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Phosphate as a control factor in cell division of Chlamydomonas reinhardti, studied in synchronous culture
Copyright All rights
(a 1973 by Academic Press, Inc. reproduction in any form reseroed
of
Experimental
PHOSPHATE IN CELL
DIVISION STUDIED
Cell Research 78 (1973) 79-88
AS A CONTROL OF CHLAMYDOMONAS IN SYNCHRONOUS
FACTOR REINHARDTI,
CULTURE
T. LIEN and G. KNUTSEN Botanical
Laboratory,
of Bergen,Bergen,Norway
University
SUMMARY Growth, cell division and time course of accumulation of macromolecules has been compared in Chlumydomonas reinhardti during synchronized vegetative growth in complete and phosphate free medium. Deprivation of Pi from the growth medium at the beginning of the life cycle (zoospore stage) led to 1. shortening of G 1 phase with earlier start of DNA synthesis, karvokinesis. cvtokinesis and _ _ sporulation (with reduction in spore number). 2. Moderate variation in protein content and oscillation of RNA content. 3. Decrease in degree of synchrony of nuclear division and sporulation. 4. Derepression of phosphatases. Re-addition of Pi at different times to cells grown in P-free medium from the beginning of the life cycle, demonstrated a transition period in the cells regarding the processes started by the removal of Pi.
During studies of derepressionof phosphatasesin synchronized culture of the green algae Chlamydomonasreinhardti [l], it became evident that culturing the zoosporesin phosphate free growth medium led to an appreciable shortening of the cell cycle. The release of spores in the P-deficient culture started 7 h after the beginning of the growth period as compared with 12 h of the control culture. These observations proposed that phosphate plays a key role in the regulation of the events leading to cell division in this organism. The present report deals with experiments performed to gain knowledge about the role of phosphate in the cell’s preparation for division. MATERIALS
AND METHODS
Culturing conditions and synchronization procedure Chlamydomonas
Algal 6-
Collection
~31818
no. 11-32 (90) from The of the University of Gottingen,
reinhardti
Germany, was grown vegetatively in the medium used by Kuhl & Lorenzen for Chlorella [2]. The cultures were held at 35°C and aerated continuously with a filtered mixture of carbon dioxide and air (1.5 % CO,). The light intensity was about 20 Klux incident on the front surface of the culturing tubes. All the culturing equipment used was identical to that reported previously [3]. The cells were synchronized by repeated shifts between 12 h light and 4 h dark (12:q), with dilution to a standard cell density of 1.5 x lo8 cells/ml at the end of each dark period [4].
Measurement of cell size and cell number These parameters were measured electronically with a Celloscope 302 particle counter (AB Lars Ljungberg, Stockholm) according to the procedure reported earlier [5].
Quantitative determination of DNA, RNA andprotein content DNA content of the cells was determined by the rapid and sensitive fluorometric method of Kissane &Robbins [6], which was modified for marine phytoplankton by Holm-Hansen et al. [ 71.The fluorescence resulting from the excitation of the reaction product of deoxyribose (DNA) and 3.5-diamino-benzoic acid dihydrochloride was measured at 530 nm in a MPS50 L Schimadzu spectrophotometer with a doublebeam fluorometry attachment (Schimadzu Ltd, JaExptl
Cell Res 78 (1973)
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T. Lien & G. Knutsen
pan). The excitation wavelength was 430 nm. The fluorescence intensity was linearly dependent on the cell amount extracted and analysed, the measurements having an S.E.M. of i. 4.0 %. For the RNA determination harvested cells (0.75 x 10s cells/sample) were extracted according to Baker & Schmidt [8] to remove interfering substances. RNA was then extracted by the method of Wanka [9] and the RNA content of the extract determined as ribose content using a modification of the DischeBorenfreud phloroglucinol method [lo]. The standard error of the mean of 10 cell sarnpies carried through the complete procedure was +-8.0%. The total protein content was determined by the method of Lowry et al. 1111 modified as reoorted previously [5]. The standard error of the mean of 10 protein determinations was +7.0 %.
Assay of phosphatases The activities of acid and alkaline phosphatases were measured in living cells according to the method of Lowry [12]. For the analysis cells contained in 2.0 ml of culture suspension were collected on a 21 mm membrane filter disc by suction filtration. The cells were washed with 1 ml of distilled water which was drawn through the filter. The filter with the cells on was then transferred to a 25 ml Erlenmever flask containing 1 .O ml 9.0 mM p-nitrophenylphosphate in 0.1 M acetate or .rdvcine/NaOH buffer of 2°C. The pH was 5.0 and 9.0fdr the assay of acid and alkaline phosphatases, respectively. The temperature was rapidly elevated to 35°C and the cells incubated with the substrate and the mixture shaken for 30 min. The incubation was stopped with the addition of 4.0 ml of 0.25 N NaOH. the mixture was filtered and the absorption of the filtrate measured at 410 nm.
Nuclear staining Cells were fixed, treated with 1 N HCI at 60°C for 5 min and the nuclei stained with 2 % brilliant cresyl blue as reported previously [3].
Reagents used Phloroalucinol and 3.5-diaminobenzoic acid were obtained -from Koch-Light Ltd., Colnbrook, UK. The hvdrochloride of 3.5-diaminobenzoic acid was prepared according to Helm-Hansen et al. [6]. The phosphatase substrate was from Sigma Chemical Co, USA, while brilliant cresyl blue was from E. Merck AG, Germany.
