Cell Biology
international
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REGULATION OF CYCLEPROGRESSION IN PLANTCELLS C. DE LA TORREl Instituto de Biologla
and G. GIMENEZ-MARTIN Celular, C.S.I.C., Madrid ABSTRACT
root Induction ofpolynucleate cells in onion meristems by inhibition of two sc,,uent'irL3 cytokineses is used to study controls operating in cell cycle progression. Triggering of both replication andmetaphase occurs synchronously in nuclei sharing a common independentoftheirploidy orintracytoplasm, cellular position. The replication rate appears to be activated by the simultaneous intracytoplasm presence of other replicating nuclei. On the other hand, central position of anucleus in the cell as well as increase in ploidy leads to slowing down of replication rate. The relative advance and lag of S periodinthe differentnuclei in a common cytoplasmispartially counter balanced by differential times of G2. Moreover prophase lengthening in the fast interphase nuclei points to a negative control exerted by the lagging nuclei mediated by cytoplasm. Finally, it could be worth emphasizing similarities in the control mechanism operating in cycle progression both in animal and plant cells.
Polynucleate cells arebeing extensively used to study both nucleus and cytoplasmrolesand autonomy in controlling metabolism.In this line,we should takeintoaccount studies based on fusionofnuclei from different species (Harris, 1965) as well as from different phases of cell cycle (Johnson and Rao, 1971).Chemicals which irreversibly inhibit cytokinesis have been amply used for induc ing homophasic polynucleate cells in plants (Kihlmany 1955; Howard and Dewey, 1960; Gimi%ez-Martin et al., 1965; Benbadis et al., 1974). Caffeine inhibits the fusion of Golgivesiclesin cytokinesis during telophase (L6pez-Sdez et al., 1966), giving rise to a binucleate cell population (2n-2n) initiating its next cycle. 1
Correspondence Inst. de3iolog /
Caffeine
Fig. 2. Analysis of cycle progression in the polynucleate cells. 3H-thymidine pulses at intervals after caffeine show kinetics of entrance andexit of S period. Previous qualitative data suggested intracellular synchrony in initiating but not in endingreplication. Central nuclei were always behind in the S/G as well as in G2/ prophase boundaries. Finally,there is2alsointracellular synchrony in the triggering of metaphase.
214
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In term tional Reports,
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radioactive precursors at given times aftercaffeinemark the kinetics of entrance and exit in S period, while studies of mitosis frequencywillindicate the kinetics of entrance in mitosis of the cell population. This fig. 2 schematically shows data which confirm previous studies (Gimenez-Martin et al., 1968 ; Gonzslez-Fer nsndez et al., 1971). There seemedtobeintracellularsynchrony both in initiationof replication and metaphase, while the endofreplication and the initiation of prophase can occur asynchronously in the different nuclei of the same cell. also the intra Apparently not only the ploidy but cellular location affects the cycle rate of a nucleus, Nuclei having a larger surrounding cytoplasm are faster than those having a more in interphase progression limited amount, since observations at S/G and G2/profastphase boundaries consistently showed that 32oththe est nuclei in 2n-2n-2n-2n and 2n-4n-4n polynucleate of the cells. As the cells were those in the extremes frequency of 2n-4n-2n cells in the meristem is greater than 2n-2n-2n-2n cells and they seem to have greater the study of G2/prophase asynchrony, we have limited intercellular andintracellularasynchrony in the fringes of the different cell compartments to 2n-4n-2n cells, comparing them to 2n-2n binucleate cells. initiating : Intracellular synchrony in G1 duration renlication. The kinetics of entry into S of binucleate and polynucleatecellswere observedbygiving3H-TdR at one hour The frequency of labelled spaced intervals after caffeine. these cells. cells provides a direct measure of G1 in Fig. 3 shows how the population of polynucleate cells initiates replication with a kinetics similar to that shown by binucleate cells. Hence, the duration of G1 in a cell is independentboth of the number of nuclei and of individual nuclear ploidies. The intracellular synchrony in initiating replication was total both in binucleate and polynucleate cells. On the other hand, Benbadis et al. (1974) pointed out an asynchrony in 3% of the binucleate cells induced by methyl 3 hydroxy 6 (lH, 3 H) quinazoline dione 2.4 in Allium sativum L. Efforts to detect asynchrony in the G1-S fringe failed in our cells. Asynchrony if any could be within the limits of resolution of labelling in autoradiography and its detection depend on background accepted level. Our data seem to support a strict nuclear synchrony in initiating replication even in 2n-4n-2n polynucleate cells which showed the greatest asynchrony in Gi,/prophase
Cell Biology
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l blnucleate
opolynucleate
215
Vol. 1, No. 3, 19 7 7
25°C
cells cells
hours
5 after
1
k
caffeine
Fig. 3. Kinetics of entering into Speriodthe binucleate and polynucleate cells by recording frequency oflabelled cells in each population at different times after caf feine. There was strict intracellular synchrony.
