Control of cell division in Paramecium tetraurelia. Effects of abrupt changes in nutrient level on accumulation of macronuclear DNA and cell mass

In the cell cycle of Paramecium there are three points of interaction between cell growth-related processes and the processes of macronuclear DNA replication and cell division: initiation of DNA synthesis, regulation of the rates of growth and DNA accumulation, and initiation of cell division. This study examines the regulation of the latter two processes by analysis of the response of each to abrupt changes in nutrient level brought about either by transferring dividing cells from a steady-state chemostat culture to medium with unlimited food, or by transferring well-fed dividing cells to exhausted medium. The rates of DNA accumulation and cell growth respond quickly to changes in nutrient level. The amounts of these cell components accumulated during the cell cycle following a shift in nutrient level are typical of those occurring during equilibrium growth under post-shift conditions. Commitment to division occurs at a fixed interval prior to fission that is similar in well-fed and nutrient-limited cells. Initiation of cell division in Paramecium is associated with accumulation of a threshold DNA increment, whose level is largely independent of nutritive conditions. The amount of DNA accumulated during the cell cycle varies with nutritional conditions because the rates of growth and DNA accumulation are affected by nutrient level; slowly growing cells accumulated relatively little DNA during the fixed interval between commitment to cell division and fission.     

The cell cycle of<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: the role of the commitment point

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.     

Downward regulation of macronuclear DNA content in Paramecium tetraurelia : Effects of excess DNA on the subsequent DNA and protein content and the cell growth rate

Paramecium cells were selected which received the entire parental macronucleus at fission and thus started the cell cycle with twice the normal post-fission DNA content. During each of the subsequent two cell cycles the cells synthesized approximately as much DNA as did control cells. The amount of excess macronuclear DNA was consequently halved during each cell cycle. The minimum pre-fission DNA content was just larger than the mean post-replication DNA amount, confirming that a similar amount of DNA, approximately equal to the mean post-fission DNA content of the non-selected population, was synthesized in macronuclei, regardless of the post-fission DNA content. These observations confirm a model for DNA content regulation previously devised for Paramecium and are inconsistent with DNA content regulation schemes proposed for other ciliates. The increased DNA content has no effect either on the subsequent total protein content of pre-fission cells, or on the rate of cell growth. This suggests that the rate of cell growth is limited by the size of the cell when the macronuclear gene-dosage is equal to or greater than that in normal cells. The results also suggest that the amount of DNA synthesized within an interfission period is also limited by the size of the cell and is proportional to the cell mass. Paramecium does not require a fixed nucleocy oplasmic ratio as a pre-condition either for cell division, or, by inference, for initiation of DNA synthesis.     

GROWTH-CONTROLLED OSCILLATION IN ACTIVITY OF HISTONE H1 KINASE DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronous cultures of the cell wall-less mutant Chlamydomonas reinhardtii Dangeard cw 15 were grown under different mean irradiances and different illumination regimes, which produced cell cycles that differed in the number of daughter cells released from one mother cell, in the length of the cell cycle, and in the growth rate. During the cell cycle, the cells reached several commitment points whose number and timing differed according to the particular pattern of the cell cycle. The cell volume was used as a growth parameter and increased in a stepwise manner. Each of the steps consisted of periods of both fast and slow growth. Growth usually stopped when the cells attained a volume twice that of the preceding step. Reaching particular commitment points was coupled with the position of these points in the enlargement of cell volume. Changes in the activity of histone H1 kinase were noted during the cell cycles of all experimental variants, and the activities were compared with the timing of various commitment points. It was found that kinase activity varied markedly within a single cell cycle, attaining maximal values when the cellular volume had doubled. Each peak in kinase activity slightly preceded the commitment to an individual sequence of reproductive events. In addition to the oscillations related to cell growth, a peak of kinase activity always occurred toward the end of the cell cycle when multiple rounds of DNA replication, mitosis, and cell division occurred.     

Commitment to Division in Ciliate Cell Cycles

Near the end of the cell cycle, ciliates commit irreversibly to cell division. The point of commitment occurs at the time of oral polykinetid assembly and micronuclear anaphase. The commitment is a checkpoint which requisites a threshold cell mass/ DNA ratio and stomatogenesis. It is also a physiological transition point, involving cdk protein kinases similar to those of other eukaryotes. Both P34 kD and P36 kD kinases, similar to the S. pombe cdc2 kinases, have been described to have activity as monomers. Subsequent to commitment to division, dramatic cytoskeletal modifications occur for separation of organelles, cortex morphogenesis and cytokinesis. Numerous mutants affecting cytoskeletal function associated with the division process have been obtained in several species. Of these, only the ccl mutant in Paramecium affects cell cycle progression prior to commitment to division. The material reviewed is used to speculate about the mechanisms of regulation of pre-fission morphogenesis and cell division related processes in ciliates.     