RESULTS Comparison of growth and division of cells in complete and phosphatefree medium Fig. 1 outlines the vegetative cell cycle of Chlamydomonaswith the G 1 period followed Exptl
Cell Res 78 (1973)
by two or more sequencesof DNA synthesiskaryokinesis-cytokinesis [4, 131. These processesresult in a number of zoospores contained within a sporangium consisting of the mother cell wall which is finally dissolved enzymatically, thus releasing the motile spores [4]. The synchronization procedure, in principle that of Lorenzen [2], gave repetitive synchronous cultures at a high degree of synchrony. At the end of each 12:4 growth and division cycle, the culture usually consisted of more than 99 % of spores. The experiments reported herein were performed with spores harvested at the end of the dark period: The cells were rapidly sedimentedin a small, continuously operating centrifuge, and washed under centrifugation with phosphatefree (P-free) medium of the sa.metemperature. Thereafter the cells were suspended in such medium giving 1.5 x lo6 cells/ml. This suspensionwas halved and phosphate added to one part to give the concentration of the normal growth medium. The harvesting and washing took less than 10 min. The cells of both suspensionswere then cultured under the same conditions as those of the stock synchronous culture, and parameters of growth and division of the control and phosphate-starved cells were recorded. The results stemming from typical experiments are given below. It should be noted that the growth and division sequenceof the control cells was identical to that of the stock culture cells Fig. 2 shows the number of cells in the control and P-free cultures at different times after the start of a synchronous cycle, together with the time courses of karyokinesis and cytokinesis in them. The cell number of the control culture was constant for 12 h, i.e. until the start of the 4 h dark period. During the two following hours all the cells sporulated, with the highest sporulation rate observed during the first hour, sporulation re-
Regulation by phosphate of cell division in Chlamydomonas suiting in a total increase in cell number of 16. The starts of nuclear and cytoplasmic divisions in the members of the control culture took place during the 3 h from 9 h onwards, and they were separated by about 15 min. In the P-free culture the sporulation started at 7 h in this particular experiment and was completed within 3 h thereafter, giving only a fourfold increase in cell number. From one experiment to another with P-free cultures, variations were found both in sporulation starting times and final cell number, with the latter varying from 4 to 8. What caused this variation is still obscure. Comparing the average generation times, i.e. the time interval from the start of illumination to the sporulation of the average cell, showed that phosphate deficiency reduced it from the 12.650.2 h of the control culture to 9.5kO.7 h of the P-free culture; a shortening of 28 y/,. Coefficient of variation calculated according to Spencer et al. [14] gave 1.6 and
Fig. 1. Vegetative life cycle of C. reinhardti. The age or developmental stages of the cells in the synchronous cultures are given in hours from the beginning of the illumination period, here defined as 0 h for each synchronous cycle. The black section on the time scale indicates the 4 h (12-16 h) long dark period.
a
80
12. IO-
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81
60
I 2( 4I 6(‘I8
c
10 12 141
0
Fin. 2. Abscissa: time (hours) from the beginning of the illumination period; ordinate: relative cell number (left), fraction of total cell number ( %, right). ( n - n . Cell number as function of time; ‘o-6, fraction of cells with one nucleus (karyokinesis); O-O, fraction of undivided cells (cytokinesis) during synchronous (a) Cells grown in vegetative growth of C. reinhardti. complete medium, (b) cells grown in P-free medium from the beginning of the illumination period.
6.9% for the former and latter cultures, respectively, showing reduction in sporulation synchrony. For the P-free culture only karyokinesis was recorded, and it can be seen from fig. 2b that the starting times of this process in the culture as a whole were found during the interval from 5 to 9 h, a longer time span than found for the sameprocessin the control culture. This shows a decreasein synchrony of nuclear division due to the phosphate removal. From the distribution of cell size in the control culture, recorded just before and after sporulation (fig. 3), it is evident that the largest cell found after sporulation was smaller than the smallest found just prior to that process, meaning that all the cells in the culture sporulated. In the same figure is also shown the size distribution of the zoospores released in the P-free cultures. These cells Exptl
Cell Res 78 (1973)
82
T. Lien & G. Knutsen
50-
40-
30-
20-
IO-
'0
2
4
6
8
101214
16
J
Fig. 3. Abscissa: diameter of cells Cum); ordinate: fraction of total cell number (%). Distribution of cell sizes. A. After and C, before the soorulation of synchronized cells grown in complete medium. B, After the sporulation of cells grown in P-free medium from the beginning of the illumination period.
were larger than the spores of the control culture, with an average diameter of 5.4 versus 4.6 pm of the control value. Mote detailed information about the progress of nuclear division in the two cultures was gained by recording with time the distribution of cells with I, 2, 4, 8 and 16 nuclei/ cell. We found, as did Bernstein [15] with Feulgen staining of C. moweusii, that a diffusely stainable state of the nucleus preceded the first one of the nuclear divisions. At all other times during the life cycle the nuclei were in a much more compact state in cells of both cultures. The distributions of cells with different numbers of nuclei are given in fig. 4a, b. The overlapping of the distributions shows that for a large proportion of the cells in each culture their second division started before the first one of other cells had commenced, and that a similar situation existed for the following divisions. The maximal frequencies of cells with 2,4 and 8 nuclei occurred 40 min apart in the control culture versus 90 min in the P-free culture for the frequencies of cells with 2 and 4 nuclei, meaning that more time was spent by the minus-P cells in two Exptl Cell Res 78 (1973)
sequences of DNA synthesis-karyokinesiscytokinesis than the control cells spent in four such sequences.This could either be due to decreasedrates of the processesinvolved, or to a combination of both effects. The maximal frequencies of cells with 8 and 16 nuclei in the control culture occurred concomitantly, as was also the casefor the frequencies of 4- and 8-nuclei containing cells in the P-free culture. It should be noted that the tight coupling between the DNA synthesisand the following division processes was found in the P-free culture as well as in the control. Accumulation of macromoleculesin control and phosphate-starved cultures The time courses of accumulation of RNA, protein and DNA in the control and phosphate-starved synchronous cultures are all shown in fig. 5. RNA The control cells accumulated RNA only during the illumination period of the syn-
Fig. 4. Abscissa: time (hours) from the beginning of the illumination period; ordinate: fraction of total cell number (%). Distribution of cells with different numbers of nuclei. (a) Cells grown in complete medium; (b) cells grown in P-free medium from the beginning of the illumination period. O-O, cells with 2 nuclei; O-O, cells with 4 nuclei; n - w , cells with 8 nuclei; o - q , cells with 16 nuclei; v - 7, cells with 32 nuclei.
Regulation by phosphate of cell division in Chlamydomonas
83
17 15 13 11 a 9 7 5 3 1 5" 4 b3 2 1 '0
L
2
4
9 / 6 8
I I j I 10 1214 16 0
I 2
I 4
I 6
I I I ( I 8 10 12 14 16 0
1 2 4
I3 I I 6 8 IO 12 14
5. Abscissa: time (hours); ordinate: rel. values. The time course of accumulation of RNA, protein and DNA during synchronous vegetative growth of C. reinhardti. (a) Cells grown in complete medium and the results from two --a--, -A--, sequential light-dark shift are presented. (b) Cells grown in P-free medium from the beginning of the illumination period. Fig.
