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: 3.07). 09/2008; 73(9):825-33. DOI: 10.1002/cyto.a.20609
Source: PubMed

ABSTRACT 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cells adjust to changes in environmental conditions using complex regulatory programs. These cellular programs are the result of an intricate interplay between gene expression, cellular growth and protein degradation. Technologies that enable simultaneous and time-resolved measurements of these variables are necessary to dissect cellular homeostatic strategies. Here we report the development of an automated flow cytometry robotic setup that enables real-time measurement of precise and simultaneous relative growth and protein synthesis rates of multiplexed microbial populations across many conditions. These measurements generate quantitative profiles of dynamically evolving protein synthesis and degradation rates. We demonstrate this setup in the context of gene regulation of the unfolded protein response (UPR) of Saccharomyces cerevisiae and uncover a dynamic and complex landscape of gene expression, growth dynamics and proteolysis following perturbations.
    Nature Methods 03/2014; DOI:10.1038/nmeth.2879 · 25.95 Impact Factor
  • [Show abstract] [Hide abstract]
    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 · 2.44 Impact Factor
  • [Show abstract] [Hide abstract]
    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; DOI:10.1016/j.gpb.2014.10.004

Full-text (2 Sources)

Available from
Jun 4, 2014