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Journal of Cell Science 102, 469-474 (1992)Printed in Great Britain The Company of Biologists Limited 1992 469

Cell cycle events in the green alga Chlamydomonas eugametos and theircontrol by environmental factorsV. ZACHLEDER1'* and H. VAN DEN ENDE21 Institute of Microbiology, Czechoslovak Academy of Sciences, Tfebon, Czechoslovakia2Department of Molecular Cell Biology, University of Amsterdam, The Netherlands*Author for correspondence: Dr. Vile'm Zachleder, Department of Autotrophic Microorganisms, Institute of Microbiology ofCzechoslovak Academy of Sciences, CS 379 81Tfeboft-Opatovicky mlyn, Czechoslovakia

SummaryA procedure for routine synchronization of largeamounts of the unicellular green alga Chlamydomonaseugametos in liquid culture by alternating light and darkperiods is described. The synchronized populations weregrown at various light intensities and tempera tures. Theeffect of these variables on the lengths of parts of the cellcycle and the number of daughter cells per cell divisionwas followed. T he cell cycle of C. eugametos started witha period in which the cells increased in size only(precommitment period). The length of this period wasdependent on both the light intensity and the tempera-ture. At the end of this period, a key point of the cellcycle (called commitment point) was attained. From thispoint, the cell were committed to divide and cellreproduction was triggered. The following period (post-commitment period), during which daughter cells wereformed, could be traversed without supply of external

energy, and without further growth of the cells.However, if sufficient energy was supplied during thisperiod, the cells were able to attain more commitmentpoints, leading to a higher number of daughter cells. Thepostcommitment period was fairly constant within acertain range of light intensity. At light intensitiesleading to more commitment points, however, thisperiod was prolonged. No evidence was found forcircadian rhythms or endogenous factors of "Zeitgeber"type playing a role in the control of growth andreproductive sequences in the cell cycle of C. eugametos.

Key words: cell cycle, Chlamydomonas eugametos,commitment to division, cell cycle length, precommitmentperiods, postcommitment periods, light intensity,temperature.

IntroductionChlamydomonas eugametos has often been used forstudies of gametogenesis and cell-cell interactions(Tomson et al. 1986; Homan et al. 1987; Musgrave andEn de, 1987; for review, see Harr is, 1989). For ex ample,it was found recently that the sexual cycle is tightlycoupled to the vegetative cell cycle, in the sense thatnewly born cells can be mating competent during thefirst part of the G, phase (Zachleder et al. 1991).However, little attention has been paid to the cell cycleof C. eugametos in comparison with that of the relatedC reinhardtii (Lien and Knutsen, 1979; Spudich andSager, 1980; Donnan and John, 1984; Donnan et al.,1985). John and his collaborators found, in correspon-dence with previous findings in the alga Scenedesmusquadricauda (Setlfk et al. 1972; Zachleder and Setlik,1988, 1990), that the cell cycle of a green alga can beseparated into two periods, a precommitment and apostcommitment period. At the end of the precommit-ment period, a commitment point is reached that isequivalent to the transition point "start" in yeast cells

(John et al. 1989). This point plays a key role in theregulation of the cell cycle, because from this point onthe cell is committed to undergo cell division. In greenalgae, an additional feature is that several commitmentpoints can follow each other, which results in multiplecell divisions. The mechanism controlling the numberand timing of the consecutive commitments is ofparticular interest and is addressed in this paper.Synchronized cultures are the best tool for this typeof research. The only attempt to synchronize C.eugametos was published by Dem ets et al. (1985), whichdid not result in a detailed analysis of the cell cycle. Inthe present paper, completely synchronized cultureswere used to study the effect of light intensity,light/dark regime and temperature on the length of thecell cycle and on cell proliferation in C. eugametos.Materials and methodsOrganismThe UTEX 10 strain of C. eugametos (mt~) was obtained