chronous cycle. The rate was slightly variable until the 8th h when it dropped abruptly almost to zero, where it stayed for 2 h. Thereafter the accumulation continued at the highest rate recorded during the whole synchronous cycle. This halt in accumulation coincided with the time period when the nucleus was in the aforementioned diffuse state. The RNA content of the P-free culture varied throughout the illumination period according to a periodic pattern which was consistent from one experiment to another. A representative time course showing the RNA content of the cells is given in fig. 5 and it shows that the RNA content increased considerably from the onset of illumination to reach a peak at 3 h, whereafter the content oscillated. Thus 3 additional peaks were found at about 5.5, 9 and 12 h with corresponding troughs at 4, 7 and 10 h, respectively. The trough at 4 h corresponds with the time of appearance of diffuse nuclei in the cells. It is not possible to tell whether this
correlation is of significance or not. The relative size of the different peaks varied strongly between experiments, but their times of appearance during the illumination period were relatively constant. The most striking difference between the two cultures with respect to RNA is the degradation which was observed in the minus P cells, but not detected in the controls. We have not yet investigated this degradation in detail and we are therefore unable to tell which RNA speciesare unstable in the treated cells. Protein Protein accumulated exponentially during the illumination period in the control culture, with no net production during the dark period. The total amount accumulated during the light-dark cycle corresponded with the number of zoospores produced. In the phosphate-starved culture protein accumulated at a varying rate, the rate being Expfl
Cell Res 78 (1973)
84
T. Lien & G. Knutsen almost zero between 3-4, 6-7, 10-11 h and after 14 h. These reductions in accumulation rate, which are significant because the same pattern was found in all experiments of this type, appeared concomitantly with the abovementioned time periods of RNA degradation. We therefore assumethat the degradation of RNA caused the temporary reduction in protein synthesis.
DNA 60 60 40 20 0
Fig. 6. Abscissa: time (hours); ordinate: relative values (a) (lejt) and (b) (right), % of cells with more than one nucleus (b) (right), activity of acid phosphatase uer ml culture measured at 35°C and uH 5.0 given as I4410.,/30 min (b, Ml. Effect of re-addition of Pi at various time points on the processes started by the removal of P, at 0 h. During the illumination period, 50 ml samples were periodically removed from the culture of cells grown in P-free medium from O-h. To the samples was added concentrated P-solution to obtain the same concentration of orthophosphate as that of the complete medium, and thereafter these cultures were illuminated and aerated as usual. At the 10th h after the start of the experiment (i.e. 10 h in the illumination period) the cell number in each of the treated cultures was measured. The recorded cell numbers, expressing the effect of re-addition of Pi, were calculated relative to the number of cells found in the “0 h” culture, i.e. the culture which had phosphate for the whole experiment. These relative cell numbers are plotted versus the time of re-addition of Pi, 0 h on the time axis denoting the time of Pi removal and start of culturing in the light. The amount of DNA which accumulated in the cells of a culture from which Pi was removed at 0 h, was measured at intervals during the 12 h illumination period. The DNA content found per ml culture is exoressed relative to the content found at the start of cuhivation (0 h). Relative cell number (a, O-U ); DNA accumulation (a, e-0); activity of acid phosphatase (b, W- n ); cells with more than one nucleus (b, A-A ); and cell number (b, q - q ); in a culture of cells growing in P-free medium from 0 h and measured in an experiment parallel with the readdition experiment.
ExptI
Cell Res 78 (1973)
DNA was synthesized during l/4 of the generation time, starting at 9 h and ending within 4 h. During this interval the DNA content increased almost 16-fold, corresponding with the number of zoospores produced. In the P-free culture the onset of DNA synthesis occurred 4 h earlier than that of the control cells, namely at 5 h. The whole synthesis period lasted for 5 h, increasing the DNA content fourfold. From this observation we conclude that removal of orthophosphate from the growth medium of zoospores led to a shortening of the Gl phase by triggering the DNA synthesis at an earlier time than in the control culture cells.
Experiments with minus-P cells To see if there was a transition point in the
cells regarding the processesstarted by the removal of Pi, Pi was re-added at different times to cells which had been cultured in Pfree medium from the zoospore stage (0 h). The effect of this readdition was recorded as the number of cells/ml of culture measured at 10 and 24 h after the start of the experiment. If a transition point existed we would expect to find that addition of P, prior to this stage would convert the cell to a normal one showing no early division and sporulation. On the contrary, addition after the transition point would have no recovering effect, and the cell would therefore sporulate early and show no further division and sporulation.
Regulation by phosphate of cell division in Chlamydomonas In the culture there would be a distribution in time of the transition points due to the degree of asynchrony and a transition period should therefore be observed in the actual experiment. The results showed the presence of such a period (fig. 6a). Addition of Pi during the first 4 h after Pi removal resulted in complete recovery of the cells. They did not divide early, but sporulated as normal cells to give the same cell number increase as the controls (fig. 7). During the next 4 h progressively fewer cells could be recovered, which is evident from the fact that progressively more cells sporulated early and fewer behaved as normal ones (figs 6a, 7). It was discovered that both the cells which divided early and the controls always gave at least four spores. The transient increase in recovery found in the middle of the transition period seems to be significant, since similar, but less dominant effects were found in other experiments. The transition period started about 2 h before the start of DNA accumulation in the P-free culture (fig. 6a) with the curve describing the time function of individual transition points in the culture almost paralleling the time course of DNA accumulation. The interval with increased recovery mentioned above was followed 2 h later by a similar interval with decreased rate of DNA accumulation. That the transition point preceded the DNA synthesis means that the phosphate-sensitive system was not DNA synthesis itself, but some unknown preparatory processes appearing about 2 h before the start of synthesis. We found earlier [I] that one of the effects of Pi removal was derepression of acid, neutral and alkaline phosphatases. In fig. 6b is shown the time course of acid p-nitrophenyl phosphatase synthesis of the P-free culture. No increase in enzyme activity was observed for the first 2 h after Pi removal, but thereafter it increased rapidly to reach a maximal level at 5 h. The main increase in
:=
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Fig. 7. Abscissa: time (hours);ordinate: relative cell numbers.Recoveryof P,deficientcells by P, addition.
During the illumination period 50 ml samples were periodically removed from the culture of cells grown in P-free medium from O-h. To the samples was added concentrated P-solution to obtain the same concentration of orthophosphate as that of the complete medium, and thereafter these cultures were illuminated and aerated as usual. Cell numbers of the subcultures were registered after O-O, 12 h of illumination, i.e. before sporulation of recovered cells, and O-O, after 12 h of darkness, to register the degree of recovery. The cell numbers are relative to that of the starting suspension at 0 h and plotted versus the time of re-addition of Pi.
activity took place before the transition period started, indicating that the formation of more or lessspecific phosphatasesmight have played a part in the preparatory processesfor DNA synthesis. Do older cells respond to Pi starvation in the same way as did zoospores? To answer this question, 4 h old cells were transferred to P-free medium and cultivated further, and their contents of acid and alkaline phosphatase determined during the following hours, together with the karyo- and cytokinesis. For comparison the sameexperiment was performed with zoospores. The results of the experiment detailed in legend to fig. Exptl
Cell Res 78 (1973)
86
T. Lien & G. Knutsen prior to P, removal, but both phosphatases increased amount before karyokinesis started, with the increase being closer to the time of division in the “4 h old cells” culture than in the “zoospore culture”. DISCUSSION
0
2
4
6
8
Fig. 8. Abscissa: time (hours); ordinate: fraction of total cell number (left, %). activity of acid and alkaline phosphatase pkr7ml cuhure measured at 35°C and pH 5.0 and 9.0 respectively (right) given as Ad14/30 min. (a) Cells grown in P-free medium from 0 h; (6) cells grown in complete medium (in stock synchronous culture) during the first 4 h of the illumination period (4 h old cells) and then transferred to and grown in P-free medium for the rest of the illumination period. The times of transferring the cells to P-free medium are indicated by arrows. Time course of increase in activity of n - n , acid- and alkaline A - A, phosphatase; O-O, of increase in divided cells; O-O, in cells with more than one nucleus.
