Name: use of time-lapse microscopy and stage-specific nuclear depletion of proteins to study meiosis in s. cerevisiae
Uploaded: 2022-10-11T14:40:00
Description: Watch this Scientific Journal Video about Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae at JoVE.com

We thank the Light Microscopy Imaging Center at Indiana University. This work was supported by a grant from the National Institutes of Health (GM105755).

1. Preparation of necessary materials
<ol>
	<li>Prepare reagents for the growth and sporulation of yeast cells. 
	NOTE: If budding yeast strains are ade2- and trp1-, supplement all media in steps 1.1.1-1.1.3 with a final concentration of 0.01% adenine and 0.01% tryptophan from 1% stocks. If sterilizing media by autoclave, add these amino acids only after the reagents have been autoclaved and allowed to cool to room temperature.
	<ol>
		<li>For vegetative growth, prepare 2x synthe...

<ol>
	<li>Tsuchiya, D., Gonzalez, C., Lacefield, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle+checkpoint+protein+Mad2+regulates+APC/C+activity+during+prometaphase+and+metaphase+of+meiosis+I+in+Saccharomyces+cerevisiae.">The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae.</a> Molecular Biology of the Cell. 22 (16), 2848-2861 (2011).</li><li>Cairo, G., MacKenzie, A. M., Lacefield, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+requirement+for+Bub1+and+Bub3+in+regulation+of+meiotic+versus+mitotic+chromosome+segregation.">Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation.</a> Journal of Cell Biology. 219 (4), 201909136 (2020).</li><li>van Werven, F. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+entry+into+gametogenesis.">Regulation of entry into gametogenesis.</a> Philosophical Transactions of the Royal Society London B: Biological Sciences. 366 (1584), 3521-3531 (2011).</li><li>Carlile, T. M., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis+I+is+established+through+division-specific+translational+control+of+a+cyclin.">Meiosis I is established through division-specific translational control of a cyclin.</a> Cell. 133 (2), 280-291 (2008).</li><li>Benjamin, K. R., Zhang, C., Shokat, K. M., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+landmark+events+in+meiosis+by+the+CDK+Cdc28+and+the+meiosis-specific+kinase+Ime2.">Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2.</a> Genes and Development. 17 (12), 1524-1539 (2003).</li><li>Xu, L., Ajimura, M., Padmore, R., Klein, C., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NDT80,+a+meiosis-specific+gene+required+for+exit+from+pachytene+in+Saccharomyces+cerevisiae.">NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.</a> Molecular and Cellular Biology. 15 (12), 6572-6581 (1995).</li><li>Chu, S., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gametogenesis+in+yeast+is+regulated+by+a+transcriptional+cascade+dependent+on+Ndt80.">Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80.</a> Molecular Cell. 1 (5), 685-696 (1998).</li><li>Haruki, H., Nishikawa, J., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anchor-away+technique:+rapid,+conditional+establishment+of+yeast+mutant+phenotypes.">The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.</a> Molecular Cell. 31 (6), 925-932 (2008).</li><li>Borek, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proteomic+landscape+of+centromeric+chromatin+reveals+an+essential+role+for+the+Ctf19(CCAN)+complex+in+meiotic+kinetochore+assembly.">The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19(CCAN) complex in meiotic kinetochore assembly.</a> Current Biology. 31 (2), 283-296 (2021).</li><li>Mehta, G. D., Agarwal, M., Ghosh, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+kinetochore+protein,+Ctf19+in+meiosis+I:+an+implication+of+differential+impact+of+Ctf19+on+the+assembly+of+mitotic+and+meiotic+kinetochores+in+Saccharomyces+cerevisiae.">Functional characterization of kinetochore protein, Ctf19 in meiosis I: an implication of differential impact of Ctf19 on the assembly of mitotic and meiotic kinetochores in Saccharomyces cerevisiae.</a> Molecular Microbiology. 91 (6), 1179-1199 (2014).</li><li>Ortiz, J., Stemmann, O., Rank, S., Lechner, 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+putative+protein+complex+consisting+of+Ctf19,+Mcm21,+and+Okp1+represents+a+missing+link+in+the+budding+yeast+kinetochore.">A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore.</a> Genes and Development. 13 (9), 1140-1155 (1999).