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470 V. Zachleder and H. van den Endefrom the Algal Collection kept at the University of Texas,Austin, USA.Culture equipment and conditionsCells were cultivated batchwise in 1,200 ml plate-parallelvessels (18 mm in width) at 30C, illuminated by two HgMIF400/D lamps (Tungsram, Budapest, Hungary). Details of theculture equipment were the same as those described byDoucha (1979). The light intensity at the surface of the culturevessels was approximately 70 W m~2 of photosyntheticallyactive radiation (400 to 720 nm). The CO 2 concentration inthe gas mixture by which the culture was aerated was 2%(v/v). The composition of the mineral nutrient solution was asdescribed by Kates and Jones (1964): 1.0 g I"1 KNO 3; 0.74 gI" 1 KH2PO 4; 0.136 g P 1 MgSO 4.7H2O; 0.05 g P^CaCl2.2H2O; 0.14 g P 1 K 2HPO 4; 0.025 g P 1 FeEDTA, andincluded 1 ml P 1 of solution of trace elements (Zachleder andSetlfk, 1982).The synchronization procedureFlooded cells from a 3-week-old agar plate were used toinoculate the batch culture that was to be synchronized.Under the conditions described above, cell division started atabout the 16th hour and the cells divided mostly into eightdaughter cells. The cells were grown for one whole cell cycleand at the beginning of the next light period they were dilutedto the initial density (lxlO 6 cells ml"'). The synchronizationitself was carried out by alternating light/dark periods, thelengths of which were chosen according to the growthparameters of the cells. The optimal time for darkening thecells was when they started their first protoplast fission. Thelength of the dark period was chosen to allow all cells of thepopulation to release their daughter cells. For the first two orthree cycles, the culture was observed by light microscopy toset the correct length for both the light and dark periods.Once the culture was synchronous, the length of the light anddark periods was kept constant.Assessment of commitment curvesTo determine when the cells were committed to divide,samples taken every 2 h were spread on agar (2% in nutrientmedium) in Petri dishes (10 cm in diameter) and incubated inthe dark at 30C. Under these conditions every cell eventuallydivided if it had passed the commitment point (Setlfk andZachleder, 1984). About 30 h after the beginning of the lightperiod, such cells had formed small colonies in which thenumber of released daughter cells per mother cell wascounted. By this method , the percentage of the cells that haddivided into 2, 4,8,16 or more daughter cells was determined.The sigmoidal "commitment curves" were obtained byplotting the cumulated number of daughters as a percentageof total cells against time of sampling.Measurement of light intensityA quantum/radiometer-photometer (LI-COR, Inc., USA)was used. Adjustment of the light intensity was achieved byinserting metal screens between the light source and theculture vessel. For exact adjustment of the required lightintensity the distance of the vessel from the light source wasvaried.ResultsThe synchronization procedureThe most convenient conditions for synchronizing a C.

eugametos culture were those optimal for cell growth,i.e. an increase in cell size, and for cell proliferation,i.e. increase in cell number. They were: 30C, lightintensity 50-70 Wm~2, and aeration using 2% of CO 2 inair. It appeared to be advantageous to place the initialcell suspension in the dark for about 15 h. During thisperiod most cells divided. Consequently, the popu-lation, which was subjected to the following synchroniz-ation procedure, was already partially synchronous (allcells were in the precommitment period). Under theconditions described above, cell division started atabout the 16th hour in the light. At that time the cellshad rounded off, were immotile and displayed the firstprotoplast division (Fig. 1, 16 h). Most of the cellsdivided m ore or less synchronously into tw o, four, eightor 16 daughters. If the cells were then transferred todarkness, daughter cells were released without furtherincrease of cell size (Fig. 1, 0 h) . The length of the darkperiod was chosen to allow all cells to release theirdaugh ters. The daug hter cells were grown further underoptimal conditions until protoplast fission was againobserved. If the cells were then put in the dark apopulation of daughter cells was produced that werehomogeneous in size and that divided more synchron-ously. In this procedure, the lengths of the imposedlight and dark period s were adjusted to the length of thecell cycle under th e given circumstances, r ather than thecell cycle being forced into the framework of a diurnalregime. The advantage of this procedure is that it couldbe used to test the effect of different conditions on theduration of the cell cycle and its components. In thefollowing experiments, a synchronous population,grown for several cell cycles under standard conditions(30C, 70W m~2,16:10 h), was divided into subculturesat the beginning of the light period and grown for threecycles at four different light intensitie s (7 .5, 15, 35 and70 W irT 2) or temperatures (20, 25, 30 and 35C). Bydiluting with fresh medium, the cell density of thesubcultures at the beginning of each cell cycle was keptat 5xlO 5 cells ml"1 in all experimental variants.Length of the pre-commitment periodAs can be seen in Fig. 2 and Fig. 4 (curve 3), there wasan inverse relationship between the length of theprecommitment period (measured as the distancebetween the beginning of the cell cycle and themidpoint of the first commitment curve) and lightintensity. This supports the idea that the main (if notonly) factor determining the timing of the commitmentto divide is the growth rate, which is determined by therate of pho tosynthesis (Spudich and Sager, 1980). It canbe assumed that the cells that had attained the firstcommitment point (curve 1 in Fig. 2) at different lightintensities were all at the same cell cycle stage, becausethey all divided without an additional increase in size,implying that all conditions for cell duplication hadbeen fulfilled.Similarly, the length of the precommitment periodwas inversely related to the temperature (from 35C to20C) as long as the light intensity was not limiting (Fig.3, Fig. 5, curve 2). In Fig. 6, the reciprocal values of the

