Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

Mutants altered in the control co-ordinating cell division with cell growth in the fission yeast Schizosaccharomyces pombe

Summary The control co-ordinating cell division with cell growth has been investigated in the fission yeast Schizosaccharomyces pombe. Twenty-five mutants altered in this control have been isolated which have the same growth rate as wild type but divide at a smaller cell size. The mutants define two genes wee 1 and wee 2, both of which are involved in a control initiating mitosis when the cell attains a critical size.     

Segregation of elongation potential inEscherichia coli mediated by thewee genetic system

Under normal conditions, the two cells within a pair ofEscherichia coli siblings elongate at a similar rate. Genetic impairment of thewee genetic system leads to significant variations in the rate of elongation of each cell within a given pair of siblings. This result is in accordance with previous reports that showed the need of active cell division for expression of the Wee phenotype. The genetic location of one element of the system,weeA, has been determined to be at min 67 of theE. coli map; this does not coincide with the previously reported genetic location. The inability to reproduce the Wee phenotype in a wild-type background by transduction ofweeA suggests that more genetic elements should participate in the segregation of elongation zones at cell division.     

Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division

In the fission yeast Schizosaccharomyces pombe, nutritional reduction of growth rate by supplying poor nitrogen, carbon or phosphate sources causes a decrease in cell size. The effect on cell division following three different nutritional shifts-up has been investigated. In all cases, about 20% of the cells divide at the original cell length, and then cell division stops for a period. Cell division then resumes at the new faster rate, cell length at division being characteristic of the new medium. Further investigation reveals that the first effect of the shift is to inhibit nuclear division rapidly and completely. These results are strongly suggestive of the operation of a cell size requirement for entry into nuclear division. The cell size necessary for nuclear division is set, or modulated, by the prevailing growth conditions. This model is confirmed by a nutritional shift-down, where nuclear division and cell division are stimulated after the shift. Cell length at division falls rapidly until the new shorter length is attained, when a new steady state is assumed at a slower growth rate. The control system is compared with that in bacteria, and its implications for various models proposed for the control of timing of mitosis are discussed.     

Pom1 and cell size homeostasis in fission yeast

Cells sense their size and use this information to coordinate cell division with cell growth to maintain a constant cell size within a given population. A model has been proposed for cell size control in the rod-shaped cells of the fission yeast, Schizosaccharomyces pombe. This involves a protein localized to the cell ends, which inhibits mitotic activators in the middle of the cell in a cell size-dependent manner. This protein, Pom1, along with another tip-localized protein, Nif1, have been implicated as direct sensors of cell size controlling the onset of mitosis. Here we have investigated cell size variability and size homeostasis at the G₂/M transition, focusing on the role of pom1 and nif1. Cells deleted for either of these 2 genes show wild-type size homeostasis both in size variability analyses and size homeostasis experiments. This indicates that these genes do not have a critical role as direct cell size sensors in the control mechanism. Cell size homeostasis also seems to be independent of Cdc2–Tyr15 phosphorylation, suggesting that the size sensing mechanism in fission yeast may act through an unidentified pathway regulating CDK activity by an unknown mechanism.     

Isolation of acdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

We have isolated a mutation in the budding yeastSaccharomyces cerevisisae CDC28 gene that allowscdc13 cells, carrying damaged DNA, to continue with the cell division cycle. Whilecdc13 mutant cells are arrested as largebudded cells at the nonpermissive temperature 37‡C, thecdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that thecdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

A unified model for the G1/S cell cycle transition

Efficient S phase entry is essential for development, tissue repair, and immune defences. However, hyperactive or expedited S phase entry causes replication stress, DNA damage and oncogenesis, highlighting the need for strict regulation. Recent paradigm shifts and conflicting reports demonstrate the requirement for a discussion of the G1/S transition literature. Here, we review the recent studies, and propose a unified model for the S phase entry decision. In this model, competition between mitogen and DNA damage signalling over the course of the mother cell cycle constitutes the predominant control mechanism for S phase entry of daughter cells. Mitogens and DNA damage have distinct sensing periods, giving rise to three Commitment Points for S phase entry (CP1-3). S phase entry is mitogen-independent in the daughter G1 phase, but remains sensitive to DNA damage, such as single strand breaks, the most frequently-occurring lesions that uniquely threaten DNA replication. To control CP1-3, dedicated hubs integrate the antagonistic mitogenic and DNA damage signals, regulating the stoichiometric cyclin: CDK inhibitor ratio for ultrasensitive control of CDK4/6 and CDK2. This unified model for the G1/S cell cycle transition combines the findings of decades of study, and provides an updated foundation for cell cycle research.     