The timing of initiation of macronuclear DNA synthesis is set during the preceding cell cycle in Paramecium tetraurelia. Analysis of the effects of abrupt changes in nutrient level

In many eukaryotic organisms, initiation of DNA synthesis is associated with a major control point within the cell cycle and reflects the commitment of the cell to the DNA replication-division portion of the cell cycle. In Paramecium, the timing of DNA synthesis initiation is established prior to fission during the preceding cell cycle. DNA synthesis normally starts at 0.25 in the cell cycle. When dividing cells are subjected to abrupt nutrient shift-up by transfer from a chemostat culture to medium with excess food, or shift-down from a well-fed culture to exhausted medium. DNA synthesis initiation in the post-shift cell cycle occurs at 0.25 of the parental cell cycle and not at either 0.25 in the post-shift cell cycle or at 0.25 in the equilibrium cell cycle produced under the post-shift conditions. The long delay prior to initiation of DNA synthesis following nutritional shift-up is not a consequence of continued slow growth because the rate of protein synthesis increases rapidly to the normal level after shift-up. Analysis of the relation between increase in cell mass and initiation of DNA synthesis following nutritional shifts indicates that increase in cell mass, per se, is neither a necessary nor a sufficient condition for initiation of DNA synthesis, in spite of the strong association between accumulation of cell mass and initiation of DNA synthesis in cells growing under steady-state conditions.     

Unbalanced cell growth and increased protein synthesis induced by chemotherapeutic agents

Unbalanced cell growth as manifested by an increase in cellular volume and in cellular dry mass following exposure to a variety of chemotherapeutic agents has been shown for neoplastic cells in vitro and human leukemic cells in vivo. The purpose of the present investigation was to test the hypothesis that unbalanced cell growth results from a disassociation of cell growth and cell division due to the blocking effect of chemotherapeutic agents. Monolayer cultures of CHO fibroblasts were studied in terms of their response to two chemotherapeutic agents that differ significantly in their mode of action, adriamycin and chlorambucil. Following exposure to these drugs, cell volume increased at a rate of from 1% to 4% per h; the total cell protein increased at a rate of from 4% to 7% per h. These changes were observed in both log and stationary phase cultures. Thus exposure to adriamycin and chlorambucil was followed by a more rapid rate of protein synthesis relative to the rate of degradation, resulting in larger cells with more protein whether or not the cells were actively in the division cycle. This is inconsistent with the hypothesis that unbalanced growth results simply from a disassociation of the cell division cycle from cell growth. These observations suggest that a final common pathway in the mode of action of chemotherapeutic agents may be the induction of unscheduled protein synthesis resulting in unbalanced cell growth.     

Calmodulin in Zajdela hepatoma cell growth

Calmodulin levels were measured in Zajdela hepatoma cells growing both in vivo and in culture, with respect to the distribution of the cells into G1 and S+G2 phases of the cell cycle and growth conditions. These levels, expressed on a per-microgram of protein basis, were significantly elevated at the G1-S boundary and maintained throughout the remainder of the cell cycle. This elevation of calmodulin took place independently of the culture conditions. Taken together with previous observations, these data suggest that a threshold concentration for calmodulin is required for progression through the cell cycle, DNA synthesis and cell division.     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.     

Regulation of macronuclear DNA content in Paramecium tetraurelia

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.     

Cell elongation and division probability during the Escherichia coli growth cycle.

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.     

Cell cycle control in normal and neoplastic cells.

Recent studies of cell cycle control suggest that cyclin-dependent protein kinases play a central role in the cell's commitment to a new division cycle in late G1. The regulation of these kinases in normal and neoplastic growth is becoming clear.     

EFFECT OF IRRADIANCE ON GROWTH AND REPRODUCTIVE PROCESSES DURING THE CELL CYCLE IN SCENEDESMUS ARMATUS (CHLOROPHYTA)1

Synchronized populations of the chlorococcal alga Scenedesmus armatus (Chod.) Chod. were grown under five irradiance levels. During the cell cycles of these populations, reproductive processes such as DNA replication, nuclear division, protoplast fission, and daughter cell release and growth processes such as RNA and protein accumulation were followed. The amount of RNA and proteins increased stepwise with a short time interval between individual steps during which the rate of RNA and protein accumulation decreased. At each of the steps, the amount of RNA and protein approximately doubled and the number of steps increased with irradiance. At the end of each of the growth steps, a commitment to trigger the sequence of reproductive events (DNA replication, nuclear division, protoplast fission) was attained. After attaining the commitment point, the cells were able to trigger and terminate the whole reproductive sequence without any further growth, that is, even in the dark when the external supply of energy was cut off. With increasing irradiance, the number of commitment points attained during one cell cycle increased from one to four. Consequently, one to four sequences of the reproductive steps were triggered, and each of them ended by doubling the reproductive structures, which resulted in the formation of 2, 4, 8, or 16 daughter cells. The length of the precommitment periods shortened with increasing irradiance as the result of an increasing rate in growth. The length of postcommitment periods showed light independence and remained constant at the range of irradiances at which the number of growth steps and, consequently, the number of sequences of reproductive events did not change. At higher irradiances, the number of sequences of reproductive events increased, which caused a prolongation of postcommitment periods. The length of the cell cycle varied as a result of this distinct effect of irradiance on pre- and postcommitment periods.     

