding (A-F)
and
synchronous division (G-L). Photographs (A) and (G) were taken at 0 min; (B) and (If), at 30 min; (C) and (I), at 65 min; (D) and (J), at 75 min; (E) and (K), at 80 min; (F) and (L), at 120 min after EHT. Note rounding cells in (C, D, E) and division (I, J, K).
of the 8th temperature-shock and exposed to 3 more temperature cycles, since a small part of the total cells tends to enter division at the second synchronous rounding unless the medium is renewed.
50
1
,'
,'
,’
Setback found in the process of synckronous rounding Fig. 4 shows the delays in the first synchronous rounding induced by 20 min exposures to 34°C or to 10 mM sodium fluoride at various stages after EHT. In both curves, the delays increase gradually from EHT to approx. 45 min after EHT, followed by a very sharp decrease. The physiological transition point is present about 45 min after EHT. After the transition point, the cells show little or no delay by the exposure to 34°C or 10 mM sodium fluoride. The setback curves in synchronous rounding as shown in fig. 4 are essentially the same as those in synchronous division [l, 10, 111. DNA, RNA and protein amounts in the induction process of synchronous rounding
Fig. 4. Abscissa: min after EHT; ordina~ec excess delay (min), o-3, heat shock at 34°C; O--O, 10 mM sodium fluoride; m, transition point. Effect of 20 min-exposure to 34°C or IOmM sodium fluoride on synchronous rounding. The plot shows the delay of the first synchronous rounding as function of cell age. The rounding period was shaded. Note the transition point about 45 min after EHT. Exptl Cell Res 68
Fig. 5 represents DNA, RNA and protein amounts of the amino acid-starved cells during HT and in the period between EHT and the end of the first synchronous rounding. When the amounts of DNA, RNA and protein per 1O6cells in log-phase are taken as 100, those of the cells kept in amino acid-deprived
Mechanism of synchrony induction. I
435
events in the induction process of synchrony and the changes underlying in the process of cell division. We have endeavored to divide independent biological phenomena, two namely “induction of synchrony” and “cell 47 division”, from the complicated synchronous 31) zc division. The synchronous rounding we found appears to provide a simpler experimental system for analysing a cause involved in the induction of synchrony. Fig. 5. Abscissa:-min after EHT; ordinate: % amount. It is certain that HT induces synchronizdC~C, DNA; y -- i, RNA; O-.-O, protein. DNA, RNA and protein amounts in the inducing tion of the cell. When amino acids are added processof synchronous rounding. DNA, RNA and protein contents per cell in log phase are taken as to the amino acid-deprived medium, starved 100, and those of amino acid-starvedcells at various cells without HT divide only asynchronously stagesare shown in percentages.Each point represents a meanvalue of six separateexperiments.Bottom: The but cells which have undergone HT show system of heat-treatment and minutes after EHT. synchronous division [2]. Disappearance of SR, the period of synchronousrounding. spherical cells at the beginning of HT suggests a phasing effect of such treatment, medium for 5 h were about 77 96. The value since dividing cells also disappear at the reflects the fact that those cells which had corresponding period in the system of synreached proximity to division under the chronous division [9]. Furthermore, the previous log-phase culture (about 30 %) must number of non-feeding cells in terms of have divided once in the starvation period trypan blue uptake increases gradually during before the heat-treatment. After that, the HT. Studies of oral morphogenesis also amounts of DNA, RNA and protein per reveal the phasing effect of HT. According cell remained constant. Especially, DNA and to Frankel’s observations [3], about IO ‘“b of RNA contents do not change at all during the cells kept standing in amino acidthe heat-treatment and in the period from deprived medium are performing so called EHT to the end of the synchronous rounding, oral replacement and the percentage increases while protein amounts per cell decreased gradually up to nearly 100~; during HT. slightly at the latter half of HT (fig. 5). Oral replacement leading to synchronous rounding corresponds to the accumulation of cells possessing “anarchic fields” (the DISCUSSION early stage of primordium) in the inducing The discovery of the method inducing synprocess of synchronous division. The result chronous division by a controlled temperature concerning oral replacement well agrees with cycle [12] provided many interesting problems that of our present experiment using trypan and developments in the solution of the blue. mechanism of cell division. However, the The role that HT plays in synchronization artificially induced synchrony of cell division may also be deduced from the setback curve is initiated by a phase of recovery from speciin synchronous rounding (fig. 4). It has been fied imposed stress, that is to say, serial accepted that cells at various stages between biochemical events toward the synchronous 0 and 45 min after EHT are returned quickly division are undoubtedly composed of the to their initial physiological condition (0 min E,q~tl Cell Rex 68
436
Y. Watanabe
after EHT) by the heat shock [13]. Whether the cells are kept in amino acid-deprived synthetic medium or in proteose-peptone medium, the setback curves induced by 34°C or sodium fluoride treatment are essentially the same. It is, therefore, concluded that both systems (synchronous rounding and synchronous division) lie on common ground. The present experimental results show that DNA, RNA and protein amounts per cell remain constant through most process of synchronization. This is one of the marked differences between synchronous rounding and synchronous division; in the latter system, the cells synthesize DNA, RNA and protein in larger quantities with no division during HT [9, 141. It is of utmost importance that the progress of the abortive cell life cycle and synchrony induction by HT are possible without doubling of DNA, RNA and protein. Moreover, it must be emphasized here that the interval between the first and second synchronous rounding after EHT is clearly shorter than the usual one cell cycle. It has sometimes been assumed that the reduction of generation time found between the first and second synchronous division is attributed to the intracellular surplus accumulation of various biomolecules produced during HT. This seems to be logical if one takes into account only the results obtained in the system of synchronous division. However, the idea is not always tenable, since the net amounts of DNA, RNA and protein per
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cell do not increase in the system of synchronous rounding. Nevertheless, even in the latter circumstances, one cannot exclude the possibility of accumulation of a particular factor(s) closely related to the reduction of one cell cycle. Further scrutiny of the factor will be discussed in succeeding papers. I am grateful to Professor K. Dan, President of Tokyo Metropolitan University, for the critical reading of the manuscript. Thanks are also due to Dr S. Tamura and Miss A. Mochizuki for their assistance in carrying out some of the experiments.
REFERENCES I. Watanabe, Y, Japan j med sci biol I6 (1963) 107. 2. Tamura, S, Toyoshima, Y & Watanabe, Y, Japan j med sci biol I9 (1966) 85. 3. Frankel, J, J exptl zoo1 173 (1970) 79. 4. Dewey, V C, Parks, R E Jr & Kidder, G W, Arch biochem 29 (1950) 281. 5. Schmidt, G & Thannhauser, S J, J biol them 161 (1945) 83. 6. Dische, Z, Mikrochem 8 (1930) 4. I. Majbaum, W, Z physiol them 25X (1939) 117. 8. Lowry, 0 H, Rosebrough, N J, Farr. A L & Randall. R J. J biol them 193 (1951) 265. 9. Zeuthen; E & Scherbaum, 0, Colston papers 7 (1954) 141. 10. Hamburger, K, Compt rend trav lab Carlsberg 32 (1962) 359. I I. Frankel, J, Compt rend trav lab Carlsberg 33 (1962) I. 12. Scherbaum, 0 & Zeuthen, E, Exptl cell res 6 (1954) 221. 13. Zeutgen, E, Synchrony in cell division and growth, p. 99. Interscience, New York, London Sydney, 1964. 14. Scherbaum, 0, Exptl cell res I3 ( 1957) 24. Received January 11, 1971 Revised version received May 24, 1971