Name: flow cytometry-based purification of s. cerevisiae zygotes
Uploaded: 2012-09-21T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

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 &amp; Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center (P30 CA43703).

1. Haploid Cell Growth
<ol>
 <li>Grow S. cerevisiae cells from the S288C background (BY4741) under standard conditions 10.</li>
 <li>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 cou...

<ol>
	<li>Herskowitz, I., Oshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Control of cell type in Saccharomyces cerevisiae: Mating type and mating-type interconversion. , 181-210 (1981).</li><li>Muller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parental+age+and+the+life-span+of+zygotes+of+Saccharomyces+cerevisiae.">Parental age and the life-span of zygotes of Saccharomyces cerevisiae.</a> Antonie Van Leeuwenhoek. 51, 1-10 (1985).</li><li>Tartakoff, A. M., Jaiswal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+fusion+and+genome+encounter+during+yeast+zygote+formation.">Nuclear fusion and genome encounter during yeast zygote formation.</a> Mol. Biol. Cell. 20, 2932-2942 (2009).</li><li>Azpiroz, R., Butow, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+mitochondrial+sorting+in+yeast+zygotes.">Patterns of mitochondrial sorting in yeast zygotes.</a> Mol. Biol. Cell. 4, 21-36 (1993).</li><li>Rose, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+fusion+in+the+yeast+Saccharomyces+cerevisiae.">Nuclear fusion in the yeast Saccharomyces cerevisiae.</a> Annu. Rev. Cell Dev. Biol. 12, 663-695 (1996).</li><li>Dujon, B., Jones, E., Strathern, J., Broach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. , 505-635 (1981).</li><li>Sprague, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+yeast+mating+reaction.">Assay of yeast mating reaction.</a> Methods Enzymol. 194, 77-93 (1991).</li><li>Ydenberg, C. A., Rose, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mating:+a+model+system+for+studying+cell+and+nuclear+fusion.">Yeast mating: a model system for studying cell and nuclear fusion.</a> Methods Mol. Biol. 475, 3-20 (2008).</li><li>Sena, E. P., Radin, D. N., Fogel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+mating+in+yeast.">Synchronous mating in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 1373-1377 (1973).</li><li>Amberg, D., Burke, D., Strathern, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition. , (2005).</li><li>Nolan, S., Cowan, A. E., Koppel, D. E., Jin, H., Grote, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FUS1+regulates+the+opening+and+expansion+of+fusion+pores+between+mating+yeast.">FUS1 regulates the opening and expansion of fusion pores between mating yeast.</a> Mol. Biol. Cell. 17, 2439-2450 (2006).</li><li>Weisman, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelles+on+the+move:+insights+from+yeast+vacuole+inheritance.">Organelles on the move: insights from yeast vacuole inheritance.</a> Nat. Rev. Mol. Cell Biol. 7, 243-252 (2006).</li></ol>

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...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Source</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>pEG220, URA3/YIp, encoding cytoplasmic GFP 11)</td>
 <td>E. Grote</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pAJ1661, URA3/CEN, encoding RPL25 fused to mCherry </td>
 <td>A. Johnston</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fisher FB120 sonicator</td>
 <td>Fisher Scientific</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene tube with strainer cap</td>
 <td>BD Falcon</td>
 <td>Ref 352235</td>
 </tr>
 <tr>
 <td>Refrigerated microfuge </td>
 <td>Tomy</td>
 <td>MRX-150</td>
 </tr>
 <tr>
 <td>Flow cytometry sheath solution</td>
 <td>Biosure</td>
 <td>1027</td>
 </tr>
 <tr>
 <td>iCyt Reflection Model Flow Cytometer</td>
 <td>Sony</td>
 <td></td>
 </tr>
 <tr>
 <td>DeltaVision deconvolution microscope</td>
 <td>Applied Precision</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright epifluorescence microscope</td>
 <td>Leica</td>
 <td></td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>Invitrogen</td>
 <td>15510-07</td>
 </tr>
 <tr>
 <td>CSM glucose formulation Notes: For agar plates, simply include 2% agar (Difco)</td>
 <td>10</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

flow cytometry-based purification of s. cerevisiae zygotes

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.

Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

Flow Cytometry-based Purification of S. cerevisiae Zygotes

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.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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