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.     

The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.     

Cell-cycle-specific initiation of replication

The following characteristics are relevant when replication of chromosomes and plasmids is discussed in relation to the cell cycle: the timing or replication, the selection of molecules for replication, and the coordination of multiple initiation events within a single cell cycle. Several fundamentally different methods have been used to study these processes: Meselson—Stahl density-shift experiments, experiments with the so-called‘baby machine', sorting of cells according to size, and flow cytometry. The evidence for precise timing and co-ordination of chromosome replication in Escherichia coli is overwhelming. Similarly, the high-copy-number plasmid ColE1 and the low-copy-number plasmids R1/R100 without any doubt replicate randomly throughout the cell cycle. Data about the low-copy-number plasmids F and P1 are conflicting. This calls for new types of experiments and for a better understanding of how these plasmids control their replication and partitioning.     

Timing of micronuclear mitosis and its relation to commitment to division inParamecium tetraurelia

This study examines the timing of micronuclear mitosis during the vegetative cell cycle and shows that mitosis begins early in the division process and coincides approximately with the earliest stages of oral morphogenesis (about 0.6 in the cell cycle in synchronous cell samples). The cc1 mutation blocks cell cycle progression prior to the point of commitment to division. Although the cc1 mutation blocks macronuclear DNA synthesis under restrictive conditions, it does not block micronuclear DNA synthesis. However, absence of functional cc1 gene product leads to blockage of micronuclear mitosis prior to completion of anaphase. This point coincides with commitment to division and is also the point at which oral morphogenesis is blocked in cc1 cells. The tim-ings of the transition points for micronuclear mitosis and oral morphogenesis in cc1 cells are closely associated in both synchronous cell samples and in asynchronous cultures. © 1992 Wiley-Liss, Inc.     

Gene conversion at different points in the mitotic cycle of Saccharomyces cerevisiae

Summary In the Saccharomyces cerevisiae mitotic cycle, the timing of radiation-induced gene conversion has been studied using thermosensitive cell division cycle mutants. The cells were found to perform conversion at different G1 or post-replication steps. A lower yield in induction is found during the G2 phase and is explained by the competition for recombinational repair between sister chromatids and homologous chromosomes. The results are discussed in relation to repair.     

Expanded progenitor populations, vitreo-retinal abnormalities, and Müller glial reactivity in the zebrafish leprechaun/patched2 retina

Background  

The roles of the Hedgehog (Hh) pathway in controlling vertebrate retinal development have been studied extensively; however, species- and context-dependent findings have provided differing conclusions. Hh signaling has been shown to control both population size and cell cycle kinetics of proliferating retinal progenitors, and to modulate differentiation within the retina by regulating the timing of cell cycle exit. While cell cycle exit has in turn been shown to control cell fate decisions within the retina, a direct role for the Hh pathway in retinal cell fate decisions has yet to be established in vivo.     

The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1

We previously identified a tight bidirectional phase coupling between the circadian clock and the cell cycle. To understand the role of the CLOCK/BMAL1 complex, representing the main positive regulator of the circadian oscillator, we knocked down Bmal1 or Clock in NIH3T3^3C mouse fibroblasts (carrying fluorescent reporters for clock and cell cycle phase) and analyzed timing of cell division in individual cells and cell populations. Inactivation of Bmal1 resulted in a loss of circadian rhythmicity and a lengthening of the cell cycle, originating from delayed G2/M transition. Subsequent molecular analysis revealed reduced levels of Cyclin B1, an important G2/M regulator, upon suppression of Bmal1 gene expression. In complete agreement with these experimental observations, simulation of Bmal1 knockdown in a computational model for coupled mammalian circadian clock and cell cycle oscillators (now incorporating Cyclin B1 induction by BMAL1) revealed a lengthening of the cell cycle. Similar data were obtained upon knockdown of Clock gene expression. In conclusion, the CLOCK/BMAL1 complex controls cell cycle progression at the level of G2/M transition through regulation of Cyclin B1 expression.     

Timing of oral morphogenesis and its relation to commitment to division in Paramecium tetraurelia

The interval between commitment to division and fission in synchronous cell samples is a constant fraction of the cell cycle (0.2) in cell cycles up to 6.5 h in duration. In longer cell cycles this interval has a fixed duration of about 80 min. The point of commitment to division is associated with the six-rowed anlage stage of oral primordium development (stage V). At this stage cells carrying the cc1 mutation are not blocked by transfer to restrictive conditions but rather proceed to division. Stage V is also the stabilization point for oral anlagen. When shifted to restrictive conditions prior to this stage, development is arrested and resorption of anlagen is initiated. The cc1 mutation also blocks contractile vacuole duplication and migration under restrictive conditions. The cc1 gene function is required continuously prior to the transition point. The timing of morphogenetic stages in asynchronous cells is roughly similar to that in synchronous cells. There are, however, significant differences in timing as estimated by the two experimental procedures.     