Ba, b, are shown in these figures. In the “4 h old cells” culture division started earlier than in the control culture (see fig. 2a), needing less time (4 h) to reach the stage of division after Pi was removed than did the zoospores (6 h). While the zoospores gave 4 new cells upon cultivation in P-free medium, the 4 h old cells yielded an average of 19 spores/cell, somewhat in excessof the control cells. Thus the higher production and the greater speed of DNA-synthesis-karyokinesis-cytokinesis of the “4 h old cells” culture must be ascribed to the 4 h of growth in complete medium before the starvation period started. Activities of alkaline phosphatase were not detected Exptl Cell Res 78 (1973)
This study shows that synchronous culturing of Chlamydomonas reinhardti is a highly reproducible experimental system for cell cycle investigation. The main observation of this study is that exclusion of phosphate from the growth medium at the zoospore stage of the organism resulted in DNA synthesis, division and sporulation after a considerably shorter time of growth than under normal conditions. The Gl period was not only shortened by this treatment, but it was also distorted relative to that of the control cells, as can be seenby comparing the time coursesfor RNA and protein accumulation of the two cultures. Especially striking is the difference between the two RNA accumulation courses. We are not at present able to evaluate the significance of the observed RNA degradation and the types of RNA degraded during the four periods of reduction of this compound. It might be the result of recycling of nucleotides, fat example for the synthesis of DNA. The two sequencesof DNA synthesis and the cell division lasted longer than four such events in the control cells, which might be due to reduced energy metabolism caused by phosphate deficiency. This was most probably also the cause of lack of further growth and division after the “premature” division. In the following we will discussthe possible control mechanism for cell division of this algae (which also applies to other sporeforming specieslike Chlorella) in relation to our findings. It is obvious that for any given speciesthere is a size range outside which the cell is not viable. The smallest living cell of a
Regulation by phosphate of cell division in Chlamydomonas species, here called “minimal cell”, contains the minimum quantity of matter and of subcellular organelles which can be integrated to give a viable unit. Thus for Chlamydomonas the smallest functional size of the chloroplast and nucleus, together with the Golgi apparatus, mitochondria, and endoplasmatic reticulum, are among the components which determine the “minimal Chlamydomonas cell”. To be able to divide, this cell must reach a size which, in terms of the just mentioned components, is at least four times the “minimal cell”. The observed tight coupling between DNA synthesis and cytokinesis, and also the seemingly obligate four-division, mean that when the cell is able to start its DNA synthesis it must also be able to divide into four viable cells. This means, furthermore, that the cell can somehow monitor and compare its capacity for replication and the cell size. When the cell “sees” that these two parameters allow for a division into at least four spores of minimal size, the cell can initiate DNA synthesis, which is then followed by the division processes.The minus-P cells recognize 4 x “minimal cell” as the division triggering size, while the normal cells do not respond to this size, but use 8 or 16 times the “minimal size” as minimum prerequisite for division. The mechanism used by the cell for monitoring the parameter values is at the moment entirely unknown. Since the inception and progress of DNA synthesis seem to be obligately followed by the complex of processesleading to chromosome replication, karyokinesis and cytokinesis, the provision for these reactions is included in the term “capacity for DNA synthesis” used above as one of the parameters monitored by the cell. At present we can only speculate about how the phosphate metabolism is interconnected with the cell’s “counting” mechanism. The large number of possibleinteractions can
87
be grouped into two categories: (a) The concentration in the cell of orthophosphate or a derivative thereof directly controls the initiation of DNA synthesis, for example by inhibiting a step in the initiation process. (b) Phosphate can indirectly control initiation through its action as a co-repressor of phosphatase synthesis. In the present case this might be the mode of action as indicated by the derepression of phosphatases after Pi removal both in zoospores and in 4 h old cells, and by the observation that the enzyme activity increased to reach a high level in the culture before DNA synthesisstarted. One or more specific phosphatasescould conceivably reduce the concentration of a hypothetic phosphate ester being an initiation inhibitor. We have shown that orthophosphate is an inhibitor of the phosphatases of this organism, and readdition of Pi to the minus-P cells would cause inhibition of the enzyme present as well as repressing the synthesis. Assuming that a certain level of phosphatase activity is required in order to initiate DNA synthesis, inhibition and repression together could keep the activity below the critical value. According to such a mechanism the transition period could be explained. The transition period does not give the exact times of occurrence of the transition points in the culture, since, due to the uptake process it takes time after the Pi addition to reach an effective phosphate level in the cells. The expert assistance of Kirsten Arnesen is greatly appreciated.
REFERENCES 1. Lien, T & Knutsen, G, Biochim biophys acta 287 (1972) 154. 2. Kuhl, A & Lorenzen, H, Methods in cell physiology (ed D M Prescott) vol. 1, p. 159. Academic Press, New York (1964). 3. Knutsen, G, Biochim biophys acta 161 (1968) 205. 4. Schlasser, U, Arch microbial 54 (1966) 129. Exptl
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5. Moberg, S, Knutsen, G & Goksoyr, J, Physiol plant 21 (1968) 390. 6. Kissane, J M & Robbins, E, J biol them 233 (1958) 184. 7. Helm-Hansen, 0, Sutcliffe, W H & Jonathan, S, Limnol oceanogr 13 (1968) 507. 8. Baker, A L & Schmidt, R R, Biochim biophys acta 82 (1964) 336. 9. Wanka, ‘F, Pianta 58 (1962) 594. 10. Boloanani. L. Coupi. G & Zambotti. ._V. Experien_ tia 17 (1961)‘67. - - . 11. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265.
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12. Lowry, 0 H, Methods in enzymology (ed S P Colowick & N 0 Kaplan) vol. 4, p. 366. Academic Press, New York (1957). 13. Jones, R F, Ann NY acad sci 175 (1970) 413. 14. Spencer, H T, Schmidt, R R, Kramer, C Y, Moore W E C & King, K W, Exptl cell res 25 (1961) 485. 15. Bernstein. E, J protozool 11 (1964) 56.
