Period Of Cell Cycle Research Articles

Meiotic and Mitotic Origin of Aneuploidies Detected in Preimplantation Genetic Testing

Over 90% of aneuploidies originate from maternal meiosis, with the only established cause of aneuploidy being maternal age. So it is not surprising that the reproductive age is the major indication for preimplantation genetic testing for chromosomal aneuploidy (PGT-A), which allows couples with advanced reproductive age to achieve their reproductive goals without risk for fetal loss or birth of an affected child. This is achieved through detecting and avoiding from transfer a considerable proportion of embryos with different abnormalities, which according to our experience is as high as 56.9% (1661 of 2922 blastocysts samples tested by NGS). Of these abnormalities, 8% (133 of 1661) were with mosaicism and 14.4% (240/1661) were found to have segmental chromosome abnormalities. Of these, 63.3% (154/240) were found to have one deletion, 8.3% (20/240) one duplication and 28.4% (66/240) multiple segmental abnormalities. The smallest abnormality identified was 5.8 Mb, while the largest was 139.6 MB. The p to q arm ratio of these abnormalities was found to be 1.03. Mosaicism of segmental abnormalities was observed in 59% of samples identified. Segmental chromosome abnormalities were most commonly found in chromosomes 1 (32/240; 13.3%), 5 (22/240; 9/2%), and 4 (21/240; 8.8%). No chromosome 22 or Y chromosome abnormalities were identified in this cohort. Only three of these segmental anomalies were of meiotic origin, detected also in parents, while others were de novo events, of which biological and clinical significance is unknown. These are clearly post-meiosis errors, but their impact cannot be evaluated. It cannot be excluded that they are technical errors, or chromosome specific differences of completion of S period of cell cycle. Difference in the replication pattern of certain chromosomes were demonstrated long time ago, depending on maternal and paternal origin of homologous chromosomes and on differences of cell type tested, so segmental errors may reflect a tissue specific differences of DNA replication. Technical errors cannot be excluded either in detection of mosaicism, as it may be affected by chromosome drifting caused by cell damage during blastocyst biopsy, or by the choice of methodology for 24-chromosome aneuploidy testing. One of the he major factors in reproductive outcome of mosaicism is their meiotic or meiotic origin. For example, the experience on the establishment of embryonic stem cell lines demonstrated a higher success rate of obtaining embryonic stem cell lines from mitotic rather than meiotic trisomics. While, it is not always possible to distinguish the origin of mosaicism, the available data on the prevalence and types of meiotic and mitotic error, to be summarized in this presentation, may be useful for evaluating their corresponding impact depending on the mosaicism origin.

Effect of down-regulation of growth arrest and DNA damage inducible protein 45β on PC9 lung adenocarcinoma cells

To explore the effect of down-regulation of growth arrest and DNA damage inducible protein 45β (GADD45β) on the PC9 lung adenocarcinoma cells. Methods: GADD45β gene siRNA sequence was designed and synthesized, which was transfected into PC9 lung adenocarcinoma cells through lentivirus transfection. Quantitative real-time PCR (qRT-PCR) and Western blot are used to examine the mRNA and protein levels of GADD45β in PC9 cells before and after the transfection. Annexin V-allophycocyanin (APC) double-staining flow cytometry was used to detect the apoptosis level after the transfection. The intracellular DNA content after transfection was detected by flow cytometry. The percentage of the cells at each period of cell cycle was calculated, and the effect of RNA interference on the cell growth were analyzed. The effects of RNA interference on the tumor-formation ability of cells were tested by counting the number of clones. MTT assay was used to test the half maximal inhibitory concentration (IC50) of PC9 cells for gefitinib. Results: The 5'-AAATCCACTTCACGCTCAT-3' sequence was identified as the effective sequence for GADD45β gene RNA interference. The mRNA and protein expression levels of GADD45β were markedly decreased (both P<0.05) at 48 h after transfection of GADD45β-siRNA, which resulted in the increased apoptosis rate (P<0.05), decreased tumor clone number (P<0.05) and increased percentage of PC9 cell at the S stage and G2/M stage (P<0.05). The IC50 for gefitinib was decreased obviously (P<0.05). Conclusion: Down-regulation of GADD45β can reduce the colony-forming ability of PC9 cells, promote the cell apoptosis, and enhance the sensitivity of PC9 cells to gefitinib.

