The dynamics of cell cycle regulation

Major events of the cell cycle--DNA synthesis, mitosis and cell division-are regulated by a complex network of protein interactions that control the activities of cyclin-dependent kinases. The network can be modeled by a set of nonlinear differential equations and its behavior predicted by numerical simulation. Computer simulations are necessary for detailed quantitative comparisons between theory and experiment, but they give little insight into the qualitative dynamics of the control system and how molecular interactions determine the fundamental physiological properties of cell replication. To that end, bifurcation diagrams are a useful analytical tool, providing new views of the dynamical organization of the cell cycle, the role of checkpoints in assuring the integrity of the genome, and the abnormal regulation of cell cycle events in mutants. These claims are demonstrated by an analysis of cell cycle regulation in fission yeast.     

Shaping Fission Yeast with Microtubules

For cell morphogenesis, the cell must establish distinct spatial domains at specified locations at the cell surface. Here, we review the molecular mechanisms of cell polarity in the fission yeast Schizosaccharomyces pombe. These are simple rod-shaped cells that form cortical domains at cell tips for cell growth and at the cell middle for cytokinesis. In both cases, microtubule-based systems help to shape the cell by breaking symmetry, providing endogenous spatial cues to position these sites. The plus ends of dynamic microtubules deliver polarity factors to the cell tips, leading to local activation of the GTPase cdc42p and the actin assembly machinery. Microtubule bundles contribute to positioning the division plane through the nucleus and the cytokinesis factor mid1p. Recent advances illustrate how the spatial and temporal regulation of cell polarization integrates many elements, including historical landmarks, positive and negative controls, and competition between pathways.One of the ultimate goals in cell biology is to understand how cells are assembled. As in the development of multicellular organisms, single cells need to form distinct spatial domains with specific form, structure, and functions. How do cells organize themselves in space to form a specific shape and size?The fission yeast Schizosaccharomyces pombe is an attractive, simple unicellular model organism for studying cell morphogenesis. These are nonmotile cells with highly invariant shape 8–14 µm long and 3 µm in diameter. The relative simplicity of the cells and the powers of genetic approaches and live cell imaging facilitate rigorous and quantitative studies.Here, we review the current understanding of spatial regulation in fission yeast. The cell defines distinct cortical domains at each of the cell tips, along the sides of cells, and at the cell division plane. Each cortical domain is characterized by different sets of molecules, which impart distinct functions. In particular, as it proceeds through its cell cycle, the cell delineates distinct actin-rich cortical regions at cell tips for polarized cell growth and at the middle for cell division. In both cases, a self-organizing network of microtubules directly or indirectly contributes to the proper localization of these markers. In cell polarity, microtubule ends transport polarity factors to the plasma membrane, where they function to recruit protein complexes involved in actin assembly. In cytokinesis, a medial cortical site is marked by an interacting system of microtubules, the nucleus, and cell tip factors, and functions to organize actin filaments into a cytokinetic ring. This reliance on microtubules contrasts with polarity mechanisms in budding yeast in which spatial cues are dependent on septins and actin, but not microtubules. As many of these processes involve conserved proteins, this work in fission yeast contributes toward understanding the more complex microtubule-based regulation of cell migration, cytokinesis, and cell shape regulation in animal cells. This work in fission yeast thus provides a paradigm for how a self-organizing system can shape a cell.     

Estimating the Stochastic Bifurcation Structure of Cellular Networks

High throughput measurement of gene expression at single-cell resolution, combined with systematic perturbation of environmental or cellular variables, provides information that can be used to generate novel insight into the properties of gene regulatory networks by linking cellular responses to external parameters. In dynamical systems theory, this information is the subject of bifurcation analysis, which establishes how system-level behaviour changes as a function of parameter values within a given deterministic mathematical model. Since cellular networks are inherently noisy, we generalize the traditional bifurcation diagram of deterministic systems theory to stochastic dynamical systems. We demonstrate how statistical methods for density estimation, in particular, mixture density and conditional mixture density estimators, can be employed to establish empirical bifurcation diagrams describing the bistable genetic switch network controlling galactose utilization in yeast Saccharomyces cerevisiae. These approaches allow us to make novel qualitative and quantitative observations about the switching behavior of the galactose network, and provide a framework that might be useful to extract information needed for the development of quantitative network models.     

Central roles of small GTPases in the development of cell polarity in yeast and beyond.