the cell types we studied. Alfert and Das were able to show 4n mononucleate cells had a G1 1.7 times longer than 2n mononucleate cells in plant autopolyploid cells. The similar Gllengthinthe different nuclei of the 2n-4n-2ncellsagreesnicelywith rapid induction of replication via cytoplasm in the 4nnucleus, andalsoagrees with results obtained in mammalian cells where replication is induced both in GIHeLa nuclei and in hen erythrocytes when fused with S HeLa cells.(Harris, 1970; Rao and Johnson, 1974). The nature ofthepossible inductor for DNA replication is unknownbut entrance of cytoplasmic protein precedes henerythrocyte stimulation (see Harris, 1974), and protein synthesis in G1 is required for replication to start in polynucleate cells (Gonzdlez-Ferndndez et al., 1974). among
(1972),
all
216
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The termination of ences in replication
International
the S period. rate of the
Reports,
Vol. 1, No. 3, 1977
Intracellular differdifferent nuclei.
.
Pulses with 'H-TdR around the S/G2fringe zoneallowed us to detect considerable asynchrony between the nuclei sharing a common cytoplasm. The general observation was anisolabelling, and 4n central nuclei always remaining labelled, i.e. had ended replication. These observations point to differential rates of DNA replication fornuclei sharing a common cytoplasm. Interphase duration. ing prophase.
Intracellular
asynchronyinenter-
When the 2n-4n-2n cell populationlabelledatS/G2 studied at the G prophase area, 4n nuclei which behind in the S/s2 f ringe still remained behind.
25°C
061
0 2n
fast
s
0
slow
s
;
fast
s
\II
& .c
was were
I
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*-,
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//
0
E
P -slow s
6 D s go.2 E IL
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A
0
: /
/
a
t-
M M fast *slow
S
I 9
11
10 hours
12 after
1’3
li
caffeine
Fig. 4. Kinetics of entering into prophase (P) and metaphase (M) the binucleate cells(contro1) as well as the Fast nuclei different nuclei of the polynucleate cell. (2n nuclei) reach prophase earlier than control nuclei, than while slow nuclei(4n) reach prophase slightly later Besides, entrance into metaphase ofpolycontrol nuclei. nucleate cell nuclei is slightly delayed with respect to control nuclei.
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Vol. 1, No. 3, 19 7 7
s1ni.s observation points to the autonomy of the differ en> nuclei sharing a common cytoplasm in G2 progression as well as in initiating mitosis. The kinetics of the differentnucleientering prophase 2n nuclei in in figure 4 (P-lines). represented are 2n-4n-2n cells reach prophase about one hour earlierthan while 4n nuclei in the of a binucleate cell, 2n nuclei 2n-4n-2ncellsreached prophase a little later than connuclei. We have represented both nuclei in binu trol in them cleate cells in a single line since asynchrony can be considered negligible. The figure could be much strict smaller taking into account difficulties in the classification of a nucleus in very early prophase.This syncould explain why Benbadis et al. (1974) reported chrony in binucleate cells in this stage.
!
25°C
/ I
/
I
I
1
1
2
3
4
5
Time
after
3H-TdR
(h)
in the nuclei of the measure of G2 timing Fig. 5. Direct nuclei and in the control 2n-4n-2n polynucleate cell 4n slow interphase nuclei shows G2 2n-2n cell nuclei. timing slightly shorter than control nuclei, while 2n fast nuclei show longer G2 than control nuclei.
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The general conclusion from these data is that inter phase duration may be shortened by the cytoplasmic duration was simenvironment a nucleus has. Since G ilar to Gl duration in binucleate &ells, the shortening must take place during S + G2. 22 duration The G2 timing in the different nuclei of the polynucleate cell 2n-4n-2n was studied and compared with that of 2n mononucleate cells. Pulses of H-TdR were given in different bulbs around the time when they were expected to be at the S/G2 fringe area. The frequency of labelled prophases was recorded at differenttimesafter nuclei in 3H-TdR labelling. Figure 5 shows 4n lagging the 2n-4n-2n cells have shorter G2 than control nuclei, 2n nuclei while 2n nuclei had a rather longer G than of the control i+ had a shorter cell even through t ey S + G duration. In z hese data nuclear progression through G2 appears to respond both to negativeandpositive control factors present in the cytoplasm of the cell supporting hetero phasic nuclei. also found that increasing Rao and Johnson (1970) the ratio G /S nuclei in a mammalian heterophasic multi nucleate ce z 1 led to an advanced entry of S nuclei into (Rao and Johnson, 1974) G2 G and later postulated works by Graves s 2 ortening on the gounds of previous (1972). Synchronous triggering of metaphase with reached metaphase Polynucleate 2n-4n-2n cells cells. a kinetics closely resembling that of control However there is a small delay in initiating metaphase hours in a cycle (around 0.4 compared to control averaging 13.5 hours). Complementation of cycle compartment requirements By integrating all our data we have been able to time required draw figure 6, which shows the minimum for each period. sort of from this The most important conclusion representation is the information it gives on duration when first of replication time (S period). The time prophase appears minus the minimum G2 duration gives Fast nuclei (2n nuclei) the timing of S/G transition. in 2n-4n-2n ccl 1 s can complete their S period in a time as short a 1.9 h versus the 4.5 hours it takes the minimum S period in control binucleate cells. It means the rate of replication can be more than doubled in these nuclei.
