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
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Dr. R. Michael Sramkoski for help with flow cytometry, Dr. Purnima Jaiswal and Dr. Di Wu for earlier input and Ilya Aylyarov for help with the illustrations. Supported by NIH Grant R01GM089872 and the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center (P30 CA43703).
Materials
| Reagent/Equipment | Source | Catalogue Number |
| 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 |
Riferimenti
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