Effects of temperature on the yeast cell cycle analyzed by flow cytometry

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.     

The dynamics of yeast telomeres and silencing proteins through the cell cycle

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.     

Stable stochastic dynamics in yeast cell cycle

Chemical reactions in cells are subject to intense stochastic fluctuations. An important question is how the fundamental physiological behavior of the cell is kept stable against those noisy perturbations. In this study, a stochastic model of the cell cycle of budding yeast was constructed to analyze the effects of noise on the cell-cycle oscillation. The model predicts intense noise in levels of mRNAs and proteins, and the simulated protein levels explain the observed statistical tendency of noise in populations of synchronous and asynchronous cells. Despite intense noise in levels of proteins and mRNAs, the cell cycle is stable enough to bring the largely perturbed cells back to the physiological cyclic oscillation. The model shows that consecutively appearing fixed points are the origin of this stability of the cell cycle.     

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

Synthesis of ribosomal proteins during the yeast cell cycle

The synthesis of ribosomal proteins during the cell division cycle of Saccharomyces cerevisiae has been examined. A technique was utilized whereby cells in unique phases of the cell cycle were selected from an asynchronous culture after the period of pulse labeling. Some of the proteins of the small and large ribosomal subunits were synthesized continuously throughout the cell cycle and there was no evidence of discontinuous synthesis for any of the ribosomal proteins.     

Protein feature based identification of cell cycle regulated proteins in yeast

DNA microarrays have been used extensively to identify cell cycle regulated genes in yeast; however, the overlap in the genes identified is surprisingly small. We show that certain protein features can be used to distinguish cell cycle regulated genes from other genes with high confidence (features include protein phosphorylation, glycosylation, subcellular location and instability/degradation). We demonstrate that co-expressed, periodic genes encode proteins which share combinations of features, and provide an overview of the proteome dynamics during the cycle. A large set of novel putative cell cycle regulated proteins were identified, many of which have no known function.     

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.     

Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.     

Flow cytometry and cell cycle kinetics in continuous and fed-batch fermentations of budding yeast.

Cell size distributions, obtained either as protein distribution by flow cytometry or as cell volume distribution by a Coulter counter, give relevant information about the growth conditions of populations of budding yeast Saccharomyces cerevisiae. We have previously found a good correlation between these distributions and the growth rate in continuous cultures (Ranzi et al., Biotechnol. Bioeng. 1986, 28, 185-190). We now present determinations of the protein distributions and cell volume distributions during different fed-batch fermentations performed with a simple on/off controller. Since during the fed-batch fermentation a true steady state is not obtained, the distributions continuously change with time, but nevertheless we observed a good correlation between the average of both distributions and the actual growth rate. The behavior of the cell size distributions can be interpreted on the basis of a two-threshold cell cycle model in which both the critical protein content at budding (Ps) and the critical protein content for cell division (Pm) are differently modulated by the growth rate. Additional findings will be presented showing that this model can be used to successfully explain the insurgence and the maintenance of oscillatory states in continuous cultures.     

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.     

Cell-cycle analysis of fission yeast cells by flow cytometry

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.     

Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate

An organism arises from the coordinate generation of different cell types and the stereotypical organization of these cells into tissues and organs. Even so, the dynamic behaviors, as well as the ultimate fates, of cells driving the morphogenesis of an organism, or even an individual organ, remain largely unknown. Continued innovations in optical imaging modalities, along with the discovery and evolution of improved genetically-encoded fluorescent protein reporters in combination with model organism, stem cell and tissue engineering paradigms are providing the means to investigate these unresolved questions. The emergence of fluorescent proteins whose spectral properties can be photomodulated is one of the most significant new developments in the field of cell biology where they are primarily used for studying protein dynamics in cells. Likewise, the use of photomodulatable fluorescent proteins holds great promise for use in developmental biology. Photomodulatable fluorescent proteins also represent attractive and emergent tools for studying cell dynamics in complex populations by facilitating the labeling and tracking of individual or defined groups of cells. Here, we review the currently available photomodulatable fluorescent proteins and their application in model organisms. We also discuss prospects for their use in mice, and by extension in embryonic stem cell and tissue engineering paradigms.     

Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate

An organism arises from the coordinate generation of different cell types and the stereotypical organization of these cells into tissues and organs. Even so, the dynamic behaviors, as well as the ultimate fates, of cells driving the morphogenesis of an organism, or even an individual organ, remain largely unknown. Continued innovations in optical imaging modalities, along with the discovery and evolution of improved genetically-encoded fluorescent protein reporters in combination with model organism, stem cell and tissue engineering paradigms are providing the means to investigate these unresolved questions. The emergence of fluorescent proteins whose spectral properties can be photomodulated is one of the most significant new developments in the field of cell biology where they are primarily used for studying protein dynamics in cells. Likewise, the use of photomodulatable fluorescent proteins holds great promise for use in developmental biology. Photomodulatable fluorescent proteins also represent attractive and emergent tools for studying cell dynamics in complex populations by facilitating the labeling and tracking of individual or defined groups of cells. Here, we review the currently available photomodulatable fluorescent proteins and their application in model organisms. We also discuss prospects for their use in mice, and by extension in embryonic stem cell and tissue engineering paradigms.Key words: fluorescent protein, photomodulation, photoactivation, photoconversion, mouse, live imaging, embryonic development, organogenesis, GFP, PA-GFP, PS-CFP, Kaede, KikGR     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

DNA measurement and cell cycle analysis by flow cytometry

Measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. Protocols for DNA measurement have been developed including Bivariate cytokeratin/DNA analysis, Bivariate BrdU/DNA analysis, and multiparameter flow cytometry measurement of cellular DNA content. This review summarises the methods for measurement of cellular DNA and analysis of the cell cycle and discusses the commercial software available for these purposes.     

Surface glycoproteins of differentiating neuroblastoma cells analyzed by lectin binding and flow cytometry

Cell surface glycoproteins of mitotic neuroblastoma cells and cells differentiated by prostaglandin cyclic adenosine monophosphate treatment were quantified by flow cytometric analysis and specific fluorescent lectins. No differences in fluorescent lectin binding were seen between suspensions of mitotically active and differentiated N2AB-1 cells following exposure to either fluorescein (FL)-labeled soy bean agglutinin (FL-SBA) specific for N acetyl galactosamine or FL-concanavalin A (FL-CON A) which binds to mannose residues. These lectins, however, were shown to bind specifically to these cells as revealed by competitive blocking studies with hapten sugars. When FL Ulex europaeus (FL-UEA) specific for fucose was reacted with control or differentiated cells, no binding was seen even with an increased dose of lectin before or after enzyme treatment. However, differentiated N2AB-1 cells, reacted with FL-wheat germ agglutinin (FL-WGA) specific for N acetyl glucosamine, bound more FL-WGA than that seen for control cultures. Furthermore, specific sites for FL-WGA were shown to be saturable and were lost upon pretreatment of cells with neuraminidase. Neuraminidase pretreatment revealed masked sites for FL-CON A and FL-SBA since binding was increased at least twofold for these lectins on mitotic and differentiated cells. These data indicate that single cell measurements of surface glycoproteins can be made on living neural cells and that differentiation induces an increase in cell surface N-acetyl glucosamine residues.