An artifact in measurement of S phase initiation and its implication for the kinetics of S phase-specific enzyme activities

It is shown that the different onset of S phase as measured by autoradiography vs cumulative thymidine uptake is an artifact. We consequently propose that S phase-specific enzyme activities may accumulate a few hours prior to the actual initiation of DNA synthesis. A “pre-S” DNA synthesis that can be readily detected only by autoradiography has been proposed. Published data show that DNA synthesis in cultured animal cells is initiated approx. 2 h later when measured by cumulative incorporation of [³H]thymidine ([³H]TdR) as compared with autoradiography. We show here that the difference is in reality an artifact, owing to not taking into account both gradual, asynchronous entry of cells into S phase, as well as time-dependent accumulation of radioactivity into each cell after it has entered S phase. Combination of these two factors leads to the conclusion that [³H]TdR should be incorporated approximately as the square of time following entry of the first cell into S. Taking this into account, the two methods then are in agreement, as predicted. This argument also applies to the enzyme activities shown to increase with DNA synthesis in synchronized cultures. Such an enzyme accumulation really could begin some time earlier than indicated by conventional plots of cumulative enzyme activity vs time and may, in fact, precede the onset of S by a few hours.     

A genome–wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.     

Regulatory system for the G1‐arrest during neuronal development in Drosophila

Neuronal network consists of many types of neuron and glial cells. This diversity is guaranteed by the constant cell proliferation of neuronal stem cells following stop cell cycle re‐entry, which leads to differentiation during development. Neuronal differentiation occurs mainly at the specific cell cycle phase, the G1 phase. Therefore, cell cycle exit at the G1 phase is quite an important issue in understanding the process of neuronal cell development. Recent studies have revealed that aberrant S phase re‐entry from the G1 phase often links cellular survival. In this review we discuss the different types of G1 arrest on the process of neuronal development in Drosophila. We also describe the issue that aberrant S phase entry often causes apoptosis, and the same mechanism might contribute to sensory organ defects, such as deafness.     

The cell cycle thermal-inactivation sensitive stage of Schizosaccharomyces pombe is independent of 2-phenylethanol-induced changes in S phase location

Synchronous cultures of the fission yeast Schizosaccharomyces pombe 972 h⁻¹ are most sensitive to killing by 15 min, 49 °C pulses during a stage stretching from nuclear division through short G1 and S phases to a point early in G2. In this work the cell cycle position of the S phase has been altered by growing the cells in the presence of 2-phenylethanol. The heat sensitivity of these cells was greater at all stages of the cell cycle compared with the cells grown without 2-phenylethanol. However, the position of the most heat sensitive stage in the cell cycle was unaltered. This heat sensitive stage did not include S phase in the cells grown with 2-phenylethanol.     

PROLIFERATION RATE OF HAEMOPOIETIC STEM CELLS AFTER DAMAGE BY SEVERAL CYTOSTATIC AGENTS

The haemopoietic tissue of mice was damaged by different cell-cycle-stage specific and cell-cycle-stage non-specific cytostatic agents. The proliferation rate among the surviving pluripotential stem cells, i.e. those cells forming colonies in spleens of lethally irradiated mice (CFUs), was then investigated. The results suggest that, at least in the CFUs population, the cells which synthesize DNA in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle. This evidence was based on the ability of three cytostatic agents, hydroxyurea, cytosine arabinoside and methotrexate, which are toxic specifically to the S phase cells to increase the proliferation in the CFUs population. This increase was quite out of proportion to the small amount of damage they caused to the population. Colchicine, which kills cells in mitosis, and ionizing irradiation, damaging cells in all stages, proved to be much weaker stimulators of proliferation. It has been suggested that a mechanism for the control of cellular proliferation might be based on the negative feedback in the cell cycle. In this feedback control loop the cells which are preparing for cell division in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle.     

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G₁, DNA synthesis, S, the second growth stage, G₂, mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4′, 6-diamidino-2-phenylindole dichloride.

Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G_q, an hypothesized side loop from G₁. All morphological stages, not only division, appear to be phased by the circadian clock.

     

Countercurrent distribution and multiple sedimentation of Chlorella pyrenoidosa during its life cycle

Synchronously and normally grown Chlorella pyrenoidosa cell populations were analysed by countercurrent distribution in aqueous two-polymer phase systems and by a multiple sedimentatation technique. Partition of cells in aqueous phases reflects the surface properties of cells (primarily surface charge) and multiple sedimentation reflects the cells' size-density parameters. It was found that:

1. 1. Synchronized cells that have just divided have the lowest partition of any in the population. Surface charge (as reflected by partition) increases with time after cell division. Cells have the highest partition just prior to division.

2. 2. Synchronized cells that have just divided are the smallest of any in the population. Since size and sedimentation rate increase with time after cell division multiple sedimentation permits the separation of cells of different ages.

3. 3. Both countercurrent distribution and multiple sedimentation studies reveal considerable heterogeneity of synchronized Chlorella populations. The increase in both surface charge and size with cell age does not appear to proceed in a continuous fashion. Rather, it seems to go in a stepwise manner.

4. 4. Non-synchronized cells examined by either countercurrent distribution or multiple sedimentation show two distinct sub-populations. One of these corresponds to the youngest, just divided cells; and the other to cells just prior to cell division. It is suggested that a lag time just prior to cell division and just after cell division explains these results.

5. 5. Countercurrent distribution in two-polymer phases and multiple sedimentation at unit gravity in a suspension medium best suited for the cell under investigation seem to be methods of choice for tracing cell changes during division, maturation and aging and for sub-fractionating such cell populations.