Unraveling the Control of Cell Cycle Periods during Intestinal Stem Cell Differentiation

During differentiation, intestinal stem cells (ISCs), a prototypical adult stem cell pool, become either secretory transit-amplifying cells, which give rise to all secretory cell types, or absorptive transit-amplifying cells, which give rise to enterocytes. These cells exhibit distinct cell cycle dynamics: ISCs cycle with a period of 24 h and absorptive transit-amplifying cells cycle with a period of ∼12 h, whereas secretory transit-amplifying cells arrest their cycle. The cell cycle dynamics of ISCs and their progeny are a systems-level property that emerges from interactions between the cell cycle control machinery and multiple regulatory pathways. Although many mathematical models have been developed to study the details of the cell cycle and related regulatory pathways, few models have been constructed to unravel the dynamic consequences of their interactions. To fill this gap, we present a simplified model focusing on the interaction between four key regulatory pathways (STAT, Wnt, Notch, and MAPK) and cell cycle control. After experimentally validating a model prediction, which showed that the Notch pathway can fine-tune the cell cycle period, we perform further model analysis that reveals that the change of cell cycle period accompanying ISC differentiation may be controlled by a design principle that has been well studied in dynamical systems theory—a saddle node on invariant circle bifurcation. Given that the mechanisms that control the cell cycle are conserved in most eukaryotic cell types, this general principle potentially controls the interplay between proliferation and differentiation for a broad range of stem cells.

Disorder-to-order transitions in the molten globule-like Golgi Reassembly and Stacking Protein

BackgroundGolgi Reassembly and Stacking Proteins (GRASPs) are widely spread among eukaryotic cells (except plants) and are considered as key components in both the stacking of the Golgi cisternae and its lateral connection. Furthermore, GRASPs were also proved essential in the unconventional secretion pathway of several proteins, even though the mechanism remains obscure. It was previously observed that the GRASP homologue in Cryptococcus neoformans has a molten globule-like behavior in solution. MethodsWe used circular dichroism, synchrotron radiation circular dichroism and steady-state as well as time-resolved fluorescence. ResultsWe report the disorder-to-order transition propensities for a native molten globule-like protein in the presence of different mimetics of cell conditions. Changes in the dielectric constant (such as those experienced close to the membrane surface) seem to be the major factor in inducing multiple disorder-to-order transitions in GRASP, which shows very distinct behavior when in conditions that mimic the vicinity of the membrane surface as compared to those found when free in solution. Other folding factors such as molecular crowding, counter ions, pH and phosphorylation exhibit lower or no effect on GRASP secondary structure and/or stability. General significanceTo the best of our knowledge, this is the first study focusing on understanding the disorder-to-order transitions of a molten globule structure without the need of any mild denaturing condition. A model is also introduced aiming at describing how the cell could manipulate the GRASP sensitivity to changes in the dielectric constant during different cell-cycle periods.

The Angel's staircase: Cell cycle, and the embryogenesis of vertebrates

I present a simple model of vertebrate embryogenesis, coupling cell division, differentiation, and morphogenesis. The model relies on a gradient of cell cycle period, in a flat shell of tissue. When the cell cycle period varies linearly between two points, a cascade of cell division occurs in the tissue, which generates a staircase of cell sizes (the Angel's staircase). The variation in cell size is associated to a stepwise variation of mechanical properties, which induces a deterministic pattern of folds. The folded shell is recognized as an animal.