</li><li>Hyland, K. M., Kingsbury, J., Koshland, D., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctf19p:+A+novel+kinetochore+protein+in+Saccharomyces+cerevisiae+and+a+potential+link+between+the+kinetochore+and+mitotic+spindle.">Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle.</a> Journal of Cell Biology. 145 (1), 15-28 (1999).</li><li>Fernius, J., Marston, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+cohesion+at+the+pericentromere+by+the+Ctf19+kinetochore+subcomplex+and+the+replication+fork-associated+factor,+Csm3.">Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3.</a> PLoS Genetics. 5 (9), 1000629 (2009).</li><li>Meyer, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipl1/Aurora-B+is+necessary+for+kinetochore+restructuring+in+meiosis+I+in+Saccharomyces+cerevisiae.">Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae.</a> Molecular Biology of the Cell. 26 (17), 2986-3000 (2015).</li><li>Elrod, S. L., Chen, S. M., Schwartz, K., Shuster, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+sporulation+conditions+for+different+Saccharomyces+cerevisiae+strain+backgrounds.">Optimizing sporulation conditions for different Saccharomyces cerevisiae strain backgrounds.</a> Methods in Molecular Biology. 557, 21-26 (2009).</li><li>Deutschbauer, A. M., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+mapped+to+single-nucleotide+resolution+in+yeast.">Quantitative trait loci mapped to single-nucleotide resolution in yeast.</a> Nature Genetics. 37 (12), 1333-1340 (2005).</li><li>Ben-Ari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+linked+genes+participate+in+controlling+sporulation+efficiency+in+budding+yeast.">Four linked genes participate in controlling sporulation efficiency in budding yeast.</a> PLoS Genetics. 2 (11), 195 (2006).</li><li>Primig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+core+meiotic+transcriptome+in+budding+yeasts.">The core meiotic transcriptome in budding yeasts.</a> Nature Genetics. 26 (4), 415-423 (2000).</li><li>Spencer, F., Gerring, S. L., Connelly, C., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+transmission+fidelity+mutants+in+Saccharomyces+cerevisiae.">Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae.</a> Genetics. 124 (2), 237-249 (1990).</li><li>Weinhandl, K., Winkler, M., Glieder, A., Camattari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+dependent+promoters+in+yeasts.">Carbon source dependent promoters in yeasts.</a> Microbial Cell Factories. 13, 5 (2014).</li><li>Byers, B., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+pachytene+arrest+of+Saccharomyces+cerevisiae+at+elevated+temperature.">Reversible pachytene arrest of Saccharomyces cerevisiae at elevated temperature.</a> Molecular and General Genetics. 187 (1), 47-53 (1982).</li><li>Winter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sum1/Ndt80+transcriptional+switch+and+commitment+to+meiosis+in+Saccharomyces+cerevisiae.">The Sum1/Ndt80 transcriptional switch and commitment to meiosis in Saccharomyces cerevisiae.</a> Microbiology and Molecular Biology Reviews. 76 (1), 1-15 (2012).</li><li>Neiman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+in+the+budding+yeast+Saccharomyces+cerevisiae.">Sporulation in the budding yeast Saccharomyces cerevisiae.</a> Genetics. 189 (3), 737-765 (2011).</li><li>Robinett, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+al.+In+vivo+localization+of+DNA+sequences+and+visualization+of+large-scale+chromatin+organization+using+lac+operator/repressor+recognition.">C. al. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.</a> Journal of Cell Biology. 135, 1685-1700 (1996).</li><li>Straight, A. F., Belmont, A. S., Robinett, C. C., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+tagging+of+budding+yeast+chromosomes+reveals+that+protein-protein+interactions+can+mediate+sister+chromatid+cohesion.">GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.</a> Current Biology. 6 (12), 1599-1608 (1996).</li><li>Sym, M., Engebrecht, J. A., Roeder, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZIP1+is+a+synaptonemal+complex+protein+required+for+meiotic+chromosome+synapsis.">ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis.</a> Cell. 72 (3), 365-378 (1993).</li><li>Scherthan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+mobility+during+meiotic+prophase+in+Saccharomyces+cerevisiae.">Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (43), 16934-16939 (2007).</li></ol>