Divide and differentiate

The elegant choreography of metazoan development demands exquisite regulation of cell-division timing, orientation, and asymmetry. In this review, we discuss studies in Drosophila and C. elegans that reveal how the cell cycle machinery, comprised of cyclin-dependent kinase (CDK) and cyclins functions as a master regulator of development. We provide examples of how CDK/cyclins: (1) regulate the asymmetric localization and timely destruction of cell fate determinants; (2) couple signaling to the control of cell division orientation; and (3) maintain mitotic zones for stem cell proliferation. These studies illustrate how the core cell cycle machinery should be viewed not merely as an engine that drives the cell cycle forward, but rather as a dynamic regulator that integrates the cell-division cycle with cellular differentiation, ensuring the coherent and faithful execution of developmental programs.     

Cell cycle inHelianthus tuberosus tuber tissue in relation to dormancy

Summary Observations are presented on the patterns of DNA synthesis and mitotic activity in medullary parenchyma cells excised from tubers ofHelianthus tuberosus in four different periods of dormancy. Dormancy break (activation) was induced byin vitro culture on media added with 2,4-dichlorophenoxyacetic acid. The cell cycle responsein vitro to different combinations of growth substances has also been investigated.The results show that remarkable changes in the timing of the first and second cell cycles and their phases occur with the progression of dormancy. With increasing time after tuber harvest, the following behaviours are observed: (i) a lengthening of the first cell cycle, chiefly due to a lengthening of the G₂ phase (G₂ is absent at the beginning of dormancy) and an increase in the time interval between the start of thein vitro culture and the onset of the first mitotic wave; (ii) an increased duration of the S phase; (iii) a remarkable reduction in the cell synchrony.These behaviours, as indicated also by their comparison with thein vitro response of the cell cycle to different hormonal treatments, seem to depend on the physiological status of the tubers at the time of explant. It is concluded that the analysis of the cell cycle is an useful tool for understanding some aspects of such a complex physiological situation as dormancy.Istituto di Mutagenesi e Differenziamento del C.N.R., Pisa, Italy, publication no. 321.     

The application of mathematical modelling to aspects of adjuvant chemotherapy scheduling

In this paper simple models for tumour growth incorporating age-structured cell cycle dynamics are considered in the presence of two non-cross-resistant S-phase specific chemotherapeutic drugs. According to the seminal work of Goldie and Coldman, if one cannot deliver two cell cycle phase non-specific, non-cross-resistant drugs simultaneously, for example due to toxicity, and both drugs are identical apart from resistance, one should alternate their delivery as rapidly as possible. However consider S-phase specific drugs. One might speculate that, for example, alternating the two drugs at intervals of T, where T is the mean cell cycle time, is better than alternating the drugs at intervals of T/2, as the latter strategy allows the possibility of a cell cycle sanctuary. Such speculation implicitly requires a sufficiently low variance of the cell cycle time, and hence it is not clear if such reasoning prevents a generalisation of the results of Goldie and Coldman. This question is addressed in this paper via a detailed modelling investigation, as motivated by suggestions for future colorectal adjuvant chemotherapy trials and developments in hepatic arterial infusion technology. It is shown that the cell cycle distribution of the resistant cell populations is strongly influenced by the chemotherapy schedule. The consequences of this can be dramatic, and can lead to chemotherapy failure at resonant chemotherapy timings, especially for a small standard deviation of the cell cycle time. The novel aspects of this observation are highlighted compared to other models in the literature exhibiting resonant behaviour in the timing of a periodic chemotherapy protocol. The above investigation also results in the principal prediction of this paper that reducing the drug alternation time to approximately a few hours, if possible, can result in substantial improvements in predicted chemotherapy outcomes. Critically, such improvements are not predicted by the Goldie Coldman model or other chemotherapy scheduling models in the literature.     

THE NUCLEAR CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYCEAE)1

The timing of replication and division of the Chlamydomonas Ehrenberg nucleus in the vegetative cell cycle and at gametogenesis was examined, using fluorescence microspectrophotometry with two fluorochromes, mithramycin and 4′,6-diamidino-2-phenylindole (DAPI). Under appropriate conditions, these bind specifically to DNA, and the fluorescence of the DNA fluorochrome complex is a quantitative measure of the DNA content. The alga is a haplont, which produces 2ⁿ daughter cells at the time of vegetative reproduction; cytokinesis and daughter cell release lag behind karyokinesis. No nucleus was found to contain more than the 2c quantity of DNA. Hence daughter cell production proceeds by doubling of the nuclear DNA followed by karyokinesis, in a repetitive sequence. As reported previously for C. reinhardtii Dangeard, the gametes of C. moewusii Gerloff contain the 1c amount of nuclear DNA. Several conflicting interpretations of the cell cycle sequence proposed in the literature were resolved.     