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219
4n nuclei was 1.2 On the other hand S phase of slow Alfert binucleate cells. time longer than S phase in and Das (1972) found that 4n mononucleate cells had an S 1.5 times longer than cells. Hence, on mononucleate the whole, nuclei in the polynucleate cell have shorter This obS phases than their independent counterparts. servation could be compatiblewitha consequent need for thresholds initiators forthechrodose of different mosomal takes place in a segments whose replication definite position of the The situation would S period.
I
CAFFEINE I I
0
: I
5
I,
I :
:I’0 1
: hours I
>
0 0 0 2n
2n
Fig. 6. Schematic representation of the minimum interphase and prophase timings for control nuclei (those in 2n-2n cells) and in the nuclei of the polynucleate 2n-4n-2n cells.The intracellular synchrony in initiating replication allows to draw the dotted common line whichrepresent the first 3H/-TdRincorporation after the caffeine labelling. There is again intracellular synchrony ininitiatingmetaphase, but the time when polynucleate cells reach this mitoticphase is slightly later than in control binucleate cells. The 2n nuclei in the 2n-4n-2n nuclei show a much shorter S period, partially compensated with a longer G2 + Prophase timing. 4n nuclei in the same cells shows longer S period, compensated by shorter G2 + Prophase trmrngs.
220
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of replication be similar to that at the very start (Gl/S boundary), where early replicons seemtobe induced by cytoplasm stimulus (Barlow, 1972). Relative autonomy in replication rate is combinedwith apparent positive control mediated by substances found in the cytoplasm.This finding of induced shortening of S period seems to contradict Graves' (1972) conclusion based on constancy of previous S timings when nuclei with short and long S periods are fused irrespective of the cytoplasm share. However, short lives of they postulated consecutive inductors combinedwiththe readiness of the target replicons in the sequential activation of different species must not be forgotten, in spite of the observation that the early postulated inductor is not species specific ( Johnson and Harris, 1969). Furthermore, Thmpson and McCarthy(l971) showed cytoplasmic extracts were able to increase DNA synthesis in an "in vitro" system. This finding will be taken as in replication meaning that the progressive inductors specifically work at the level of DNA polymerase the template DNA complex. Our data also show how natural desynchronization between nuclei in a common cytoplasm during interphase appears mostly owe to an increase in replication rate. replicating nuclei Intracellular presence of multiple speeds up this cellular function, which appears to be under positive control. The shorter S period in these fast 2n nuclei is compensated by both a lengthening of their G2 as well as lengthening of prophase. The length of G2 appears controls in to respond both to positive and negative slow and fast nuclei of the 2n-4n-2n trinucleate cell. is not shortened more than the time However, prophase fast it takes in control cells, while the prophase of nuclei is nearly 3 times longer than inother remaining still nuclei. The negative control of the 4n nucleus in G2 prevents the triggering of metaphase oftheother fellow nuclei. controls It is hard to adscribe these differential G (positive and negative) operating in GI (positive) points control and prophase (negative ) to t i+e three mapped in each of these which have been respectively where protein stages (Gl, early G2 and early prophase) synthesis is necessary to allow nuclear progression on but they must not be overlooked to next cycle stage, 1974; Garcia-Herdugo etal., (Gonzdlez-Fern6ndez et al., fact that stressed by the 1974). Their importance is resting meristems are always stopped atproteincontrol points: 1973). either GI or G (Van't Hof et al., results the most in $ eresting finding these Finally,
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suggest is that cell cycle in animal and plant cells. cesibility to experimental stressing for such studies.
221
are quite similar controls The plant offer better acmanipulation which is worth
MATERIAL AND METHODS The material used was the root meristemof Allium cepa bulbs were grown in L. The onion the dark a a constant temperature of 25°C f 0.5 incylindricalglass re,ceptacles of 90ml capacity, in filtered tapwaterwhich was renewed every 24 h and aerated continuously by bubbling air at lo-20 ml/ml. place that only their The bulbs were so bases remained submerged in the water. The roots we fixed in a 3:l mixture of ethanol-acetic acid and the specimens were preparedby staining the root with acetic orcein. Induction of the bipolynucleate cells was and accomplished by placing the bulbs in 0.1% caffeine solution in otherwise identical conditions ofr the one hour treatment periods. 3H-thymidine was carried out in a simLabelling with periods of 15 min, at ilar way, in different bulbs for different times after caffeine. 3H-thymidine at 17.8 Ci/mmol specific activity was used at 10 pCi/ml final concentration ( Radiochemical Centre, Amersham, United Kingdom). Squashes of the root meristems were prepared on subbed slides. They were processed for autoradiography with AR-10 Kodak stripping film. Development was done with D-19 Kodak after 2-3 weeks' exposure time. Acknowledgments.by the Comision Tgcnica (grant
This work has been partially Asesora para Investigacisn no 613/4).