This protocol combines the NDT80-in system to synchronize cells, the anchor away technique to deplete proteins from the nucleus, and fluorescence time-lapse microscopy to image budding yeast cells during meiosis. The NDT80-in system is a method for meiotic cell cycle synchronization that utilizes a prophase I arrest and release4,8. Although individual cells will vary slightly in the amount of time spent in each of the subsequent meiotic stages, ...

Time-lapse fluorescence microcopy is a valuable tool for studying the dynamics of meiotic chromosome segregation in budding yeast1,2. Budding yeast cells can be induced to undergo meiosis through starvation of key nutrients3. During meiosis, cells undergo one round of chromosome segregation followed by two divisions to create four meiotic products that are packaged into spores (Figure 1). Individual cells can be visualized throughout each stage of meiosis, which generates spatial and temporal data that can be easily missed by fixed-cell imaging. This protocol shows how combining time-lapse fluorescence microscopy with two previously established methods, the inducible NDT80 system (NDT80-in) and the anchor away technique, can be used to study the function of specific proteins in distinct meiotic stages.
The NDT80-in system is a powerful tool for meiotic cell cycle synchronization that relies on the inducible expression of the middle meiosis transcription factor NDT804,5. NDT80 expression is required for prophase I exit6,7. With the NDT80-in system, NDT80 is under the control of the GAL1-10 promoter in cells expressing the Gal4 transcription factor fused to an estrogen receptor (Gal4-ER)4,5. Because Gal4-ER only enters the nucleus when bound to &#946;-estradiol, NDT80-in cells arrest in prophase I in the absence of &#946;-estradiol, which allows the synchronization of cells in prophase I (Figure 1). &#946;-estradiol addition promotes the translocation of the Gal4-ER transcription factor into the nucleus, where it binds GAL1-10 to drive expression of NDT80, leading to synchronous entry into the meiotic divisions. Although time-lapse microscopy can be performed without synchronization, the advantage of using synchronization is the ability to add an inhibitor or a drug while cells are at a specific stage of meiosis.
The anchor away technique is an inducible system by which a protein can be depleted from the nucleus with the addition of rapamycin8. This technique is ideal for studying nuclear proteins during cell division in budding yeast because yeast cells undergo closed mitosis and meiosis, in which the nuclear envelope does not break down. Furthermore, this technique is very useful for proteins that have multiple functions throughout meiosis. Unlike for deletions, mutant alleles, or meiotic null alleles, the removal of a target protein from the nucleus at a specific stage does not compromise target protein activity at earlier stages, allowing for a more accurate interpretation of results. The anchor away system utilizes the shuttling of ribosomal subunits between the nucleus and cytoplasm that occurs upon ribosomal maturation8. To deplete the target protein from the nucleus, the target protein is tagged with the FKBP12-rapamycin-binding domain (FRB) in a strain in which the ribosomal subunit Rpl13A is tagged with FKBP12. Without rapamycin, FRB and FKBP12 do not interact, and the FRB-tagged protein remains in the nucleus. Upon rapamycin addition, the rapamycin forms a stable complex with FKBP12 and FRB, and the complex is shuttled out of the nucleus due to the interaction with Rpl13A (Figure 1). To prevent cell death upon rapamycin addition, cells harbor the tor1-1 mutation of the TOR1 gene. Additionally, these cells contain fpr1&#916;, a null allele of the S. cerevisiae FKBP12 protein, which prevents endogenous Fpr1 from out-competing Rpl13A-FKBP12 for FRB and rapamycin binding. The anchor away background mutations, tor1-1 and fpr1&#916;, do not affect meiotic timings or chromosome segregation2.
