Optimization of yeast cell cycle analysis and morphological characterization by multispectral imaging flow cytometry

Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
Cytometry Part A (Impact Factor: 2.93). 09/2008; 73(9):825-33. DOI: 10.1002/cyto.a.20609
Source: PubMed


Budding yeast Saccharoymyces cerevisiae is a powerful model system for analyzing eukaryotic cell cycle regulation. Yeast cell cycle analysis is typically performed by visual analysis or flow cytometry, and both have limitations in the scope and accuracy of data obtained. This study demonstrates how multispectral imaging flow cytometry (MIFC) provides precise quantitation of cell cycle distribution and morphological phenotypes of yeast cells in flow. Cell cycle analysis of wild-type yeast, nap1Delta, and yeast overexpressing NAP1, was performed visually, by flow cytometry and by MIFC. Quantitative morphological analysis employed measurements of cellular length, thickness, and aspect ratio in an algorithm to calculate a novel feature, bud length. MIFC demonstrated reliable quantification of the yeast cell cycle compared to morphological and flow cytometric analyses. By employing this technique, we observed both the G2/M delay and elongated buds previously described in the nap1Delta strain. Using MIFC, we demonstrate that overexpression of NAP1 causes elongated buds yet only a minor disruption in the cell cycle. The different effects of NAP1 expression level on cell cycle and morphology suggests that these phenotypes are independent. Unlike conventional yeast flow cytometry, MIFC generates complete cell cycle profiles and concurrently offers multiple parameters for morphological analysis.

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Available from: Joanne Lannigan, Oct 10, 2015
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    • "DAPI/EdU flow cytometry [15] is a good alternative to hiMAC for cell cycle profiling and can be combined with the measurement of whole-cell fluorescence intensities; however, it lacks the spatial resolution required for the analysis of subcellular structures. Microscopic analysis of cells immobilized after sorting by flow cytometry, imaging flow cytometry [16], and combination of flow cytometry with cell fractionation [17] are potential alternatives to hiMAC in particular for simple readouts such as overall protein levels. The major benefit of these approaches when compared with hiMAC is the high throughput achieved by integrating flow cytometry. "
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    ABSTRACT: Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically powerful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular localization at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3-4- day protocol, which can be adjusted to any other cell cycle stage-dependent analysis. Copyright © 2014. Production and hosting by Elsevier Ltd.
    Genomics Proteomics & Bioinformatics 11/2014; 12(6). DOI:10.1016/j.gpb.2014.10.004
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    • "These cells can be observed as a 'sub-G 0/1 ' peak in a DNA histogram. In contrast, necrotic cells generally do not show an immediate reduction in DNA staining, and sub-G 0/1 cannot be discriminated from a histogram DNA content analysis of live cells (Darzynkiewicz et al., 1992, 2010; Dive et al., 1992; Riccardi & Nicoletti, 2006; Calvert et al., 2008). In Fig. 7i–j, about 20% of the population are in sub-G 0/1 region and are comprised of apoptotic cells, apoptotic bodies and debris. "
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    ABSTRACT: Candida glabrata cells suspended in water are under hypo-osmotic stress and undergo cell death in 1-2 days, unless they are at a density of more than 10(5 ) CFU mL(-1) . The dying cells exhibit FITC-annexin V staining, indicative of programmed cell death (apoptosis). In a higher cell density, cells are protected and survive at least for 4 days. Filtrates from cells at high density can protect those at lower density, indicating that cells release substances, amounting to c. 5 mg L(-1) of cell suspension, that protect each other against hypo-osmotic stress. In a concentrated form, the released materials can support growth, indicating that the protective material includes carbon and nitrogen sources, as well as vitamins that are required by C. glabrata for growth. We conclude that cell death from hypo-osmotic stress can be alleviated by small amounts of nutrients.
    FEMS Yeast Research 11/2013; 14(3). DOI:10.1111/1567-1364.12122 · 2.82 Impact Factor
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    • "To make a distinction between G2 and S-phase cells within populations having a 2N DNA content, we used multispectral imaging flow cytometry according to a previously published protocol [71], but with the following changes. Sytox-green-stained cells were resuspended in phosphate-buffered saline at a concentration of 107 cells/ml prior to loading them into an Imagestream imaging flow cytometer (Amnis Corporation). "
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    ABSTRACT: In the model eukaryote, Saccharomyces cerevisiae, previous experiments have identified those genes that exert the most significant control over cell growth rate. These genes are termed HFC for high flux control. Such genes are overrepresented within pathways controlling the mitotic cell cycle. We postulated that the increase/decrease in growth rate is due to a change in the rate of progression through specific cell cycle steps. We extended and further developed an existing logical model of the yeast cell cycle in order elucidate how the HFC genes modulated progress through the cycle. This model can simulate gene dosage-variation and calculate the cycle time, determine the order and relative speed at which events occur, and predict arrests and failures to correctly execute a step. To experimentally test our model's predictions, we constructed a tetraploid series of deletion mutants for a set of eight genes that control the G2/M transition. This system allowed us to vary gene copy number through more intermediate levels than previous studies and examine the impact of copy-number variation on growth, cell-cycle phenotype, and response to different cellular stresses. For the majority of strains, the predictions agreed with experimental observations, validating our model and its use for further predictions. Where simulation and experiment diverged, we uncovered both novel tetraploid-specific phenotypes and a switch in the determinative execution point of a key cell-cycle regulator, the Cdc28 kinase, from the G1/S to the S/G2 boundaries.
    BMC Genomics 10/2013; 14(1):744. DOI:10.1186/1471-2164-14-744 · 3.99 Impact Factor
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