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Cell cycle control in C. eugametos 471

8 h

Fig. 1. Photomicrographs of the cells during their cell cyclein synchronous populations of Chlamydomonas eugametos.Growth conditions: 70 W m" 2, 30C, 2% CO 2. The age ofthe cells in hours is indicated in the upper left-hand corner .Bar, 10 fim.length of precommitment periods are plotted againstlight intensity (Fig. 6A) or temperature (Fig. 6B). It canbe seen that the effects of temperature and lightintensity cannot be judged separately. At low lightintensities, the energy supply limited growth at highertemperatures (Fig. 6B, curve at 7.5 W m~ 2). Conse-quently, the length of the precommitment period did

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Fig. 2. Time courses of commitments to nuclear andcellular divisions and termination of these processes insynchronous populations of Chlamydomonas eugametos Wrown at various light intensities. (A) 70 W m , (B) 35 VrrT2; (C) 15 W m"*; (D) 7.5 W m~2. Curves 1, 2, 3, 4, 5,percentage of the cells that attained commitment for thefirst, second, third, fourth and fifth nuclear divisions,respectively. Curve 6, percentage of the cells in which thefirst protoplast fission occurred. Curve 7, percentage of thecells that released their daughter cells. Light and darkperiods are indicated by white and black strips abovepanels and separated by vertical lines.not change at various temperatures, giving the im-pression that the length of the cell cycle is temperature-insensitive. On the other hand, at low temperatures(below 20C), growth processes were so slow that evenlow light intensities were sufficient to saturate theirphotosynthetic demands. So the length of the cell cyclebecame seemingly light-insensitive (Fig. 6A, curve at20C). We conclude that the length of the precommit-ment period is only dependent on the rate of assimi-lation, and is not determined by endogenous timingmechanisms.Length of the postcommitment periodThe events after the first commitment point do notrequire external energy, because they can be performedin the dark and are typically temperature-dependent(Fig. 3B,C,D). The length between the first commit-

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472 V. Zachleder and H. van den Ende100

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Fig. 3. Time courses of commitments to nuclear andcellular divisions and termination of these processes insynchronous populations of Chlamydomonas eugametosgrown at various temperatures. (A) 35C; (B) 30C; (C)25C; (D) 20C. Curves 1, 2, 3, 4, 5, percentage of thecells that attained commitment for the first, second, third,fourth and fifth nuclear divisions, respectively. Curve 6,percentage of the cells that released their daughter cells.Light and dark periods are indicated by white and blackstrips above panels and separated by vertical lines.

ment point and the first protoplast fission (henceforthcal led the postcommitment period) was constantregardless of light intensity applied (Fig. 2C,D).Ho wev er, with intensities higher than 15 W m~ 2 morecommitment points were attained, resulting in a largernumber of daughter cells (Fig. 2A to C). Accordingly,this postcommitment period increased, which was onlypartially compensated by the overlap of the commit-ment curves (Fig. 2 A,B compare with C or D; Fig. 4,curve 2). As long as the number of commitments didnot change, the length of the postcommitment periodwas independent of the light intensity (Fig. 2 C,D; Fig.4, curve 2). On the other hand, a rise in temperaturecould also result in an increase in the number ofcommitments. Insert ing new commitments prolongedthe postcommitment period and thus part ial ly compen-sated for the shortening of the postcommitment periodat the higher temperature (compare Fig. 3B and 3D).The present results strongly suggest that the number

101 102log irradiance (W m~2)

Fig. 4. The length of the cell cycle (curve 1),precommitment (curve 2) and postcommitment (curve 3)periods in synchronous populations of Chlamydomonaseugametos at various light intensities.