Received June 26, 1972 Revised version received September 21, 1972
(a 1973 by Academic Press, Inc. reproduction in any form reseroed
of
Experimental
PHOSPHATE IN CELL
DIVISION STUDIED
Cell Research 78 (1973) 79-88
AS A CONTROL OF CHLAMYDOMONAS IN SYNCHRONOUS
FACTOR REINHARDTI,
CULTURE
T. LIEN and G. KNUTSEN Botanical
Laboratory,
of Bergen,Bergen,Norway
University
SUMMARY Growth, cell division and time course of accumulation of macromolecules has been compared in Chlumydomonas reinhardti during synchronized vegetative growth in complete and phosphate free medium. Deprivation of Pi from the growth medium at the beginning of the life cycle (zoospore stage) led to 1. shortening of G 1 phase with earlier start of DNA synthesis, karvokinesis. cvtokinesis and _ _ sporulation (with reduction in spore number). 2. Moderate variation in protein content and oscillation of RNA content. 3. Decrease in degree of synchrony of nuclear division and sporulation. 4. Derepression of phosphatases. Re-addition of Pi at different times to cells grown in P-free medium from the beginning of the life cycle, demonstrated a transition period in the cells regarding the processes started by the removal of Pi.
During studies of derepressionof phosphatasesin synchronized culture of the green algae Chlamydomonasreinhardti [l], it became evident that culturing the zoosporesin phosphate free growth medium led to an appreciable shortening of the cell cycle. The release of spores in the P-deficient culture started 7 h after the beginning of the growth period as compared with 12 h of the control culture. These observations proposed that phosphate plays a key role in the regulation of the events leading to cell division in this organism. The present report deals with experiments performed to gain knowledge about the role of phosphate in the cell’s preparation for division. MATERIALS
AND METHODS
Culturing conditions and synchronization procedure Chlamydomonas
Algal 6-
Collection
~31818
no. 11-32 (90) from The of the University of Gottingen,
reinhardti
Germany, was grown vegetatively in the medium used by Kuhl & Lorenzen for Chlorella [2]. The cultures were held at 35°C and aerated continuously with a filtered mixture of carbon dioxide and air (1.5 % CO,). The light intensity was about 20 Klux incident on the front surface of the culturing tubes. All the culturing equipment used was identical to that reported previously [3]. The cells were synchronized by repeated shifts between 12 h light and 4 h dark (12:q), with dilution to a standard cell density of 1.5 x lo8 cells/ml at the end of each dark period [4].
Measurement of cell size and cell number These parameters were measured electronically with a Celloscope 302 particle counter (AB Lars Ljungberg, Stockholm) according to the procedure reported earlier [5].
Quantitative determination of DNA, RNA andprotein content DNA content of the cells was determined by the rapid and sensitive fluorometric method of Kissane &Robbins [6], which was modified for marine phytoplankton by Holm-Hansen et al. [ 71.The fluorescence resulting from the excitation of the reaction product of deoxyribose (DNA) and 3.5-diamino-benzoic acid dihydrochloride was measured at 530 nm in a MPS50 L Schimadzu spectrophotometer with a doublebeam fluorometry attachment (Schimadzu Ltd, JaExptl
Cell Res 78 (1973)
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T. Lien & G. Knutsen
pan). The excitation wavelength was 430 nm. The fluorescence intensity was linearly dependent on the cell amount extracted and analysed, the measurements having an S.E.M. of i. 4.0 %. For the RNA determination harvested cells (0.75 x 10s cells/sample) were extracted according to Baker & Schmidt [8] to remove interfering substances. RNA was then extracted by the method of Wanka [9] and the RNA content of the extract determined as ribose content using a modification of the DischeBorenfreud phloroglucinol method [lo]. The standard error of the mean of 10 cell sarnpies carried through the complete procedure was +-8.0%. The total protein content was determined by the method of Lowry et al. 1111 modified as reoorted previously [5]. The standard error of the mean of 10 protein determinations was +7.0 %.
Assay of phosphatases The activities of acid and alkaline phosphatases were measured in living cells according to the method of Lowry [12]. For the analysis cells contained in 2.0 ml of culture suspension were collected on a 21 mm membrane filter disc by suction filtration. The cells were washed with 1 ml of distilled water which was drawn through the filter. The filter with the cells on was then transferred to a 25 ml Erlenmever flask containing 1 .O ml 9.0 mM p-nitrophenylphosphate in 0.1 M acetate or .rdvcine/NaOH buffer of 2°C. The pH was 5.0 and 9.0fdr the assay of acid and alkaline phosphatases, respectively. The temperature was rapidly elevated to 35°C and the cells incubated with the substrate and the mixture shaken for 30 min. The incubation was stopped with the addition of 4.0 ml of 0.25 N NaOH. the mixture was filtered and the absorption of the filtrate measured at 410 nm.
Nuclear staining Cells were fixed, treated with 1 N HCI at 60°C for 5 min and the nuclei stained with 2 % brilliant cresyl blue as reported previously [3].
Reagents used Phloroalucinol and 3.5-diaminobenzoic acid were obtained -from Koch-Light Ltd., Colnbrook, UK. The hvdrochloride of 3.5-diaminobenzoic acid was prepared according to Helm-Hansen et al. [6]. The phosphatase substrate was from Sigma Chemical Co, USA, while brilliant cresyl blue was from E. Merck AG, Germany.
