Flow Cytometry-based Purification of S. cerevisiae Zygotes

Summary

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

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 ^3-8. 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 cytometry-based 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.

Protocol

1. Haploid Cell Growth

1. Grow S. cerevisiae cells from the S288C background (BY4741) under standard conditions ¹⁰.
2. Transform haploids to express either of two conspicuous fluorescent markers ¹⁰. The markers of choice are soluble GFP expressed in the cytoplasm (pEG220, URA3/YIp, linearized with BglII ¹¹) and a single ribosomal protein, RPL25, fused with mCherry (pAJ1661, URA3/CEN, from A. Johnston). We expect that equivalent two-color purification protocols could make use of other brightly fluorescent proteins that are synthesized by the haploid parental cells. Alternatively, the cell wall could be labeled w....

Discussion

The availability of purified zygotes should facilitate biochemical studies including transcriptomics, proteomics and lipidomics, as well as investigation of mechanisms by which zygotes undergo cell cycle re-entry, cell wall remodeling, budding and inheritance characteristics, etc. Alternatively, zygotes could be generated starting with strains carrying characteristic mutations in one or both parents. Moreover, the purified zygotes, as for haploid or diploid cells, can be frozen in medium containing 15% glycerol and later.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pEG220, URA3/YIp, encoding cytoplasmic GFP 11)	E. Grote
pAJ1661, URA3/CEN, encoding RPL25 fused to mCherry	A. Johnston
Fisher FB120 sonicator	Fisher Scientific
Polypropylene tube with strainer cap	BD Falcon	Ref 352235
Refrigerated microfuge	Tomy	MRX-150
Flow cytometry sheath solution	Biosure	1027
iCyt Reflection Model Flow Cytometer	Sony
DeltaVision deconvolution microscope	Applied Precision
Upright epifluorescence microscope	Leica
UltraPure Agarose	Invitrogen	15510-07
CSM glucose formulation Notes: For agar plates, simply include 2% agar (Difco)	10