20 25 30 35Temperature (C)

Fig. 5. The length of the cell cycle (curve 1),precommitment (curve 3) and postcommitment (curve 2)periods in synchronous populations of Chlamydomonaseugametos at various temperatures.

of commitments is only determined by the assimilativeactivity during this period, which is favoured by highlight intensity and tem pera ture. N o evidence was foundfor the idea that the number of commitments isdetermined by the size of the cells, at the time ofcommitment, which is determined by synthetic activityduring the precommitment period. For example, a highl ight intensi ty during the preco mm itment period , whichfavoured cell growth, did not necessarily result in anincrease in the number of consecutive cell divisions.Length of the total cell cycleThe length of the total cell cycle can be considered asthe sum of the lengths of the pre- and postcommitmentperiods, and thus shows a complex dependence onexternal variables, such as light (Figs 2, 5) andtemperature (Figs 3,5). For example, a temperature of

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Fig. 6. Growth rate (expressed as reciprocal value of thelength of precommitment period, in h~') in synchronouspopulations of Chlamydomonas eugametos at variouscombinations of temperature and light intensity.30C was found to be optimal for both growth and cellproliferation, resulting in the shortest cell cycle dur-ation and the highest number of daughter cells (Figs2A, 3B). A higher temperature slowed down cellproliferation, without affecting growth (Fig. 3A). As aresult, th e cell cycle length was increased (Fig. 3A, F ig.5, curve 1) and very large cells were formed.DiscussionThis paper shows that the length of the precommitmentperiod in C. eugametos is only dependent on the ra te ofassimilation. When the input of energy is limiting, thelength of this period is independent of the temperature.On th e other hand, its length is light-independent at lowtemperatures. Thus the growth rate of the cells is theonly determining factor for this part of the Gj phase, aswas shown earlier by Spudich and Sager (1980) forChlamydomonas reinhardtii and by Zachleder andSetlfk (1990) for Scenedesmus quadricauda. Theseresults are, however, in contrast to those of Donnanand John (1983), who observed a constant precommit-ment period at different growth rates in Chlamy-domonas reinhardtii. Only when energy limitationslowed growth was the period extended. The authorstherefore postulated that commitment to divide is

Cell cycle control in C. eugame tos 473under the control of a temperature-compensated timer.We have also been unable to confirm another con-clusion of Donnan and John (1983), namely that the sizeof the cell at commitment determines the number ofconsecutive divisions within each cycle. While it is clearthat entry into mitosis can only take place when cellshave attained a critical mass, and the rate at which a cellaccumulates that mass determines the overall timing ofthe cell cycle, the occurrence of more commitments todivide is only dependent on the input of energy after thefirst commitment. It seems as if Chlamydomonas thenrapidly executes a number of cell cycles with a veryshort Gi phase, which nevertheless require energy forcompletion of each cycle. It has been noted (Donnan etal., 1985) that in C. reinhardtii, the commitments thatdetermine multiple cell divisions lie close together,whereas in Scenedesmus (Setlfk and Zachleder, 1984)and C. eugametos they are more widely spaced. Thismay be the reason why in C. reinhardtii the firstcommitment point seems to be the major point at whichthe cell cycle is controlled in response to cell size andexternal conditions (as in 5. cerevisiae; Forsburg andNurse, 1991), while in Scenedesmus and C. eugametosthis control is distributed over later stages of the cellcycle (as in fission yeast). The p henom enon of multiplecommitments and cell divisions in the reproduction of asingle cell is reminiscent of the post-fertilization wave ofcell divisions in metazoan cells. The refore , the cell cycleof Chlamydomonas remains worthy of further investi-gation.