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

Filovirus Mimics Deliver Effectively to Irreversibly Control Carcinoma Cell Fate

Filoviruses are microns long, but biophysical advantages for such encapsulating/enveloped viruses are obscure. Flexible ‘filomicelles’ have been self-assembled from amphiphilic block copolymer (PEG-PCL) and demonstrate effective delivery of two very different hydrophobic compounds. Retinoic acid (RA) and other retinoids regulate RA receptor transcription factors that induce differentiation and arrest proliferation of many cell types, including cancer cells. Lamin-A, transcriptionally regulated by RA receptors, is a structural protein surrounding chromatin and can affect differentiation and karyokinesis. Paclitaxel, on the other hand, stabilizes microtubules and induces aneuploidy by blocking mitosis, which greatly increases cell death. When cancer cells are treated with either of the drugs alone, over several periods of the normal cell cycle, they revert back to the original proliferative state, consistent with relapse commonly seen after conventional chemotherapy. On the other hand, combining RA with select chemotherapeutics has for several decades produced durable cures of select cancers, notably pro-myeloblastic leukemia (PML) where RA differentiates cells while chemotherapeutic kills the cancer stem cell. With carcinoma lines, we find dual drug treatment with RA plus Paclitaxel increases aneuploidy, and cell death beyond those achieved by either drug single-handedly, with effects being durable. A month after treatment, relapse rates are low for RA-Paclitaxel treated cells (15%), compared to almost all (92%) for cells treated with Paclitaxel alone. Reduction in levels of key cell cycle factor Cyclin-D1 and proliferation marker Ki-67 help clarify the basis for this synergy. These effects are greatly enhanced by loading the drugs into filomicelles. Co-loading the drugs into filomicelles lead to a more potent system compared to separate loading, with no loss in the integration efficiency of drugs. Notably, relapse rates were ∼2% three months after treatment, highlighting the improvement offered by filomicelles. The combination retains its potency across multiple cell lines (liver, lung and bone) despite varying responsiveness to RA alone. Preliminary tests in vivo demonstrate sustained delivery for days as well as efficacy in shrinking tumors. Injections of free RA-Paclitaxel combination are able to arrest growth of subcutaneous lung tumors for over 15 days, a feat not achieved by Paclitaxel alone. Drug loaded filomicelles are able to control the growth of subcutaneous lung and liver tumors. These results highlight the irreversible synergy of killing cancerous cells while driving differentiation. Flexible filomicelles circulate longer and accumulate better in the tumors, thus increasing the efficacy of delivery and treatment.

Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle.

The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal, and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. J. Cell. Physiol. 232: 1254-1257, 2017. © 2016 Wiley Periodicals, Inc.

Computational Modeling of Lipid Metabolism in Yeast.

Lipid metabolism is essential for all major cell functions and has recently gained increasing attention in research and health studies. However, mathematical modeling by means of classical approaches such as stoichiometric networks and ordinary differential equation systems has not yet provided satisfactory insights, due to the complexity of lipid metabolism characterized by many different species with only slight differences and by promiscuous multifunctional enzymes. Here, we present an object-oriented stochastic model approach as a way to cope with the complex lipid metabolic network. While all lipid species are treated objects in the model, they can be modified by the respective converting reactions based on reaction rules, a hybrid method that integrates benefits of agent-based and classical stochastic simulation. This approach allows to follow the dynamics of all lipid species with different fatty acids, different degrees of saturation and different headgroups over time and to analyze the effect of parameter changes, potential mutations in the catalyzing enzymes or provision of different precursors. Applied to yeast metabolism during one cell cycle period, we could analyze the distribution of all lipids to the various membranes in time-dependent manner. The presented approach allows to efficiently treat the complexity of cellular lipid metabolism and to derive conclusions on the time- and location-dependent distributions of lipid species and their properties such as saturation. It is widely applicable, easily extendable and will provide further insights in healthy and diseased states of cell metabolism.

Leptin promotes proliferation and metastasis of human gallbladder cancer through OB-Rb leptin receptor.