To demonstrate the usefulness of this technique, the kinetochore protein Ctf19 was depleted at different timepoints throughout meiosis. Ctf19 is a component of the kinetochore that is dispensable in mitosis but required for proper chromosome segregation in meiosis9,10,11,12,13. In meiosis, the kinetochore is shed in prophase I, and Ctf19 is important for kinetochore re-assembly9,14. For this protocol, cells with the NDT80-in system were synchronized, and the anchor away technique was used to deplete the target protein Ctf19 from the nucleus before and after the release from prophase I, and after meiosis I chromosome segregation (Figure 1). This protocol can be adapted to deplete other proteins of interest at any stage of meiosis and mitosis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-estradiol</td>
			<td>Millipore Sigma</td>
			<td>E8875</td>
			<td>Make 1mM stocks in 95% EtOH</td>
		</tr>
		<tr>
			<td>0.22 uM Threaded Bottle-top Filter</td>
			<td>Millipore Sigma</td>
			<td>S2GPT02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>100% EtOH</td>
			<td>Fisher Scientific</td>
			<td>22-032-601</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Fisher Scientific</td>
			<td>BP399500</td>
			<td>Dilute 1:10 to use as solvent for ConA</td>
		</tr>
		<tr>
			<td>24 mm x 50 mm coverslip No. 1.5</td>
			<td>VWR North American</td>
			<td>48393241</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm x 75 mm microscope slides</td>
			<td>VWR North American</td>
			<td>48300-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine hemisulfate salt</td>
			<td>Millipore Sigma</td>
			<td>A9126</td>
			<td>To supplement SC, SCA, and 1% Kac</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavialin A</td>
			<td>Mllipore Sigma</td>
			<td>C2010</td>
			<td>Make as 1mg/mL in 1X PBS</td>
		</tr>
		<tr>
			<td>CoolSNAP HQ2 CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td>Used in Section 4.3</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Yeast Nitrogen Base w/o Amino Acids</td>
			<td>BD</td>
			<td>291920</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Millipore Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti2 inverted-objective micrscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Used in Section 4.4</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>NIH</td>
			<td></td>
			<td>Download from https://fiji.sc/</td>
		</tr>
		<tr>
			<td>GE Personal DeltaVision Microscope</td>
			<td>Applied Precision</td>
			<td></td>
			<td>Used in Section 4.3</td>
		</tr>
		<tr>
			<td>L-Tryptophan&#160;</td>
			<td>Millipore Sigma</td>
			<td>T0254</td>
			<td>To supplement SC, SCA, and 1% Kac</td>
		</tr>
		<tr>
			<td>Modeling Clay</td>
			<td>Crayola</td>
			<td>&#160;2302880000</td>
			<td>To secure coverslip in slide holder</td>
		</tr>
		<tr>
			<td>NIS-Elements AR 5.30.04 Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td>Used in Section 4.4</td>
		</tr>
		<tr>
			<td>ORCA-Fustion BT Camera</td>
			<td>Hamamatsu</td>
			<td>C15440-20UP</td>
			<td>Used in Section 4.4</td>
		</tr>
		<tr>
			<td>Plastic pipette tip holder</td>
			<td>Dot Scientific</td>
			<td>LTS1000-HR</td>
			<td>Cut a 4 square x 4 square section of the rack portion of this product.&#160;</td>
		</tr>
		<tr>
			<td>Pottassium Acetate</td>
			<td>Fisher Scientific</td>
			<td>BP264</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapamycin</td>
			<td>Fisher Scientific</td>
			<td>BP29631</td>
			<td>Make 1mg/mL stocks in DMSO</td>
		</tr>
		<tr>
			<td>Silicone Sealant</td>
			<td>Aqueon</td>
			<td>100165001</td>
			<td>Also known as aquarium glue.</td>
		</tr>
		<tr>
			<td>SoftWorx7.0.0&#160; Imaging Software</td>
			<td>Applied Precision</td>
			<td></td>
			<td>Used in Section 4.3</td>
		</tr>
		<tr>
			<td>Synthetic Complete Mixture (Kaiser)&#160;</td>
			<td>Formedium</td>
			<td>DSCK2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Type N immersion oil&#160;</td>
			<td>Nikon</td>
			<td>MXA22166</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of time-lapse microscopy and stage-specific nuclear depletion of proteins to study meiosis in s. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

To monitor chromatin segregation, histone protein Htb2 was tagged with mCherry. In prophase I, the chromatin appears as a single Htb2 mass. After homologous chromosomes segregate in the first meiotic division, the chromatin appears as two distinct masses (Figure 3A). After the sister chromatids segregate, the chromatin appears as four masses. If some chromosomes fail to attach to spindle microtubules, additional masses can be seen after meiosis I or meiosis II.
Th...