INTERACTIONS OF LIGHT/DARK CYCLES AND NITROGEN PULSES ON THE TIMING OF CELL DIVISION IN THE NITROGEN-LIMITED MARINE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)1

Cell division in most eukaryotic algae grown on alternating periods of light and dark (LD) is synchronized or phased so that cell division occurs only during a restricted portion of the LD cycle. However, the phase angle of the cell division gate, the time of division relative to the beginning of the light period, is known to be affected by growth conditions such as nutrient status and temperature. In this study, it is shown that the phase angle of cell division in a diatom, Cylindrotheca fusiformis Reimann and Lewin, is affected by the N-limited growth rate; cell division occurred later in the dark period (12:12 h LD cycle) when the growth rate was infradian (D = 0.42 d^?1) than when it was ultradian (D = 1.0 d^?1). Nitrogen-pulses did not affect the phase angle of the division gate, but could shift the time of peak cell division activity within the division gate. The effects, if any, of N-pulses were dependent upon the growth rate and the time of day that the pulses were administered. These responses indicate that the timing of cell division in this diatom is not determined solely by the zeitgeber from the LD cycle, but rather that a LD cycle control mechanism and a N-mediated control mechanism are both involved and are somewhat interdependent. In addition, an increase in protein was observed immediately after administering a N-pulse to C. fusiformis in the ultradian growth mode indicating that the accumulation of protein can be uncoupled from the cell division cycle.     

Hormonal Control of Gene Expression During Reactivation of the Cell Cycle in Tobacco Mesophyll Protoplasts

The roles of auxin and cytokinin in cell cycle reactivation were studied during the first 48 h of culture of mesophyll protoplasts of Nicotiana tabacum. Using hormone delay and withdrawal studies we found that auxin was required by 0–4 h of culture, whereas cytokinin was not required until hour 10–12, which is 6–10 h before S phase. Cycloheximide blocks division, indicating that protein synthesis is required. In an effort to detect a molecular response to either hormone, we examined the expression of the cell cycle marker, cdc2. Cdc2 expression was detected by 12 h of culture, coincident with the timing of the cytokinin requirement and well before the entry into S. However, cdc2 was partially induced by either auxin or cytokinin alone, suggesting that cdc2 expression is not the primary target of either hormone. Our hormone delay experiments suggest that there are separate signal transduction pathways leading from auxin and from cytokinin to reactivation of the cell cycle and that these pathways converge before S. The underlying mechanisms for these distinct pathways remain to be elucidated. Received November 4, 1997; accepted October 7, 1998     

EFFECTS OF PHOTOCYCLES AND PERIODIC AMMONIUM SUPPLY ON THREE MARINE PHYTOPLANKTON SPECIES. II. AMMONIUM UPTAKE AND ASSIMILATION1

The relative influence of the photoperiod and of periodic ammonium pulses in entraining the cell division cycle in nitrogen-limited cyclostat cultures differs dramatically in Hymenomonas carterae Braarud and Fagerl, Amphidinium carteri Hulburt and Thalassiosira weissflogii Grun. We examined how each species processes an NH₄⁺ pulse at various times during the cell cycle and the L/D cycle. Rates of NH₄⁺ uptake and changes in cellular concentrations of NH₄⁺, free amino acids, and protein were examined after the addition of an NH₄⁺ pulse. Depletion of NH₄⁺ from the medium occurred earlier when the pulse was given at the beginning of the light period than at the beginning of the dark period in H. carterae and A. carteri. Depletion took longer in the T. weissflogii cultures and the kinetics were similar during both stages of the photocycle in this species. Similarly, the temporal phasing and maximum pool sizes varied with timing of the NH₄⁺ pulse in H. carterae and A. carteri but complete assimilation was relatively rapid. More persistent pools of NH₄⁺ and free amino acids accumulated in T. weissflogii, and the patterns of assimilation varied little as a function of the timing of the pulse with respect to the photocycle. Although nitrogen metabolism occurred rapidly in nitrogen-limited H. carterae and A. carteri, the entrainment of the cell division cycle by the photoperiod resulted in a large degree of uncoupling between completion of nitrogen assimilation and cell division. It is hypothesized that the strong entrainment of the cell division cycle of T. weissflogii by NH₄⁺ pulses results from a relatively slow rate of nitrogen metabolism.     

INFLUENCE OF ENVIRONMENTAL FACTORS AND POPULATION COMPOSITION ON THE TIMING OF CELL DIVISION IN THALASSIOSIRA FLUVIATILIS (BACILLARIOPHYCEAE) GROWN ON LIGHT/DARK CYCLES1

Cell division patterns in Thalassiosira fluviatilis grown in a cyclostat were analyzed as a function of temperature, photoperiod, nutrient limitation and average cell size of the population. Typical cell division patterns in populations doubling more than once per day had multiple peaks in division rate each day, with the lowest rates always being greater than zero. Division bursts occurred in both light and dark periods with relative intensities depending on growth conditions. Multiple peaks in division rate were also found, when population growth rates were reduced to less than one doubling per day by lowering temperature, nutrients, or photoperiod and the degree of division phasing was not enhanced. Temperature and nutrient limitation shifted the timing of the major division burst relative to the light/dark cycle. Average cell volume of the inoculum was found to be a significant determinant of the average population growth rate and the timing and magnitude of the peaks in division rate. The results are interpreted in the context of a cell cycle model in which generation times are “quantized” into values separated by a constant time interval.