Emerging evidence has shown that leptin, an adipocyte-derived cytokine that is closely associated with obesity, play a significant role in carcinogenesis and tumorigenesis. However, its impact on gallbladder cancer (GBC) remains unclear. In this study, we firstly found that leptin and its functional receptor OB-Rb were significantly co-expressed in human GBC tissues and cell lines, the content of which were higher than those in normal human gallbladder tissues. Treatment with leptin promoted the proliferation, migration and invasion of GBC cells, which were attenuated by OB-Rb shRNA. Blocking in the G2/M period of cell cycle, increasing of MMP3 and MMP9, increasing of VEGF-C/D, activation of SOCS3/JAK2/p-STAT3 pathway was demonstrated after treatment with leptin. All of these positive responses were attenuated by OB-Rb receptor shRNA. Taken together, our findings suggest that leptin promoted the proliferation, migration and invasion of GBC cells by increasing OB-Rb expression through the SOCS3/JAK2/p-STAT3 signal pathway. Targeting the leptin/OB-Rb axis could be an attractive therapeutic strategy for treatment of GBC.

Filovirus Mimics Deliver Effectively

Filoviruses such as Ebola are microns long but biophysical advantages for such encapsulating/enveloped viruses have remained obscure. Flexible ‘filomicelles’ have been made from amphiphilic block copolymers and demonstrate effective delivery of two very different hydrophobic compounds. Retinoic acid (RA) and other retinoids regulate RA receptor transcription factors that induce differentiation and arrest proliferation of many cell types, including cancer cells. Lamin-A is transcriptionally regulated by RA receptors, and as a structural protein surrounding chromatin, lamin-A can affect differentiation and karyokinesis as well as nuclear viscosity. Paclitaxel, on the other hand, stabilizes microtubules and induces aneuploidy by blocking mitosis at the metaphase-anaphase transition, which greatly increases cell death. When cancer cells are treated with either of the drugs alone over several periods of the normal cell cycle, cancer cell populations revert back to the original proliferative state, consistent with relapse commonly seen after conventional chemotherapy. On the other hand, combining RA with select chemotherapeutics has for several decades produced durable cures of select cancers, notably pro-myeloblastic leukemia (PML) where RA differentiates cells while chemotherapeutic kills the cancer stem cell. With carcinoma lines, we find dual treatment with RA plus Paclitaxel increases lamin-A levels, aneuploidy, and cell death beyond those achieved by either drug single-handedly, with effects appearing irreversible. Trends with the key cell cycle factor Cyclin-D1 and proliferation marker Ki-67 help clarify the basis for drug synergy. These effects are greatly enhanced by loading the drugs into filomicelles self-assembled from degradable di-block copolymers of Polyethylene glycol-Polybenzyl caprolactone (PEG-PBCL). Preliminary tests in vivo demonstrate sustained delivery for days as well as efficacy in shrinking tumors. These results highlight the irreversible synergy of killing cancerous cells while driving differentiation.

Morphokinetic behavior of euploid and aneuploid embryos analyzed by time-lapse in embryoscope.

BACKGROUND:Embryonic aneuploidy may result in miscarriage, implantation failure, or birth defects. Thus, it is clinically necessary to avoid the selection of aneuploid embryos during in vitro fertilization treatment.AIM:The aim of this study was to identify the morphokinetic differences by analyzing the development of euploid and aneuploid embryos using a time-lapse technology. We also checked the accuracy of a previously described model for selection of euploid embryos based on morphokinetics in our study population.MATERIALS AND METHODS:It is a retrospective study of 29 cycles undergoing preimplantation genetic screening from October 2013 to April 2015 at our center. Of 253 embryos, 167 suitable for biopsy embryos were analyzed for their chromosomal status using array-comparative genome hybridization (CGH). The morphokinetic behavior of these embryos was further analyzed in embryoscope using time-lapse technology.RESULTS:Among the analyzed embryos, 41 had normal and 126 had abnormal chromosome content. No significant difference in morphokinetics was found between euploid and aneuploid embryos. The percentage of embryos with blastulation was similar in the euploid (65.85%, 27/41) and aneuploid (60.31%, 76/126) embryos (P = 0.76). Although hard to define, majority of the chromosomal defects might be due to meiotic errors. On applying embryo selection model from Basile et al., embryos falling within optimal ranges for time to division to 5 cells (t5), time period of the third cell cycle (CC3), and time from 2 cell division to 5 cell division (t5-t2) exhibited greater proportion of normal embryos than those falling outside the optimal ranges (28.6%, 25.9%, and 26.7% vs. 17.5%, 20.8%, and 14.3%).CONCLUSION:Keeping a track of time interval between two stages can help us recognize aneuploid embryos at an earlier stage and prevent their selection of transfer. However, it cannot be used as a substitute for array CGH to select euploid embryos for transfer.