supported Cientzfica y
BIBLIOGRAPHY Alfert,M. and Das,N.K.(1969)Evidence for control of the rate of nuclear DNA synthesis by the nuclear membrane in eukaryotic cells. Proceedings, National Academic Science 63, 123-128. Barlow,P.W.(1972)The ordered replication of chromosomal DNA: a review and aproposal for its control. Cytobios 5, 55-80. Benbadis,M.C .,Ribsztejn,M. and Deysson,G.(1974)The mode of nuclear DNA synthesis in experimentally induced binucleate cells of root meristems.Chromosoma 46,1-11. Garcia-Herdugo,G.,Fer&ndez-6, M.E., Hidalgo>. and L6pez-Sdez,J.F.(1974)Effects of protein synthesis
222
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inhibition during plant mitosis. Experimental Cell Research 89, 336-342. Gimgnez-Martln,F., Gonzslez-Ferndndez,A. and Lbpez-Sdez, J.F.(1965)A new method of labeling cells. Journal of Cell Biology 26, 305-309. Gimenez-Martln,G.,znz6lez-Ferndndez,A. and L6pez-S$ez, J.F.(1966)Duration of the division cycle in diploid, binucleate and tetraploid cells. Experimental Cell Research 43, 293-300. Gimenez-Marsn,G.,L6pez-S&ez,J.F.,Moreno,P. and Gonzdlez-Ferndndez,A.(1968)0n the triggering of mitosis and the division cycle of polynucleate cells. Chromosoma 25, 282-296. Gonzdlez-Ferndndez,A.,Gim~nez-Martfn,G.,D~ez,J.L.,De la Torre, C. and L6pez-S6ez, J.F. (1971) Interphase development and beginning of mitosis in the different nuclei of polynucleate homokaryotic cells.Chromosoma 36, 100-111. Gonzalez-Ferndndez,A .,Gim&nez-Martfn,G.,Fernandez-G6mez, M.E. and De la Torre, C.(1974) Protein synthesis requirements at specific points in the interphase of meristematic cells. Experimental Cell Research 88, 163-170. Graves,J.A.M.(1972)DNA synthesisinheterocaryons formed by fusion of mammalian cells from different species. Experimental Cell Research 72, 393-403. Harris, H.(1965) Behaviour of-differentiated nuclei in heterokaryons of animal cells from different species. Nature 206, 583-585. Harris,H.(1970)Cellfusion.The Dunham Lectures.Clarendom Press, Oxford, U.K. and cytoplasm. 3rd. Edition Harris,H.(1974) Nucleus Clarendon Press, Oxford, U.K. Variation in the period Howard, A. and Dewey,D.L.(1960) in bean preceding deoxyribonucleic acid synthesis Butterroot cells. in The Cell Nucleus p. 155-162, worth & Co. Pub. Ltd., London. Johnson,R.T. and Harris,H.(1969)DNA synthesis andmitosis Journal of Cell in fused cells.I.HeLa homokaryons. Sciene 2, 645-697. Johnson, R.T. and Rao, P.J. (1971) Nucleo-cytoplasmic interactions in the achievement of nuclear synchrony cells. in DNA synthesis and mitosis in multinucleate Biological Reviews 46,97-155. Kihlman, ‘B. (1955) Chromosome breakage in Alliumby8Experimental Cell Research ethoxycaffeine and X-rays. &, 345-368. L6pez-S6ez, J.F., Risuefio, M.C. and Gimenez-Martln,G. (1966) Inhibition of cytokinesis in plant cells. Journal Ultrastructural Research 14, 85-94. Mammalian cell fusion: Rao, P.N. and Johnson,R.T.(1970)
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studies on the regulation of DNA synthesis and mitosis. Nature 225, 159-164. R.T. (1974) Regulation of cell Rao, P.N. and Johnson, of proliferation cycle in hybrid cells. in Control Cold Spring Harbor Labin animal cells. p. 785-800. oratory. Thompson, L.R. and McCarthy,B.J. (1968) Stimulation of nuclear DNA and RNA synthesis by cytoplasmic extracts comin vitro. Biochemical and Biophysical Research munication 30, 166-172. and Yagi, S. (1973) Cell Van't Hof, J.,Hoppin, D.P. arrest in G and G of the mitotic cycle of Vicia faba root m&ristemg. American Journal of Botany 60, 889-895.