Summary: The establishment of cell polarity is critical for the development of many organisms and for the function of many cell types. A large number of studies of diverse organisms from yeast to humans indicate that the conserved, small-molecular-weight GTPases function as key signaling proteins involved in cell polarization. The budding yeast Saccharomyces cerevisiae is a particularly attractive model because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of S. cerevisiae undergo polarized growth during various phases of their life cycle, such as during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon deprivation of nutrition such as nitrogen. Substantial progress has been made in deciphering the molecular basis of cell polarity in budding yeast. In particular, it becomes increasingly clear how small GTPases regulate polarized cytoskeletal organization, cell wall assembly, and exocytosis at the molecular level and how these GTPases are regulated. In this review, we discuss the key signaling pathways that regulate cell polarization during the mitotic cell cycle and during mating.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VIII. Genes for PI3K signaling and cell cycle

Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.     

Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.     

Silence is golden: combining RNAi and live cell imaging to study cell cycle regulatory genes during Caenorhabditis elegans development

Much of the pioneering work on the genetics of cell cycle regulation was accomplished using budding and fission yeast. The relative simplicity of these single-celled organisms allowed investigators to readily identify and assign roles to individual genes. While the molecular mechanisms worked out in yeast are more or less identical to those operating in higher organisms, additional layers of control must exist in multicellular organisms to coordinate the timing of developmental events occurring in different cells and tissues. Here we discuss experimental approaches for studying cell cycle processes in the nematode Caenorhabditis elegans.     

When plant cells decide to divide

Progression through the cell cycle is central to cell proliferation and fundamental to the growth and development of all multicellular organisms, including higher plants. The periodic activation of complexes containing cyclins and cyclin-dependent kinases mediates the temporal regulation of the cell-cycle transitions. Here, we highlight recent advances in the molecular controls of the cell cycle in plant cells, with special emphasis on how hormonal signals can modulate the regulation of cyclin-dependent kinases.     

Cell-cycle-regulatory elements and the control of cell differentiation in the budding yeast

The stable differentiation of cells into other cell types typically involves dramatic reorganization of cellular structures and functions. This often includes remodeling of the cell cycle and the apparatus that controls it. Here we review our understanding of the role and regulation of cell cycle control elements during cell differentiation in the yeast, Saccharomyces cerevisiae. Although the process of differentiation may be more overtly obvious in metazoan organisms, those systems are by nature more difficult to study at a mechanistic level. We consider the relatively well-understood mechanisms by which mating-type switching and the pheromone-induced differentiation of gametes are coupled to the cell cycle as well as the more obscure mechanisms that govern the remodeling of the cell cycle during meiosis and filamentous growth. In some cases, the cell cycle is a primary stimulus for differentiation whereas, in other cases, the signals that promote differentiation alter the cell cycle. Thus, despite relative simplicity of these processes in yeast, the nature of the interplay between the cell cycle and differentiation is diverse.     

The RAM network in pathogenic fungi

The regulation of Ace2 and morphogenesis (RAM) network is a protein kinase signaling pathway conserved among eukaryotes from yeasts to humans. Among fungi, the RAM network has been most extensively studied in the model yeast Saccharomyces cerevisiae and has been shown to regulate a range of cellular processes, including daughter cell-specific gene expression, cell cycle regulation, cell separation, mating, polarized growth, maintenance of cell wall integrity, and stress signaling. Increasing numbers of recent studies on the role of the RAM network in pathogenic fungal species have revealed that this network also plays an important role in the biology and pathogenesis of these organisms. In addition to providing a brief overview of the RAM network in S. cerevisiae, we summarize recent developments in the understanding of RAM network function in the human fungal pathogens Candida albicans, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Pneumocystis spp.     

Central Roles of Small GTPases in the Development of Cell Polarity in Yeast and Beyond

Summary: The establishment of cell polarity is critical for the development of many organisms and for the function of many cell types. A large number of studies of diverse organisms from yeast to humans indicate that the conserved, small-molecular-weight GTPases function as key signaling proteins involved in cell polarization. The budding yeast Saccharomyces cerevisiae is a particularly attractive model because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of S. cerevisiae undergo polarized growth during various phases of their life cycle, such as during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon deprivation of nutrition such as nitrogen. Substantial progress has been made in deciphering the molecular basis of cell polarity in budding yeast. In particular, it becomes increasingly clear how small GTPases regulate polarized cytoskeletal organization, cell wall assembly, and exocytosis at the molecular level and how these GTPases are regulated. In this review, we discuss the key signaling pathways that regulate cell polarization during the mitotic cell cycle and during mating.     