The Modifying Effect of Co-Mutagens on the Frequency and Spectrum of Radiation-Induced Chromosome Aberrations in Human Cells

We have studied the co-mutagenic effect of verapamil and ascorbic acid on formation of radiation-induced chromosomal aberrations in T-lymphocytes of peripheral blood (PBL) of healthy donors. Test system of PBL treated in vitro with subsequent metaphase analysis of chromosomal aberrations has been used. Cells were exposed to γ-ray radiation in G0- and G2- periods of cell cycle and treated with verapamil (1.5; 2 and 4.0 μg/ml of blood) and ascorbic acid (20; 40 and 80 μg/ml of blood). The post-irradiation treatment of PBL with ascorbic acid in concentrations of 40 and 80 μg/ml, which exceeded therapeutic concentration value 2 and 4 times, increased overall chromosome aberrations frequency in 1.4 times compared to low dose (0.3 Gy) radiation effect. Verapamil at a concentration of 4.0μg/ml also increased the damaging effect of low dose of radiation in 1.5 times. Modification of radiation-induced cytogenetic effects in human PBL treated with co-mutagens was dependent on the concentration of the drugs, the absorbed dose of radiation, as well as cell radiosensitivity. It could be concluded that high concentrations of comutagens potentiate the damaging effect of low doses of ionizing radiation.

Visualization of cell-cycle modification by ionizing irradiation in single HeLa cells using fluorescent ubiquitination-based cell-cycle indicator

It has been known that cell cycle is retarded or arrested when the cells are exposed to ionizing radiation. The cell-cycle modifications are thought to be controlled by check point mechanisms to ensure the time for DNA repair. Linear energy transfer (LET) dependence of cell-cycle modifications, however, has not been fully revealed. Considerably less is known about detailed cell-cycle arrest for a single-cell level after exposure. Our purpose is to explore high LET radiation effects on the mammalian cell cycle. To examine high LET radiation effects on mammalian cell cycle, it would be essential to track single cells as live cell images observed by time-lapse imaging technique. HeLa cells expressing fluorescent ubiquitination-based cell-cycle indicator (FUCCI) are one of the useful model cell lines to visualize cell cycle because their nuclei show different colors; orange indicating G1 (Cdt1 expression); green indicating S/G2 (Geminin expression) [ 1]. In order to establish a novel assay system to study cell-cycle modification by high LET irradiation such as ion beams, we have developed time-lapse protocol for the HeLa-FUCCI cells irradiated.As a preliminary experiment using conventional X-rays instead of high LET ion beams, we observed the cell cycles of the irradiated HeLa cells. Figure 1 shows a typical time-lapse dynamics of unirradiated cells acquired for 48 h. We establish a new method to decide the time of one cell cycle as shown in Fig. 1. We obtained an evidence that the irradiated (5 Gy) cells show prolonged cell-cycle period when compared with that of control cells (Table 1). We also revealed that the delay is mainly caused in the G2 (Geminin expressing) phase. These results suggest that S/G2, G2/M or M checkpoint mechanism regulate the cell cycle in the irradiated HeLa-FUCCI cells. In conclusion, single FUCCI cell exposure and live cell imaging are a powerful method to trace the single-cell effect of high LET irradiation on the cell cycle in future. Fig. 1.Typical time-lapse dynamics of an unirradiated HeLa-FUCCI cell monitored by geminin fluorescent intensity of the cell nucleus. Table 1.Distribution of the number of cells showing a specific cell cycle period.Cell cycle period, hNumber of cellsControl (unirradiated)5 Gy irradiated>146214–168316–1812918–20221620–2215822–245824–265726–281628–301030 <08Total7567

