Article

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.

Download full-text

Full-text

Available from: Joanne Lannigan, Aug 24, 2015
0 Followers
 · 
111 Views
  • Source
    Cytometry Part A 09/2008; 73(9):777-8. DOI:10.1002/cyto.a.20625 · 3.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: J. Inst. Brew. 115(3), 253–258, 2009 An expeditious method of yeast age estimation was developed based on selective bud scar staining (Alexa Fluor 488-labelled wheat-germ agglutinin) and subsequent fluorescence intensity measurement by flow cytometry. The calibration curve resulting from the cytometric determination of average bud scar fluores-cence intensities vs. microscopically counted average bud scar numbers of the same cell populations showed a good correlation and allowed routine cell age estimation by flow cytometry. The developed method was applied for yeast age control in tradi-tional batch and continuous beer fermentations. At the pitching rates used in industrial beer fermentations, our results support former findings by locating a gradient of increasing yeast age from the top to the bottom zone of the fermenter cone. The re-sults also indicate that in continuous beer fermentation, the in-creasing bud scar fluorescence of immobilized cells could help to schedule the replacement of aged biomass, prior to loss of viability or deterioration of process performance and product quality.
    01/2009; 115(3). DOI:10.1002/j.2050-0416.2009.tb00377.x
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under laboratory conditions. Using microarray techniques and proteomics, the four flocculation genes Lg-FLO1, FLO1, FLO5, FLO9, and the protein Lg-Flo1p were identified as factors of major importance for flocculation. The expression of the genes was several times higher in flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to quantify the proportions of powdery and flocculent yeast cells in suspensions under defined cultivation conditions. However, differentiation under industrial conditions will require mRNA and protein expression profiling.
    Cytometry Part A 02/2009; 75(2):140-7. DOI:10.1002/cyto.a.20661 · 3.07 Impact Factor
Show more