international
Reports,
211
Vol. 1, No. 3, 1977
REGULATION OF CYCLEPROGRESSION IN PLANTCELLS C. DE LA TORREl Instituto de Biologla
and G. GIMENEZ-MARTIN Celular, C.S.I.C., Madrid ABSTRACT
root Induction ofpolynucleate cells in onion meristems by inhibition of two sc,,uent'irL3 cytokineses is used to study controls operating in cell cycle progression. Triggering of both replication andmetaphase occurs synchronously in nuclei sharing a common independentoftheirploidy orintracytoplasm, cellular position. The replication rate appears to be activated by the simultaneous intracytoplasm presence of other replicating nuclei. On the other hand, central position of anucleus in the cell as well as increase in ploidy leads to slowing down of replication rate. The relative advance and lag of S periodinthe differentnuclei in a common cytoplasmispartially counter balanced by differential times of G2. Moreover prophase lengthening in the fast interphase nuclei points to a negative control exerted by the lagging nuclei mediated by cytoplasm. Finally, it could be worth emphasizing similarities in the control mechanism operating in cycle progression both in animal and plant cells.
Polynucleate cells arebeing extensively used to study both nucleus and cytoplasmrolesand autonomy in controlling metabolism.In this line,we should takeintoaccount studies based on fusionofnuclei from different species (Harris, 1965) as well as from different phases of cell cycle (Johnson and Rao, 1971).Chemicals which irreversibly inhibit cytokinesis have been amply used for induc ing homophasic polynucleate cells in plants (Kihlmany 1955; Howard and Dewey, 1960; Gimi%ez-Martin et al., 1965; Benbadis et al., 1974). Caffeine inhibits the fusion of Golgivesiclesin cytokinesis during telophase (L6pez-Sdez et al., 1966), giving rise to a binucleate cell population (2n-2n) initiating its next cycle. 1
Correspondence Inst. de3iolog /
Caffeine
Fig. 2. Analysis of cycle progression in the polynucleate cells. 3H-thymidine pulses at intervals after caffeine show kinetics of entrance andexit of S period. Previous qualitative data suggested intracellular synchrony in initiating but not in endingreplication. Central nuclei were always behind in the S/G as well as in G2/ prophase boundaries. Finally,there is2alsointracellular synchrony in the triggering of metaphase.
214
Cell Biology
In term tional Reports,
Vol. 1, No. 3, 19 7 7
radioactive precursors at given times aftercaffeinemark the kinetics of entrance and exit in S period, while studies of mitosis frequencywillindicate the kinetics of entrance in mitosis of the cell population. This fig. 2 schematically shows data which confirm previous studies (Gimenez-Martin et al., 1968 ; Gonzslez-Fer nsndez et al., 1971). There seemedtobeintracellularsynchrony both in initiationof replication and metaphase, while the endofreplication and the initiation of prophase can occur asynchronously in the different nuclei of the same cell. also the intra Apparently not only the ploidy but cellular location affects the cycle rate of a nucleus, Nuclei having a larger surrounding cytoplasm are faster than those having a more in interphase progression limited amount, since observations at S/G and G2/profastphase boundaries consistently showed that 32oththe est nuclei in 2n-2n-2n-2n and 2n-4n-4n polynucleate of the cells. As the cells were those in the extremes frequency of 2n-4n-2n cells in the meristem is greater than 2n-2n-2n-2n cells and they seem to have greater the study of G2/prophase asynchrony, we have limited intercellular andintracellularasynchrony in the fringes of the different cell compartments to 2n-4n-2n cells, comparing them to 2n-2n binucleate cells. initiating : Intracellular synchrony in G1 duration renlication. The kinetics of entry into S of binucleate and polynucleatecellswere observedbygiving3H-TdR at one hour The frequency of labelled spaced intervals after caffeine. these cells. cells provides a direct measure of G1 in Fig. 3 shows how the population of polynucleate cells initiates replication with a kinetics similar to that shown by binucleate cells. Hence, the duration of G1 in a cell is independentboth of the number of nuclei and of individual nuclear ploidies. The intracellular synchrony in initiating replication was total both in binucleate and polynucleate cells. On the other hand, Benbadis et al. (1974) pointed out an asynchrony in 3% of the binucleate cells induced by methyl 3 hydroxy 6 (lH, 3 H) quinazoline dione 2.4 in Allium sativum L. Efforts to detect asynchrony in the G1-S fringe failed in our cells. Asynchrony if any could be within the limits of resolution of labelling in autoradiography and its detection depend on background accepted level. Our data seem to support a strict nuclear synchrony in initiating replication even in 2n-4n-2n polynucleate cells which showed the greatest asynchrony in Gi,/prophase
Cell Biology
International
06
Reports,
l blnucleate
opolynucleate
215
Vol. 1, No. 3, 19 7 7
25°C
cells cells
hours
5 after
1
k
caffeine
Fig. 3. Kinetics of entering into Speriodthe binucleate and polynucleate cells by recording frequency oflabelled cells in each population at different times after caf feine. There was strict intracellular synchrony.