Modelling the coupling between intracellular calcium release and the cell cycle during cortical brain development

Most neocortical neurons formed during embryonic brain development arise from radial glial cells which communicate, in part, via ATP mediated calcium signals. Although the intercellular signalling mechanisms that regulate radial glia proliferation are not well understood, it has recently been demonstrated that ATP dependent intracellular calcium release leads to an increase of nearly 100% in overall cellular proliferation. It has been hypothesised that cytoplasmic calcium accelerates entry into S phase of the cell cycle and/or acts to recruit otherwise quiescent cells onto the cell cycle. In this paper we study this cell cycle acceleration and recruitment by forming a differential equation model for ATP mediated calcium-cell cycle coupling via Cyclin D in a single radial glial cell.Bifurcation analysis and numerical simulations suggest that the cell cycle period depends only weakly on cytoplasmic calcium. Therefore, the accelerative impact of calcium on the cell cycle can only account for a small fraction of the large increase in proliferation observed experimentally. Crucially however, our bifurcation analysis reveals that stable fixed point and stable limit cycle solutions can coexist, and that calcium dependent Cyclin D dynamics extend the oscillatory region to lower Cyclin D synthesis rates, thus rendering cells more susceptible to cycling. This supports the hypothesis that cycling glial cells recruit quiescent cells (in G0 phase) onto the cell cycle, via a calcium signalling mechanism, and that this may be the primary means by which calcium augments proliferation rates at the population scale. Numerical simulations of two coupled cells demonstrate that such a scenario is indeed feasible.

Periodic mRNA synthesis and degradation co‐operate during cell cycle gene expression

During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is achieved by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. Here, we used metabolic RNA labeling and comparative dynamic transcriptome analysis (cDTA) to derive mRNA synthesis and degradation rates every 5min during three cell cycle periods of the yeast Saccharomyces cerevisiae. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation generally follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter.

Time Scales in Epigenetic Dynamics and Phenotypic Heterogeneity of Embryonic Stem Cells

A remarkable feature of the self-renewing population of embryonic stem cells (ESCs) is their phenotypic heterogeneity: Nanog and other marker proteins of ESCs show large cell-to-cell variation in their expression level, which should significantly influence the differentiation process of individual cells. The molecular mechanism and biological implication of this heterogeneity, however, still remain elusive. We address this problem by constructing a model of the core gene-network of mouse ESCs. The model takes account of processes of binding/unbinding of transcription factors, formation/dissolution of transcription apparatus, and modification of histone code at each locus of genes in the network. These processes are hierarchically interrelated to each other forming the dynamical feedback loops. By simulating stochastic dynamics of this model, we show that the phenotypic heterogeneity of ESCs can be explained when the chromatin at the Nanog locus undergoes the large scale reorganization in formation/dissolution of transcription apparatus, which should have the timescale similar to the cell cycle period. With this slow transcriptional switching of Nanog, the simulated ESCs fluctuate among multiple transient states, which can trigger the differentiation into the lineage-specific cell states. From the simulated transitions among cell states, the epigenetic landscape underlying transitions is calculated. The slow Nanog switching gives rise to the wide basin of ESC states in the landscape. The bimodal Nanog distribution arising from the kinetic flow running through this ESC basin prevents transdifferentiation and promotes the definite decision of the cell fate. These results show that the distribution of timescales of the regulatory processes is decisively important to characterize the fluctuation of cells and their differentiation process. The analyses through the epigenetic landscape and the kinetic flow on the landscape should provide a guideline to engineer cell differentiation.