Clonal heterogeneity in specific growth rate of Succhuromyces cerevisiue cells

The cell volume increase in individual clones of cells of the yeast Saccharomyces cerevisiae has been measured using time lapse cinematography in populations showing steady state balanced exponential growth. There were significant differences in clonal specific growth rates within the population in each of 10 experiments using different strains on different media supporting different growth rates. The results suggest that specific growth rates of cells which are either genetically identical or very closely related can be different and this difference can be propagated over at least three generations. Since the proliferation rate in yeast is determined by growth rate, these observed differences provide an additional source of cell cycle variability for yeast cells that has not been considered before. The implications for the theoretical analysis of cell cycle kinetics are examined.     

Prospects of yeast systems biology for human health: integrating lipid, protein and energy metabolism

The yeast Saccharomyces cerevisiae is a widely used model organism for studying cell biology, metabolism, cell cycle and signal transduction. Many regulatory pathways are conserved between this yeast and humans, and it is therefore possible to study pathways that are involved in disease development in a model organism that is easy to manipulate and that allows for detailed molecular studies. Here, we briefly review pathways involved in lipid metabolism and its regulation, the regulatory network of general metabolic regulator Snf1 (and its human homologue AMPK) and the proteostasis network with its link to stress and cell death. All the mentioned pathways can be used as model systems for the study of homologous pathways in human cells and a failure in these pathways is directly linked to several human diseases such as the metabolic syndrome and neurodegeneration. We demonstrate how different yeast pathways are conserved in humans, and we discuss the possibilities of using the systems biology approach to study and compare the pathways of relevance with the objective to generate hypotheses and gain new insights.     

Mathematical Model for Growth Regulation of Fission Yeast Schizosaccharomyces pombe

Regulation of polarised cell growth is essential for many cellular processes including spatial coordination of cell morphology changes during the division cycle. We present a mathematical model of the core mechanism responsible for the regulation of polarised growth dynamics during the fission yeast cell cycle. The model is based on the competition of growth zones localised at the cell tips for a common substrate distributed uniformly in the cytosol. We analyse the bifurcations in this model as the cell length increases, and show that the growth activation dynamics provides an explanation for the new-end take-off (NETO) as a saddle-node bifurcation at which the cell sharply switches from monopolar to bipolar growth. We study the parameter sensitivity of the bifurcation diagram and relate qualitative changes of the growth pattern, e.g. delayed or absent NETO, to previously observed mutant phenotypes. We investigate the effects of imperfect asymmetric cell division, and show that this leads to distinct growth patterns that provide experimentally testable predictions for validating the presented competitive growth zone activation model. Finally we discuss extension of the model for describing mutant cells with more than two growth zones.     

Modeling and stochastic simulation of the Ras/cAMP/PKA pathway in the yeast Saccharomyces cerevisiae evidences a key regulatory function for intracellular guanine nucleotides pools

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of metabolism and cell cycle progression. The pathway is tightly regulated by several control mechanisms, as the feedback cycle ruled by the activity of phosphodiesterase. Here, we present a discrete mathematical model for the Ras/cAMP/PKA pathway that considers its principal cytoplasmic components and their mutual interactions. The tau-leaping algorithm is then used to perform stochastic simulations of the model. We investigate this system under various conditions, and we test how different values of several stochastic reaction constants affect the pathway behaviour. Finally, we show that the level of guanine nucleotides, GTP and GDP, could be relevant metabolic signals for the regulation of the whole pathway.     

Drf1, a novel regulatory subunit for human Cdc7 kinase

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.     

Translational control by 80S formation and 60S availability: The central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis

Ribosome biogenesis and translation can be simplified as the processes of generating ribosomes and their use for decoding mRNA into a protein. Ribosome biogenesis has been efficiently studied in unicellular organisms like the budding yeast, allowing us a deep and basic knowledge of this process in growing cells. Translation has been modeled in vitro and in unicellular organisms. These studies have given us an important insight into the mechanisms and evolutionarily conserved aspects of ribosome biology. However, we advocate the need of the direct study of these processes in multicellular organisms. Analysis of ribosome biogenesis and translation in vivo in Metazoa and mammalian models is emerging and unveils the unexpected consequences of perturbed ribosome biogenesis and translation. Here, we will describe how one factor, eIF6, plays a crucial role both in the generation of the large ribosomal subunit and its availability for translation. From there, we will make specific conclusions on the physiological relevance of eIF6 in 80S formation, cell cycle progression and disease, raising the point that the control of gene expression may occur at the unexpected level of the large ribosomal subunit. In the future, the modulation of eIF6 binding to the 60S may be pharmacologically exploited to reduce the growth of cancer cells or ameliorate the phenotype of SDS syndrome.