the cell types we studied. Alfert and Das were able to show 4n mononucleate cells had a G1 1.7 times longer than 2n mononucleate cells in plant autopolyploid cells. The similar Gllengthinthe different nuclei of the 2n-4n-2ncellsagreesnicelywith rapid induction of replication via cytoplasm in the 4nnucleus, andalsoagrees with results obtained in mammalian cells where replication is induced both in GIHeLa nuclei and in hen erythrocytes when fused with S HeLa cells.(Harris, 1970; Rao and Johnson, 1974). The nature ofthepossible inductor for DNA replication is unknownbut entrance of cytoplasmic protein precedes henerythrocyte stimulation (see Harris, 1974), and protein synthesis in G1 is required for replication to start in polynucleate cells (Gonzdlez-Ferndndez et al., 1974). among
(1972),
all
216
Cell Biology
The termination of ences in replication
International
the S period. rate of the
Reports,
Vol. 1, No. 3, 1977
Intracellular differdifferent nuclei.
.
Pulses with 'H-TdR around the S/G2fringe zoneallowed us to detect considerable asynchrony between the nuclei sharing a common cytoplasm. The general observation was anisolabelling, and 4n central nuclei always remaining labelled, i.e. had ended replication. These observations point to differential rates of DNA replication fornuclei sharing a common cytoplasm. Interphase duration. ing prophase.
Intracellular
asynchronyinenter-
When the 2n-4n-2n cell populationlabelledatS/G2 studied at the G prophase area, 4n nuclei which behind in the S/s2 f ringe still remained behind.
25°C
061
0 2n
fast
s
0
slow
s
;
fast
s
\II
& .c
was were
I
P
*-,
o+control /’
//
0
E
P -slow s
6 D s go.2 E IL
cant rol -----,
A
0
: /
/
a
t-
M M fast *slow
S
I 9
11
10 hours
12 after
1’3
li
caffeine
Fig. 4. Kinetics of entering into prophase (P) and metaphase (M) the binucleate cells(contro1) as well as the Fast nuclei different nuclei of the polynucleate cell. (2n nuclei) reach prophase earlier than control nuclei, than while slow nuclei(4n) reach prophase slightly later Besides, entrance into metaphase ofpolycontrol nuclei. nucleate cell nuclei is slightly delayed with respect to control nuclei.
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Vol. 1, No. 3, 19 7 7
s1ni.s observation points to the autonomy of the differ en> nuclei sharing a common cytoplasm in G2 progression as well as in initiating mitosis. The kinetics of the differentnucleientering prophase 2n nuclei in in figure 4 (P-lines). represented are 2n-4n-2n cells reach prophase about one hour earlierthan while 4n nuclei in the of a binucleate cell, 2n nuclei 2n-4n-2ncellsreached prophase a little later than connuclei. We have represented both nuclei in binu trol in them cleate cells in a single line since asynchrony can be considered negligible. The figure could be much strict smaller taking into account difficulties in the classification of a nucleus in very early prophase.This syncould explain why Benbadis et al. (1974) reported chrony in binucleate cells in this stage.
!
25°C
/ I
/
I
I
1
1
2
3
4
5
Time
after
3H-TdR
(h)
in the nuclei of the measure of G2 timing Fig. 5. Direct nuclei and in the control 2n-4n-2n polynucleate cell 4n slow interphase nuclei shows G2 2n-2n cell nuclei. timing slightly shorter than control nuclei, while 2n fast nuclei show longer G2 than control nuclei.
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The general conclusion from these data is that inter phase duration may be shortened by the cytoplasmic duration was simenvironment a nucleus has. Since G ilar to Gl duration in binucleate &ells, the shortening must take place during S + G2. 22 duration The G2 timing in the different nuclei of the polynucleate cell 2n-4n-2n was studied and compared with that of 2n mononucleate cells. Pulses of H-TdR were given in different bulbs around the time when they were expected to be at the S/G2 fringe area. The frequency of labelled prophases was recorded at differenttimesafter nuclei in 3H-TdR labelling. Figure 5 shows 4n lagging the 2n-4n-2n cells have shorter G2 than control nuclei, 2n nuclei while 2n nuclei had a rather longer G than of the control i+ had a shorter cell even through t ey S + G duration. In z hese data nuclear progression through G2 appears to respond both to negativeandpositive control factors present in the cytoplasm of the cell supporting hetero phasic nuclei. also found that increasing Rao and Johnson (1970) the ratio G /S nuclei in a mammalian heterophasic multi nucleate ce z 1 led to an advanced entry of S nuclei into (Rao and Johnson, 1974) G2 G and later postulated works by Graves s 2 ortening on the gounds of previous (1972). Synchronous triggering of metaphase with reached metaphase Polynucleate 2n-4n-2n cells cells. a kinetics closely resembling that of control However there is a small delay in initiating metaphase hours in a cycle (around 0.4 compared to control averaging 13.5 hours). Complementation of cycle compartment requirements By integrating all our data we have been able to time required draw figure 6, which shows the minimum for each period. sort of from this The most important conclusion representation is the information it gives on duration when first of replication time (S period). The time prophase appears minus the minimum G2 duration gives Fast nuclei (2n nuclei) the timing of S/G transition. in 2n-4n-2n ccl 1 s can complete their S period in a time as short a 1.9 h versus the 4.5 hours it takes the minimum S period in control binucleate cells. It means the rate of replication can be more than doubled in these nuclei.