Dynamics of the cell-cycle network under genome-rewiring perturbations

The cell-cycle progression is regulated by a specific network enabling its ordered dynamics. Recent experiments supported by computational models have shown that a core of genes ensures this robust cycle dynamics. However, much less is known about the direct interaction of the cell-cycle regulators with genes outside of the cell-cycle network, in particular those of the metabolic system. Following our recent experimental work, we present here a model focusing on the dynamics of the cell-cycle core network under rewiring perturbations. Rewiring is achieved by placing an essential metabolic gene exclusively under the regulation of a cell-cycle's promoter, forcing the cell-cycle network to function under a multitasking challenging condition; operating in parallel the cell-cycle progression and a metabolic essential gene. Our model relies on simple rate equations that capture the dynamics of the relevant protein–DNA and protein–protein interactions, while making a clear distinction between these two different types of processes. In particular, we treat the cell-cycle transcription factors as limited ‘resources’ and focus on the redistribution of resources in the network during its dynamics. This elucidates the sensitivity of its various nodes to rewiring interactions. The basic model produces the correct cycle dynamics for a wide range of parameters. The simplicity of the model enables us to study the interface between the cell-cycle regulation and other cellular processes. Rewiring a promoter of the network to regulate a foreign gene, forces a multitasking regulatory load. The higher the load on the promoter, the longer is the cell-cycle period. Moreover, in agreement with our experimental results, the model shows that different nodes of the network exhibit variable susceptibilities to the rewiring perturbations. Our model suggests that the topology of the cell-cycle core network ensures its plasticity and flexible interface with other cellular processes, without a need for an optimal setting of the kinetic parameters.

Dissecting genealogy and cell cycle as sources of cell-to-cell variability in MAPK signaling using high-throughput lineage tracking

Cells, even those having identical genotype, exhibit variability in their response to external stimuli. This variability arises from differences in the abundance, localization, and state of cellular components. Such nongenetic differences are likely heritable between successive generations and can also be influenced by processes such as cell cycle, age, or interplay between different pathways. To address the contribution of nongenetic heritability and cell cycle in cell-to-cell variability we developed a high-throughput and fully automated microfluidic platform that allows for concurrent measurement of gene expression, cell-cycle periods, age, and lineage information under a large number of temporally changing medium conditions and using multiple strains. We apply this technology to examine the role of nongenetic inheritance in cell heterogeneity of yeast pheromone signaling. Our data demonstrate that the capacity to respond to pheromone is passed across generations and that the strength of the response correlations between related cells is affected by perturbations in the signaling pathway. We observe that a ste50Δ mutant strain exhibits highly heterogeneous response to pheromone originating from a unique asymmetry between mother and daughter response. On the other hand, fus3Δ cells were found to exhibit an unusually high correlation between mother and daughter cells that arose from a combination of extended cell-cycle periods of fus3Δ mothers, and decreased cell-cycle modulation of the pheromone pathway. Our results contribute to the understanding of the origins of cell heterogeneity and demonstrate the importance of automated platforms that generate single-cell data on several parameters.

Direct visualization of the quadruplex structures in human chromosome using FRET: Application of quadruplex stabilizer and duplex-binding fluorophore

The G-quadruplex structures in the telomere of a chromosome can not only protect the internal chromosome sequences by preventing the improper activation of DNA-damage-response pathways but also become targets for cancer treatments. In this manuscript, we wish to prove the existence of G-quadruplex structure formation, rather than G-quadruplex sequence, in chromosome of human cancer cells. Based on our studies, the fluorescent mapping of G-quadruplex structures in the chromosome is possible with the combination of G-quadruplex targeting fluorophore (BMVC, 3, 6-bis-(1-methyl-4-vinylpyridinium)-carbazole diiodide) and duplex-binding fluorophores (Hoechst or propidium iodide). By means of an applicable incubation time between cell cycle period and proper staining procedure to the chromosome, FRET (fluorescence resonance energy transfer) between G-quadruplex targeting fluorophore and duplex-binding fluorophore can increase the signal contrast of the fluorescent color and the fluorescent mapping of quadruplex structures can be easily observed using fluorescence microscopy. These observations are further supported by basic spectral analysis, titration binding assay, gel electrophoresis binding competition assay and confocal microscopy.