Effects of Chromosome Underreplication on Cell Division in Escherichia coli

The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39°C (normal DNA/mass ratio) and shifted to 38 and 37°C. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.     

Pachytene Arrest and Other Meiotic Effects of the Start Mutations in Saccharomyes Cerevisiae

Mutations in the Start class of cell division cycle genes (CDC28, CDC36 and CDC39) define the point in the G1 phase of the vegetative cycle at which the cell becomes committed to completing another round of cell division. Genetic, cytological and biochemical data demonstrate that these mutations cause meiotic cells to become arrested at pachytene following completion of both chromosomal DNA replication and spindle pole body (SPB) duplication. In contrast these mutations have previously been found to cause arrest of the mitotic cell cycle prior to either of these landmark events, so the role of the Start genes in these events during vegetative growth must be indirect. Our observations are consistent with the hypothesis that CDC28, CDC36 and CDC39 are required for irreversible commitment to nuclear division in both the mitotic and meiotic pathways. CDC28 was additionally found to be required for the SPB separation that precedes spindle formation in preparation for the second meiotic division. Cytological and genetic analyses of this requirement revealed both that such separation may fail independently at either SPB and that ascospore formation can proceed independently of SPB separation.     

Dependence of HL-60 myeloid cell differentiation on continuous and split retinoic acid exposures: Precommitment memory associated with altered nuclear structure

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca²⁺ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.     

Effects of Radiation,Antibiotics and Alkylating Agents on Cell Division and Growth in the Ciliate Spathidium spathula*

SYNOPSIS. Spathidium spathula is very sensitive to division inhibition after X-irradiation. Five kr delivered to animals 1 hr into the cell cycle prolong the period until the next division to about 2 times the normal length. The next 2 cell cycles, however, are shorter than normal, and by the 4th division irradiated cells have recovered the normal division rate. During this division delay, scanning interference microscopy shows that growth in dry mass continues; at the 1st post-irradiation division the cells average 3 times the normal dry mass. After the 2nd post-irradiation division, dry mass is 1.5 times the normal amount. Dry mass measurements were not made beyond the 2nd division. Giant cells produced by X-rays have enlarged macronuclei, indicating that DNA synthesis is not inhibited by a dose of X-rays that blocks division. Mitomycin C and triethylene melamine, agents which attack or damage DNA, also produce division blockage and giantism in Spathidium. This suggests that damage to DNA in either the macronucleus, the micronucleus or other organelles may be much more effective in delaying cell division than cell growth.     

Cell volume increase in Escherichia coli after shifts to richer media.

Synchronous cultures of Escherichia coli 15-THU and WP2s, which were selected by velocity sedimentation from exponential-phase cultures growing in an acetate-minimal salts medium, were shifted to richer media at various times during the cell cycle by the addition of glucose or nutrient broth. Cell numbers and mean cell volumes were measured electronically. The duration of the division cycle of the shifted generation was not altered significantly by the addition of either nutrient. Growth rates, measured as rates of cell volume increase, were constant throughout the cycle in unshifted acetate control cultures. When glucose was added, growth rates also remained unchanged during the remainder of the cell cycle and then increased abruptly at or after cell division. When nutrient broth was added, growth rates remained unchanged from periods of 0.2 to 0.4 generations and then increased abruptly to their final values. In all cases, the cell volume increase was linear both before and after the growth rate transition. The strongest support for a linear cell volume increase during the cell cycle of E. coli in slowly growing acetate cultures, however, was obtained in unshifted cultures, in complete agreement with earlier observations of cell volumes at much more rapid growth rates. Although cell growth and division are under the control of the synthesizing machinery in the cell responsible for RNA and protein synthesis, the results indicate that growth is also regulated by membrane-associated transport systems.     

Control of the bacterial cell cycle

New insights into the control of DNA replication through growth, hemimethylated DNA and DnaA protein have been described. Fundamental shifts in thinking have resulted in the identification of new cell cycle genes with potential roles in initiation of DNA replication, chromosomal segregation and division. Excitingly, this trend may also narrow the apparent differences between the prokaryotic and eukaryotic cell cycles.