Optimization of yeast cell cycle analysis and morphological characterization by multispectral imaging flow cytometry.
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.
Calvert
Meredith E K
ME
Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
Lannigan
Joanne A
JA
Pemberton
Lucy F
LF
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D019044:Image Cytometry / Q000379:methods*; D009687:Nuclear Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; C408226:NBP1 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins