Commitment to the mitotic cell cycle in yeast in relation to meiosis

Under restrictive vegetative conditions, cells of cell-division cycle (cdc) temperature-sensitive mutants arrest at specific points in the cycle. Meiotic and mitotic behaviour of such arrested cells was examined under permissive sporulation conditions. Those mutants which were committed to mitosis at their specific point of arrest finished the cell cycle and could only then go into meiosis. It was found that commitment to mitosis occurred early in the cell cycle, prior to DNA replication, and that this commitment was dependent upon the gene function of cdc4.     

Logical analysis of the budding yeast cell cycle

The budding yeast Saccharomyces cerevisiae is a model organism that is commonly used to investigate control of the eukaryotic cell cycle. Moreover, because of the extensive experimental data on wild type and mutant phenotypes, it is also particularly suitable for mathematical modelling and analysis. Here, I present a new Boolean model of the budding yeast cell cycle. This model is consistent with a wide range of wild type and mutant phenotypes and shows remarkable robustness against perturbations, both to reaction times and the states of component genes/proteins. Because of its simple logical nature, the model is suitable for sub-network analysis, which can be used to identify a four node core regulatory circuit underlying cell cycle regulation. Sub-network analysis can also be used to identify key sub-dynamics that are essential for viable cell cycle control, as well as identifying the sub-dynamics that are most variable between different mutants.     

The spindle cycle in budding yeast

The mitotic spindle of the budding yeast Saccharomyces cerevisiae will probably be the first such organelle to be understood in molecular detail. Here we describe the mitotic spindle cycle of budding yeast using electron-microscope-derived structures and dynamic live-cell imaging. Recent work has revealed that many general aspects of mitosis are conserved, making budding yeast an excellent model for the study of mitosis.     

蛋白质的磷酸化作用和泛肽化降解作用与芽殖酵母细胞周期的调控

在芽殖酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）细胞中，Ｇ１期的三种ｃｙｃｌｉｎｓ和Ｓ、Ｍ期的五种ｃｙｃｌｉｎｓ之周期性的合成和分解调节着Ｃｄｃ２８的活性，驱动细胞周期的正常运转。除了ＣＤＫ的磷酸化作用外，蛋白质的泛肽化降解作用间接或直接调控细胞周期：ＣＤＣ３４泛肽化途径通过降解Ｃｄｃ２８的专一抑制子而起始ＤＮＡ复制；ＡＰＣ泛肽化途径通过降解Ｍ期后期的抑制子和Ｍ期ｃｙｃｌｉｎｓ，使姐妹染色体分离和Ｍ期终止。     

Stage-dependent density effect in the cell cycle of budding yeast

Yeasts in culture media grow exponentially in early period but eventually stop growing. The saturation of population growth is due to "density effect". The budding yeast, Saccharomyces cerevisiae, is known to exhibit a stage-dependent cell division. Daughter cell, which gives no birth, has longer generation time than mother, because daughter needs maturity time. So far, investigations have been restricted in exponential or non-crowding state; very little is known for the stage dependence of density effect. Here we present a lattice gas model to explore the population dynamics of crowding period. We compare theoretical results with experimental data, and find a stage-dependent density effect. Although small daughter cells can develop to a critical size, the reproduction of large daughter cells suddenly stops when the total density exceeds some critical level. Our results imply the existence of an inhibitor that specifically halts the reproduction of matured daughter cell.     

Integrative analysis of cell cycle control in budding yeast

The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo.     

A cell cycle checkpoint monitors cell morphogenesis in budding yeast

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.     

Improved flow cytometric analysis of the budding yeast cell cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.     

Effect of tunicamycin on cell cycle progression in budding yeast

Tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharides, prevents bud formation and growth in Saccharomyces cerevisiae cells that are either growing exponentially or recovering from different cell cycle arrests at start. Analysis of tunicamycin-treated cells by flow microfluorometry clearly shows that cells have a postsynthetic DNA content, but there is no evidence of an increase in binucleate cells. Therefore tunicamycin affects bud emergence and initiation of DNA synthesis, two events correlated under physiological conditions, in different ways. A bulk glycoprotein synthesis is shown to be required for bud emergence and localized chitin deposition, probably to sustain directional secretory vesicle transport, which allows polar growth of the bud. No evidence for a glycoprotein requirement for entrance into the S phase is obtained from the present experiments.     

Phosphoproteome dynamics during mitotic exit in budding yeast

The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin‐dependent kinase (Cdk) activity reaches its peak, the anaphase‐promoting complex/cyclosome (APC/C) is activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk‐counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3,456 phosphosites on 1,101 proteins with high temporal resolution as cells progress synchronously through mitosis. This reveals that successive inactivation of S and M phase Cdks and of the mitotic kinase Polo contributes to order these dephosphorylation events. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. These findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases contribute to order both protein dephosphorylation and phosphorylation.     

Altered states: programmed proteolysis and the budding yeast cell cycle

The recent identification of an essential RING-H2 finger protein in the SCF E3 ubiquitin ligase complex of budding yeast has uncovered a family of related E3 enzymes, including the other main cell cycle E3 complex, the anaphase promoting complex (APC). Recent insights into APC-dependent proteolysis include a novel protease activity that dissolves cohesion between sister chromatids at anaphase, and a crucial phosphatase, Cdc14, whose release from the nucleolus eliminates cyclin-dependent kinase activity and thereby drives exit from mitosis.     

Fission yeast genes which disrupt mitotic chromosome segregation when overexpressed.

An interference assay has been devised in Schizosaccharomyces pombe to rapidly identify and clone genes involved in chromosome segregation. Random S.pombe cDNAs were overexpressed from an inducible promoter in a strain carrying an additional, non-essential minichromosome. Overexpression of cDNAs derived from four genes, two known (nda3+and ubc4+, encoding beta-tubulin and a ubiquitin conjugating enzyme, respectively) and two unknown, named mlo2+ and mlo3+ (missegregation & lethal when over expressed) caused phenotypes consistent with a failure to segregate chromosomes. Full overexpression of all four cDNAs was lethal. Cells overexpressing nda3+ and ubc4+ cDNAs arrested with condensed unsegregated chromosomes and cells overexpressing mlo2+ displayed an asymmetric distribution of nuclear chromatin. Sublethal levels of overexpression of nda3+, ubc4+ and mlo2+ cDNAs caused elevated rates of minichromosome loss. A third cDNA mlo3+, displayed no increase in the frequency of minichromosome loss at sublethal levels of overexpression but full overexpression caused a complete failure to segregate chromosomes. Our results confirm the assumption that beta-tubulin overexpression is lethal in S.pombe, implicate ubc4+ in the control of metaphase-anaphase transition in fission yeast and finally identify two new genes, mlo2+and mlo3+, likely to play an important role for chromosome transmission fidelity in mitosis.