RESULTS Comparison of growth and division of cells in complete and phosphatefree medium Fig. 1 outlines the vegetative cell cycle of Chlamydomonaswith the G 1 period followed Exptl
Cell Res 78 (1973)
by two or more sequencesof DNA synthesiskaryokinesis-cytokinesis [4, 131. These processesresult in a number of zoospores contained within a sporangium consisting of the mother cell wall which is finally dissolved enzymatically, thus releasing the motile spores [4]. The synchronization procedure, in principle that of Lorenzen [2], gave repetitive synchronous cultures at a high degree of synchrony. At the end of each 12:4 growth and division cycle, the culture usually consisted of more than 99 % of spores. The experiments reported herein were performed with spores harvested at the end of the dark period: The cells were rapidly sedimentedin a small, continuously operating centrifuge, and washed under centrifugation with phosphatefree (P-free) medium of the sa.metemperature. Thereafter the cells were suspended in such medium giving 1.5 x lo6 cells/ml. This suspensionwas halved and phosphate added to one part to give the concentration of the normal growth medium. The harvesting and washing took less than 10 min. The cells of both suspensionswere then cultured under the same conditions as those of the stock synchronous culture, and parameters of growth and division of the control and phosphate-starved cells were recorded. The results stemming from typical experiments are given below. It should be noted that the growth and division sequenceof the control cells was identical to that of the stock culture cells Fig. 2 shows the number of cells in the control and P-free cultures at different times after the start of a synchronous cycle, together with the time courses of karyokinesis and cytokinesis in them. The cell number of the control culture was constant for 12 h, i.e. until the start of the 4 h dark period. During the two following hours all the cells sporulated, with the highest sporulation rate observed during the first hour, sporulation re-
Regulation by phosphate of cell division in Chlamydomonas suiting in a total increase in cell number of 16. The starts of nuclear and cytoplasmic divisions in the members of the control culture took place during the 3 h from 9 h onwards, and they were separated by about 15 min. In the P-free culture the sporulation started at 7 h in this particular experiment and was completed within 3 h thereafter, giving only a fourfold increase in cell number. From one experiment to another with P-free cultures, variations were found both in sporulation starting times and final cell number, with the latter varying from 4 to 8. What caused this variation is still obscure. Comparing the average generation times, i.e. the time interval from the start of illumination to the sporulation of the average cell, showed that phosphate deficiency reduced it from the 12.650.2 h of the control culture to 9.5kO.7 h of the P-free culture; a shortening of 28 y/,. Coefficient of variation calculated according to Spencer et al. [14] gave 1.6 and
Fig. 1. Vegetative life cycle of C. reinhardti. The age or developmental stages of the cells in the synchronous cultures are given in hours from the beginning of the illumination period, here defined as 0 h for each synchronous cycle. The black section on the time scale indicates the 4 h (12-16 h) long dark period.
a
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12. IO-
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I 2( 4I 6(‘I8
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10 12 141
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Fin. 2. Abscissa: time (hours) from the beginning of the illumination period; ordinate: relative cell number (left), fraction of total cell number ( %, right). ( n - n . Cell number as function of time; ‘o-6, fraction of cells with one nucleus (karyokinesis); O-O, fraction of undivided cells (cytokinesis) during synchronous (a) Cells grown in vegetative growth of C. reinhardti. complete medium, (b) cells grown in P-free medium from the beginning of the illumination period.
6.9% for the former and latter cultures, respectively, showing reduction in sporulation synchrony. For the P-free culture only karyokinesis was recorded, and it can be seen from fig. 2b that the starting times of this process in the culture as a whole were found during the interval from 5 to 9 h, a longer time span than found for the sameprocessin the control culture. This shows a decreasein synchrony of nuclear division due to the phosphate removal. From the distribution of cell size in the control culture, recorded just before and after sporulation (fig. 3), it is evident that the largest cell found after sporulation was smaller than the smallest found just prior to that process, meaning that all the cells in the culture sporulated. In the same figure is also shown the size distribution of the zoospores released in the P-free cultures. These cells Exptl
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50-
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101214
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Fig. 3. Abscissa: diameter of cells Cum); ordinate: fraction of total cell number (%). Distribution of cell sizes. A. After and C, before the soorulation of synchronized cells grown in complete medium. B, After the sporulation of cells grown in P-free medium from the beginning of the illumination period.
were larger than the spores of the control culture, with an average diameter of 5.4 versus 4.6 pm of the control value. Mote detailed information about the progress of nuclear division in the two cultures was gained by recording with time the distribution of cells with I, 2, 4, 8 and 16 nuclei/ cell. We found, as did Bernstein [15] with Feulgen staining of C. moweusii, that a diffusely stainable state of the nucleus preceded the first one of the nuclear divisions. At all other times during the life cycle the nuclei were in a much more compact state in cells of both cultures. The distributions of cells with different numbers of nuclei are given in fig. 4a, b. The overlapping of the distributions shows that for a large proportion of the cells in each culture their second division started before the first one of other cells had commenced, and that a similar situation existed for the following divisions. The maximal frequencies of cells with 2,4 and 8 nuclei occurred 40 min apart in the control culture versus 90 min in the P-free culture for the frequencies of cells with 2 and 4 nuclei, meaning that more time was spent by the minus-P cells in two Exptl Cell Res 78 (1973)
sequences of DNA synthesis-karyokinesiscytokinesis than the control cells spent in four such sequences.This could either be due to decreasedrates of the processesinvolved, or to a combination of both effects. The maximal frequencies of cells with 8 and 16 nuclei in the control culture occurred concomitantly, as was also the casefor the frequencies of 4- and 8-nuclei containing cells in the P-free culture. It should be noted that the tight coupling between the DNA synthesisand the following division processes was found in the P-free culture as well as in the control. Accumulation of macromoleculesin control and phosphate-starved cultures The time courses of accumulation of RNA, protein and DNA in the control and phosphate-starved synchronous cultures are all shown in fig. 5. RNA The control cells accumulated RNA only during the illumination period of the syn-
Fig. 4. Abscissa: time (hours) from the beginning of the illumination period; ordinate: fraction of total cell number (%). Distribution of cells with different numbers of nuclei. (a) Cells grown in complete medium; (b) cells grown in P-free medium from the beginning of the illumination period. O-O, cells with 2 nuclei; O-O, cells with 4 nuclei; n - w , cells with 8 nuclei; o - q , cells with 16 nuclei; v - 7, cells with 32 nuclei.
Regulation by phosphate of cell division in Chlamydomonas
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17 15 13 11 a 9 7 5 3 1 5" 4 b3 2 1 '0
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I I j I 10 1214 16 0
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5. Abscissa: time (hours); ordinate: rel. values. The time course of accumulation of RNA, protein and DNA during synchronous vegetative growth of C. reinhardti. (a) Cells grown in complete medium and the results from two --a--, -A--, sequential light-dark shift are presented. (b) Cells grown in P-free medium from the beginning of the illumination period. Fig.
chronous cycle. The rate was slightly variable until the 8th h when it dropped abruptly almost to zero, where it stayed for 2 h. Thereafter the accumulation continued at the highest rate recorded during the whole synchronous cycle. This halt in accumulation coincided with the time period when the nucleus was in the aforementioned diffuse state. The RNA content of the P-free culture varied throughout the illumination period according to a periodic pattern which was consistent from one experiment to another. A representative time course showing the RNA content of the cells is given in fig. 5 and it shows that the RNA content increased considerably from the onset of illumination to reach a peak at 3 h, whereafter the content oscillated. Thus 3 additional peaks were found at about 5.5, 9 and 12 h with corresponding troughs at 4, 7 and 10 h, respectively. The trough at 4 h corresponds with the time of appearance of diffuse nuclei in the cells. It is not possible to tell whether this
correlation is of significance or not. The relative size of the different peaks varied strongly between experiments, but their times of appearance during the illumination period were relatively constant. The most striking difference between the two cultures with respect to RNA is the degradation which was observed in the minus P cells, but not detected in the controls. We have not yet investigated this degradation in detail and we are therefore unable to tell which RNA speciesare unstable in the treated cells. Protein Protein accumulated exponentially during the illumination period in the control culture, with no net production during the dark period. The total amount accumulated during the light-dark cycle corresponded with the number of zoospores produced. In the phosphate-starved culture protein accumulated at a varying rate, the rate being Expfl
Cell Res 78 (1973)
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T. Lien & G. Knutsen almost zero between 3-4, 6-7, 10-11 h and after 14 h. These reductions in accumulation rate, which are significant because the same pattern was found in all experiments of this type, appeared concomitantly with the abovementioned time periods of RNA degradation. We therefore assumethat the degradation of RNA caused the temporary reduction in protein synthesis.
