Flow Cytometry-based Purification of <em>S. cerevisiae</em> Zygotes

Abstract

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1 . S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2 . Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells [3][4][5][6][7][8] . Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9 ; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometrybased sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell. Video LinkThe video component of this article can be found at

Quantitation of yeast cell–cell fusion using multicolor flow cytometry

“…Our method combines innocuous ConA–fluorophore conjugate staining of haploid cells and BiFC to define fused and unfused mating pair subpopulations in a complex mating mixture. In addition, FCM‐based purification protocols of S. cerevisiae zygotes could be adapted to obtain mating pair subpopulations by cell sorting for further analysis.…”

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest

“…They were then overlaid with a coverslip that was sealed with Vaseline [21] and were examined in a Deltavision RT epifluorescence microscope with an automated stage (Applied Precision, Inc). The microscope was equipped with a 100x oil immersion objective (Olympus UPlanApo 100x/1.40; 0.17/FN26.5) and images were recorded without binning.…”

Cell cycle arrest of S. cerevisiae in conjunction with labeling of the cell wall

“…Add agarose to medium (1.5% final), bring to a boil, vortex, and apply samples to the surface of microscope slides that are immediately overlaid with a second slide ( Zapanta Rinonos et al., 2012 ). These slides can be kept 1–2 days in a refrigerator.…”