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4n nuclei was 1.2 On the other hand S phase of slow Alfert binucleate cells. time longer than S phase in and Das (1972) found that 4n mononucleate cells had an S 1.5 times longer than cells. Hence, on mononucleate the whole, nuclei in the polynucleate cell have shorter This obS phases than their independent counterparts. servation could be compatiblewitha consequent need for thresholds initiators forthechrodose of different mosomal takes place in a segments whose replication definite position of the The situation would S period.
I
CAFFEINE I I
0
: I
5
I,
I :
:I’0 1
: hours I
>
0 0 0 2n
2n
Fig. 6. Schematic representation of the minimum interphase and prophase timings for control nuclei (those in 2n-2n cells) and in the nuclei of the polynucleate 2n-4n-2n cells.The intracellular synchrony in initiating replication allows to draw the dotted common line whichrepresent the first 3H/-TdRincorporation after the caffeine labelling. There is again intracellular synchrony ininitiatingmetaphase, but the time when polynucleate cells reach this mitoticphase is slightly later than in control binucleate cells. The 2n nuclei in the 2n-4n-2n nuclei show a much shorter S period, partially compensated with a longer G2 + Prophase timing. 4n nuclei in the same cells shows longer S period, compensated by shorter G2 + Prophase trmrngs.
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of replication be similar to that at the very start (Gl/S boundary), where early replicons seemtobe induced by cytoplasm stimulus (Barlow, 1972). Relative autonomy in replication rate is combinedwith apparent positive control mediated by substances found in the cytoplasm.This finding of induced shortening of S period seems to contradict Graves' (1972) conclusion based on constancy of previous S timings when nuclei with short and long S periods are fused irrespective of the cytoplasm share. However, short lives of they postulated consecutive inductors combinedwiththe readiness of the target replicons in the sequential activation of different species must not be forgotten, in spite of the observation that the early postulated inductor is not species specific ( Johnson and Harris, 1969). Furthermore, Thmpson and McCarthy(l971) showed cytoplasmic extracts were able to increase DNA synthesis in an "in vitro" system. This finding will be taken as in replication meaning that the progressive inductors specifically work at the level of DNA polymerase the template DNA complex. Our data also show how natural desynchronization between nuclei in a common cytoplasm during interphase appears mostly owe to an increase in replication rate. replicating nuclei Intracellular presence of multiple speeds up this cellular function, which appears to be under positive control. The shorter S period in these fast 2n nuclei is compensated by both a lengthening of their G2 as well as lengthening of prophase. The length of G2 appears controls in to respond both to positive and negative slow and fast nuclei of the 2n-4n-2n trinucleate cell. is not shortened more than the time However, prophase fast it takes in control cells, while the prophase of nuclei is nearly 3 times longer than inother remaining still nuclei. The negative control of the 4n nucleus in G2 prevents the triggering of metaphase oftheother fellow nuclei. controls It is hard to adscribe these differential G (positive and negative) operating in GI (positive) points control and prophase (negative ) to t i+e three mapped in each of these which have been respectively where protein stages (Gl, early G2 and early prophase) synthesis is necessary to allow nuclear progression on but they must not be overlooked to next cycle stage, 1974; Garcia-Herdugo etal., (Gonzdlez-Fern6ndez et al., fact that stressed by the 1974). Their importance is resting meristems are always stopped atproteincontrol points: 1973). either GI or G (Van't Hof et al., results the most in $ eresting finding these Finally,
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suggest is that cell cycle in animal and plant cells. cesibility to experimental stressing for such studies.
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are quite similar controls The plant offer better acmanipulation which is worth
MATERIAL AND METHODS The material used was the root meristemof Allium cepa bulbs were grown in L. The onion the dark a a constant temperature of 25°C f 0.5 incylindricalglass re,ceptacles of 90ml capacity, in filtered tapwaterwhich was renewed every 24 h and aerated continuously by bubbling air at lo-20 ml/ml. place that only their The bulbs were so bases remained submerged in the water. The roots we fixed in a 3:l mixture of ethanol-acetic acid and the specimens were preparedby staining the root with acetic orcein. Induction of the bipolynucleate cells was and accomplished by placing the bulbs in 0.1% caffeine solution in otherwise identical conditions ofr the one hour treatment periods. 3H-thymidine was carried out in a simLabelling with periods of 15 min, at ilar way, in different bulbs for different times after caffeine. 3H-thymidine at 17.8 Ci/mmol specific activity was used at 10 pCi/ml final concentration ( Radiochemical Centre, Amersham, United Kingdom). Squashes of the root meristems were prepared on subbed slides. They were processed for autoradiography with AR-10 Kodak stripping film. Development was done with D-19 Kodak after 2-3 weeks' exposure time. Acknowledgments.by the Comision Tgcnica (grant
This work has been partially Asesora para Investigacisn no 613/4).
supported Cientzfica y
BIBLIOGRAPHY Alfert,M. and Das,N.K.(1969)Evidence for control of the rate of nuclear DNA synthesis by the nuclear membrane in eukaryotic cells. Proceedings, National Academic Science 63, 123-128. Barlow,P.W.(1972)The ordered replication of chromosomal DNA: a review and aproposal for its control. Cytobios 5, 55-80. Benbadis,M.C .,Ribsztejn,M. and Deysson,G.(1974)The mode of nuclear DNA synthesis in experimentally induced binucleate cells of root meristems.Chromosoma 46,1-11. Garcia-Herdugo,G.,Fer&ndez-6, M.E., Hidalgo>. and L6pez-Sdez,J.F.(1974)Effects of protein synthesis
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inhibition during plant mitosis. Experimental Cell Research 89, 336-342. Gimgnez-Martln,F., Gonzslez-Ferndndez,A. and Lbpez-Sdez, J.F.(1965)A new method of labeling cells. Journal of Cell Biology 26, 305-309. Gimenez-Martln,G.,znz6lez-Ferndndez,A. and L6pez-S$ez, J.F.(1966)Duration of the division cycle in diploid, binucleate and tetraploid cells. Experimental Cell Research 43, 293-300. Gimenez-Marsn,G.,L6pez-S&ez,J.F.,Moreno,P. and Gonzdlez-Ferndndez,A.(1968)0n the triggering of mitosis and the division cycle of polynucleate cells. Chromosoma 25, 282-296. Gonzdlez-Ferndndez,A.,Gim~nez-Martfn,G.,D~ez,J.L.,De la Torre, C. and L6pez-S6ez, J.F. (1971) Interphase development and beginning of mitosis in the different nuclei of polynucleate homokaryotic cells.Chromosoma 36, 100-111. Gonzalez-Ferndndez,A .,Gim&nez-Martfn,G.,Fernandez-G6mez, M.E. and De la Torre, C.(1974) Protein synthesis requirements at specific points in the interphase of meristematic cells. Experimental Cell Research 88, 163-170. Graves,J.A.M.(1972)DNA synthesisinheterocaryons formed by fusion of mammalian cells from different species. Experimental Cell Research 72, 393-403. Harris, H.(1965) Behaviour of-differentiated nuclei in heterokaryons of animal cells from different species. Nature 206, 583-585. Harris,H.(1970)Cellfusion.The Dunham Lectures.Clarendom Press, Oxford, U.K. and cytoplasm. 3rd. Edition Harris,H.(1974) Nucleus Clarendon Press, Oxford, U.K. Variation in the period Howard, A. and Dewey,D.L.(1960) in bean preceding deoxyribonucleic acid synthesis Butterroot cells. in The Cell Nucleus p. 155-162, worth & Co. Pub. Ltd., London. Johnson,R.T. and Harris,H.(1969)DNA synthesis andmitosis Journal of Cell in fused cells.I.HeLa homokaryons. Sciene 2, 645-697. Johnson, R.T. and Rao, P.J. (1971) Nucleo-cytoplasmic interactions in the achievement of nuclear synchrony cells. in DNA synthesis and mitosis in multinucleate Biological Reviews 46,97-155. Kihlman, ‘B. (1955) Chromosome breakage in Alliumby8Experimental Cell Research ethoxycaffeine and X-rays. &, 345-368. L6pez-S6ez, J.F., Risuefio, M.C. and Gimenez-Martln,G. (1966) Inhibition of cytokinesis in plant cells. Journal Ultrastructural Research 14, 85-94. Mammalian cell fusion: Rao, P.N. and Johnson,R.T.(1970)
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studies on the regulation of DNA synthesis and mitosis. Nature 225, 159-164. R.T. (1974) Regulation of cell Rao, P.N. and Johnson, of proliferation cycle in hybrid cells. in Control Cold Spring Harbor Labin animal cells. p. 785-800. oratory. Thompson, L.R. and McCarthy,B.J. (1968) Stimulation of nuclear DNA and RNA synthesis by cytoplasmic extracts comin vitro. Biochemical and Biophysical Research munication 30, 166-172. and Yagi, S. (1973) Cell Van't Hof, J.,Hoppin, D.P. arrest in G and G of the mitotic cycle of Vicia faba root m&ristemg. American Journal of Botany 60, 889-895.