DNA 60 60 40 20 0
Fig. 6. Abscissa: time (hours); ordinate: relative values (a) (lejt) and (b) (right), % of cells with more than one nucleus (b) (right), activity of acid phosphatase uer ml culture measured at 35°C and uH 5.0 given as I4410.,/30 min (b, Ml. Effect of re-addition of Pi at various time points on the processes started by the removal of P, at 0 h. During the illumination period, 50 ml samples were periodically removed from the culture of cells grown in P-free medium from O-h. To the samples was added concentrated P-solution to obtain the same concentration of orthophosphate as that of the complete medium, and thereafter these cultures were illuminated and aerated as usual. At the 10th h after the start of the experiment (i.e. 10 h in the illumination period) the cell number in each of the treated cultures was measured. The recorded cell numbers, expressing the effect of re-addition of Pi, were calculated relative to the number of cells found in the “0 h” culture, i.e. the culture which had phosphate for the whole experiment. These relative cell numbers are plotted versus the time of re-addition of Pi, 0 h on the time axis denoting the time of Pi removal and start of culturing in the light. The amount of DNA which accumulated in the cells of a culture from which Pi was removed at 0 h, was measured at intervals during the 12 h illumination period. The DNA content found per ml culture is exoressed relative to the content found at the start of cuhivation (0 h). Relative cell number (a, O-U ); DNA accumulation (a, e-0); activity of acid phosphatase (b, W- n ); cells with more than one nucleus (b, A-A ); and cell number (b, q - q ); in a culture of cells growing in P-free medium from 0 h and measured in an experiment parallel with the readdition experiment.
ExptI
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DNA was synthesized during l/4 of the generation time, starting at 9 h and ending within 4 h. During this interval the DNA content increased almost 16-fold, corresponding with the number of zoospores produced. In the P-free culture the onset of DNA synthesis occurred 4 h earlier than that of the control cells, namely at 5 h. The whole synthesis period lasted for 5 h, increasing the DNA content fourfold. From this observation we conclude that removal of orthophosphate from the growth medium of zoospores led to a shortening of the Gl phase by triggering the DNA synthesis at an earlier time than in the control culture cells.
Experiments with minus-P cells To see if there was a transition point in the
cells regarding the processesstarted by the removal of Pi, Pi was re-added at different times to cells which had been cultured in Pfree medium from the zoospore stage (0 h). The effect of this readdition was recorded as the number of cells/ml of culture measured at 10 and 24 h after the start of the experiment. If a transition point existed we would expect to find that addition of P, prior to this stage would convert the cell to a normal one showing no early division and sporulation. On the contrary, addition after the transition point would have no recovering effect, and the cell would therefore sporulate early and show no further division and sporulation.
Regulation by phosphate of cell division in Chlamydomonas In the culture there would be a distribution in time of the transition points due to the degree of asynchrony and a transition period should therefore be observed in the actual experiment. The results showed the presence of such a period (fig. 6a). Addition of Pi during the first 4 h after Pi removal resulted in complete recovery of the cells. They did not divide early, but sporulated as normal cells to give the same cell number increase as the controls (fig. 7). During the next 4 h progressively fewer cells could be recovered, which is evident from the fact that progressively more cells sporulated early and fewer behaved as normal ones (figs 6a, 7). It was discovered that both the cells which divided early and the controls always gave at least four spores. The transient increase in recovery found in the middle of the transition period seems to be significant, since similar, but less dominant effects were found in other experiments. The transition period started about 2 h before the start of DNA accumulation in the P-free culture (fig. 6a) with the curve describing the time function of individual transition points in the culture almost paralleling the time course of DNA accumulation. The interval with increased recovery mentioned above was followed 2 h later by a similar interval with decreased rate of DNA accumulation. That the transition point preceded the DNA synthesis means that the phosphate-sensitive system was not DNA synthesis itself, but some unknown preparatory processes appearing about 2 h before the start of synthesis. We found earlier [I] that one of the effects of Pi removal was derepression of acid, neutral and alkaline phosphatases. In fig. 6b is shown the time course of acid p-nitrophenyl phosphatase synthesis of the P-free culture. No increase in enzyme activity was observed for the first 2 h after Pi removal, but thereafter it increased rapidly to reach a maximal level at 5 h. The main increase in
:=
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Fig. 7. Abscissa: time (hours);ordinate: relative cell numbers.Recoveryof P,deficientcells by P, addition.
During the illumination period 50 ml samples were periodically removed from the culture of cells grown in P-free medium from O-h. To the samples was added concentrated P-solution to obtain the same concentration of orthophosphate as that of the complete medium, and thereafter these cultures were illuminated and aerated as usual. Cell numbers of the subcultures were registered after O-O, 12 h of illumination, i.e. before sporulation of recovered cells, and O-O, after 12 h of darkness, to register the degree of recovery. The cell numbers are relative to that of the starting suspension at 0 h and plotted versus the time of re-addition of Pi.
activity took place before the transition period started, indicating that the formation of more or lessspecific phosphatasesmight have played a part in the preparatory processesfor DNA synthesis. Do older cells respond to Pi starvation in the same way as did zoospores? To answer this question, 4 h old cells were transferred to P-free medium and cultivated further, and their contents of acid and alkaline phosphatase determined during the following hours, together with the karyo- and cytokinesis. For comparison the sameexperiment was performed with zoospores. The results of the experiment detailed in legend to fig. Exptl
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T. Lien & G. Knutsen prior to P, removal, but both phosphatases increased amount before karyokinesis started, with the increase being closer to the time of division in the “4 h old cells” culture than in the “zoospore culture”. DISCUSSION
0
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Fig. 8. Abscissa: time (hours); ordinate: fraction of total cell number (left, %). activity of acid and alkaline phosphatase pkr7ml cuhure measured at 35°C and pH 5.0 and 9.0 respectively (right) given as Ad14/30 min. (a) Cells grown in P-free medium from 0 h; (6) cells grown in complete medium (in stock synchronous culture) during the first 4 h of the illumination period (4 h old cells) and then transferred to and grown in P-free medium for the rest of the illumination period. The times of transferring the cells to P-free medium are indicated by arrows. Time course of increase in activity of n - n , acid- and alkaline A - A, phosphatase; O-O, of increase in divided cells; O-O, in cells with more than one nucleus.