This research was supported by the Nederlandse Organisa-tie voor Wetenschappelijk Onderzoek (NWO) in the form of avisitors grant to V.Z.ReferencesDemets, R., Tomson, A. M., Ran, E. T. H ., Sigon, C. A. M., Stegwee,D. and van den Ende, H. (1985). Synchronization of the cell divisioncycle of Chlamyd omona s eugametos. J. Gen. Microbiol. 131, 2919-2924.Donnan, L., Carvill, E. P., Gilliland, T. J. and John, P. C. L. (1985).The cell cycles of Chlamydomonas an d Chlorella. New Phylol. 99.1-40.Donnan, L. and John, P. L. C. (1983). Cell cycle control by tim er andsizer in Chlamydomonas. Nature 304, 630-633.Donnan, L. and John, P. L. C. (1984). Timer and sizer controls in thecell cycles of Chlamydomonas an d Chlorella. In The Microbial CellCycle (ed. P. Nurse and E. Streiblova), pp. 231-251. Boca Raton,Flor ida: CRC Press .Doucha, J. (1979). Continuous cultures of algae. In Algal Assays andMonitoring Eutrophication (ed. P. Marvan, S. Pfibil and O.Lhotsk; / ) , pp. 181-191. Stuttgart: Schweitzerbart 'scheVerlagsbuchhandlung.Forsburg, S. L. and Nurse, P. (1991). Identification of a Gl typecyclin pucl + in the fission yeast Schizosaccharomyces pombe.Nature 351, 245-248.Harris, E . H. (1989). Th e Chlamydomonas Sourcebook. AComp rehensive Guide to Biology and Laboratory Use, pp. 780. SanDiego, Sant iago, New York, Berkeley, Boston, London, Sydney,Tokyo, Toronto: Academic Press , Inc . , Harcourt BruceJovanovich Publishers.Homan, W., Sigon, C , van den Briel, W. , Wagter, R., de Nobel, H.,Mesland, D., Musgrave, A. and van den Ende, H. (1987). Transportof membrane receptors and the mechanics of sexual cell fusion inChlamydomonas eugametos. FEBS Lett. 215, 323-326.

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474 V. Zachleder and H. van den EndeJohn, P. C. L., Sek, F. J. and Lee, M. G. (1989). A homolog of thecell cycle control protein p34cdc2 participates in the division cycleof Chlamydomonas and a similar protein is detectable in higherplants and remote taxa. Plant Cell 1, 1185-1193.Kates, J. R. and Jones, R. F. (1964). The control of gameticdifferentiatio n in liquid cultu res of Chlamyd omona s. J. Cell.Comp. Physiol. 63, 157-164.Lien, T. and Knutsen, G. (1979). Synchronous growth ofChlamydomonas reinhardtii (Chlorophyceae): A review of optimalcondit ions . J. Phycol. 15, 191-200.Musgrave, A. and van den Ende, H. (1987). How Chlamydomonascourt their partners. Trends Biochem. Sci. 12, 470-473.etlik, I., Berkova, E., Doucha, J., Kubin, S., Vendlova, J. andZachleder, V. (1972). The coupling of synthetic and reproductionprocesses in Scenedesmus quadricauda. Arch. Hydrobiol. (Suppl.41) Algolog. Stud. 7. 172-213.Setlik, I. and Zachleder, V. (1984). The multiple fission cellreproductive patterns in algae. In The Microbial Cell Cycle (cd. P.Nurse and E. StreiblovS), pp. 253-279. Boca Raton, Florida: CRCPress.Spudich, J. L. and Sager, R. (1980). Regulation of the

Chlamydomonas cell cycle by light and dark. J. Cell Biol. 85 , 136-145.Tomson, A. M., Demets, R., Simon, C. A. M., Stegwee, D. and vanden Ende, H. (1986). Cellular interactions during the matingprocess in Chlamydo mona s eugametos. Plant Physiol. 81 , 522-526.Zachleder, V., Jakobs, M. and van den Ende, H. (1991). Relationshipbetwee n gam etic differentiation and the cell cycle in the green algaChlamydo mona s eugametos. J. Gen. Microbiol. 137, 1333-1339.Zachleder, V. and Setlfk, I. (1982). Effect of irradiance on theScenedesmus quadricauda. Biol. Plant. (Praha) 24, 341-353.Zachleder, V. and Setlik, I. (1988). Distinct controls of DNAreplication and of nuclear division in the cell cycle of thechlorococcal alga Scenedesmus quadricauda . J. Cell Sci. 91 . 531-539.Zachleder, V. and Setlik, I. (1990). Timing of events in overlappingcell reproductive sequences and their mutual interactions in thealga Scenedesmus quadricauda. J. Cell Sci. 97, 631-638.

(Received 14 January 1992 - Accepted, in revised form,9 April 1992)