Ba, b, are shown in these figures. In the “4 h old cells” culture division started earlier than in the control culture (see fig. 2a), needing less time (4 h) to reach the stage of division after Pi was removed than did the zoospores (6 h). While the zoospores gave 4 new cells upon cultivation in P-free medium, the 4 h old cells yielded an average of 19 spores/cell, somewhat in excessof the control cells. Thus the higher production and the greater speed of DNA-synthesis-karyokinesis-cytokinesis of the “4 h old cells” culture must be ascribed to the 4 h of growth in complete medium before the starvation period started. Activities of alkaline phosphatase were not detected Exptl Cell Res 78 (1973)
This study shows that synchronous culturing of Chlamydomonas reinhardti is a highly reproducible experimental system for cell cycle investigation. The main observation of this study is that exclusion of phosphate from the growth medium at the zoospore stage of the organism resulted in DNA synthesis, division and sporulation after a considerably shorter time of growth than under normal conditions. The Gl period was not only shortened by this treatment, but it was also distorted relative to that of the control cells, as can be seenby comparing the time coursesfor RNA and protein accumulation of the two cultures. Especially striking is the difference between the two RNA accumulation courses. We are not at present able to evaluate the significance of the observed RNA degradation and the types of RNA degraded during the four periods of reduction of this compound. It might be the result of recycling of nucleotides, fat example for the synthesis of DNA. The two sequencesof DNA synthesis and the cell division lasted longer than four such events in the control cells, which might be due to reduced energy metabolism caused by phosphate deficiency. This was most probably also the cause of lack of further growth and division after the “premature” division. In the following we will discussthe possible control mechanism for cell division of this algae (which also applies to other sporeforming specieslike Chlorella) in relation to our findings. It is obvious that for any given speciesthere is a size range outside which the cell is not viable. The smallest living cell of a
Regulation by phosphate of cell division in Chlamydomonas species, here called “minimal cell”, contains the minimum quantity of matter and of subcellular organelles which can be integrated to give a viable unit. Thus for Chlamydomonas the smallest functional size of the chloroplast and nucleus, together with the Golgi apparatus, mitochondria, and endoplasmatic reticulum, are among the components which determine the “minimal Chlamydomonas cell”. To be able to divide, this cell must reach a size which, in terms of the just mentioned components, is at least four times the “minimal cell”. The observed tight coupling between DNA synthesis and cytokinesis, and also the seemingly obligate four-division, mean that when the cell is able to start its DNA synthesis it must also be able to divide into four viable cells. This means, furthermore, that the cell can somehow monitor and compare its capacity for replication and the cell size. When the cell “sees” that these two parameters allow for a division into at least four spores of minimal size, the cell can initiate DNA synthesis, which is then followed by the division processes.The minus-P cells recognize 4 x “minimal cell” as the division triggering size, while the normal cells do not respond to this size, but use 8 or 16 times the “minimal size” as minimum prerequisite for division. The mechanism used by the cell for monitoring the parameter values is at the moment entirely unknown. Since the inception and progress of DNA synthesis seem to be obligately followed by the complex of processesleading to chromosome replication, karyokinesis and cytokinesis, the provision for these reactions is included in the term “capacity for DNA synthesis” used above as one of the parameters monitored by the cell. At present we can only speculate about how the phosphate metabolism is interconnected with the cell’s “counting” mechanism. The large number of possibleinteractions can
87
be grouped into two categories: (a) The concentration in the cell of orthophosphate or a derivative thereof directly controls the initiation of DNA synthesis, for example by inhibiting a step in the initiation process. (b) Phosphate can indirectly control initiation through its action as a co-repressor of phosphatase synthesis. In the present case this might be the mode of action as indicated by the derepression of phosphatases after Pi removal both in zoospores and in 4 h old cells, and by the observation that the enzyme activity increased to reach a high level in the culture before DNA synthesisstarted. One or more specific phosphatasescould conceivably reduce the concentration of a hypothetic phosphate ester being an initiation inhibitor. We have shown that orthophosphate is an inhibitor of the phosphatases of this organism, and readdition of Pi to the minus-P cells would cause inhibition of the enzyme present as well as repressing the synthesis. Assuming that a certain level of phosphatase activity is required in order to initiate DNA synthesis, inhibition and repression together could keep the activity below the critical value. According to such a mechanism the transition period could be explained. The transition period does not give the exact times of occurrence of the transition points in the culture, since, due to the uptake process it takes time after the Pi addition to reach an effective phosphate level in the cells. The expert assistance of Kirsten Arnesen is greatly appreciated.
REFERENCES 1. Lien, T & Knutsen, G, Biochim biophys acta 287 (1972) 154. 2. Kuhl, A & Lorenzen, H, Methods in cell physiology (ed D M Prescott) vol. 1, p. 159. Academic Press, New York (1964). 3. Knutsen, G, Biochim biophys acta 161 (1968) 205. 4. Schlasser, U, Arch microbial 54 (1966) 129. Exptl
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5. Moberg, S, Knutsen, G & Goksoyr, J, Physiol plant 21 (1968) 390. 6. Kissane, J M & Robbins, E, J biol them 233 (1958) 184. 7. Helm-Hansen, 0, Sutcliffe, W H & Jonathan, S, Limnol oceanogr 13 (1968) 507. 8. Baker, A L & Schmidt, R R, Biochim biophys acta 82 (1964) 336. 9. Wanka, ‘F, Pianta 58 (1962) 594. 10. Boloanani. L. Coupi. G & Zambotti. ._V. Experien_ tia 17 (1961)‘67. - - . 11. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265.
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12. Lowry, 0 H, Methods in enzymology (ed S P Colowick & N 0 Kaplan) vol. 4, p. 366. Academic Press, New York (1957). 13. Jones, R F, Ann NY acad sci 175 (1970) 413. 14. Spencer, H T, Schmidt, R R, Kramer, C Y, Moore W E C & King, K W, Exptl cell res 25 (1961) 485. 15. Bernstein. E, J protozool 11 (1964) 56.
Received June 26, 1972 Revised version received September 21, 1972