Watch this Scientific Journal Video about Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae at JoVE.com

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...

By synchronizing cells in prophase one and then depleting the target protein at a particular stage, this method can identify temporarily distinct functions of specific proteins. Conditionally depleting proteins at specific stages of myosis prevents the residual effects a traditional myotic mutant can have on later stages. This method could be used to study budding yeast mitosis and other organisms that do not undergo nuclear envelope breakdown.In addition, this method can be used in other organisms to study cell cycle events prior to nuclear envelope breakdown. To begin, make an agar pad that will be used to create a monolayer of cells for imaging. Cut off and discard the cap and bottom one third of a 1.5-milliliter microcentrifuge tube to create a cylinder that will serve as a mold for the agar pad.Place the cut microcentrifuge tube cylinder on a clean glass slide with the top of the tube upside down, sitting on the slide. Make six milliliters of a 5%agar solution in a 50-milliliter beaker. Cut off the tip of the pipette to make a larger opening and pipette approximately 500 microliters of melted agar into the microcentrifuge tube.Let it sit at room temperature until the agar solidifies. To prepare yeast cells, spin down 200 microliters of the sporulation culture at 800 G for two minutes in one-milliliter microcentrifuge tubes. Discard 180 microliters of the supernatant.Resuspend the pellet in the remaining supernatant by swirling and flicking the tube. Pipette six microliters of the concentrated cells onto the cover slip in the middle of the chamber on the spot with ConA. Hold the cylinder with the agar pad and carefully slide it off the glass slide.Make sure the agar is completely flat at the bottom and using the bottom of the pipette tip, apply a slight amount of pressure to the microcentrifuge mold such that the agar pad is pushed out slightly above the boundary of the tube. Next, invert the mold such that the agar pad is facing down towards the chamber. Use forceps to gently place the agar pad on top of the cells and the pipette tip to gently slide the agar pad around the chamber 10 to 20 times to create a monolayer of cells on the cover slip.Keep the agar pad in the chamber for 12 to 15 minutes. Next, transfer two milliliters of sporulation culture to two microcentrifuge tubes and spin at 15, 700 G for two minutes. After transferring the supernatant into clean microcentrifuge tubes, spin again at the same condition and collect the supernatant to clean microcentrifuge tubes.Add two milliliters of the supernatant dropwise to the chamber containing the agar pad. Once the liquid has reached the top of the chamber, the agar pad will most likely float. Remove the agar pad gently with forceps and discard it.Place a 24 by 50 millimeter cover slip on the top of the chamber to prevent evaporation during imaging. To set up the movie on the microscope, fit the cover slip inside the slide holder. Adhere the molding clay to the side of the cover slip to keep it secure in the slide holder.Open the image acquisition software. Use coarse and fine adjustment knobs to focus the cells using DIC or bright field. On the main menu of the image acquisition software, click on File, select Acquire, and four windows will pop up.In the window named Resolve 3D, click on the Erlenmeyer flask icon. This will open a window titled Design Run Experiment, which contains the controls for setting up an experiment to set up a time-lapse movie. Under the Design tab, navigate to the tab labeled Sectioning.Select the box next to Z sectioning and set the optical section spacing to one micrometer and the number of optical sections to five. Next, under the Channels tab, click on the plus icon and select the appropriate channel. Then, select the box next to Reference Image and set the Z position to the middle of the sample from the dropdown menu.Select a value of transmittance and exposure time from the dropdown menu. Under the Time-lapse tab, select the box next to Time-lapse and enter values for minutes and hours to identify the time interval of image acquisition. Select the box next to Maintain Focus with Ultimate Focus to prevent stage drift during the movie.And under the Points tab, select the box next to Visit Point List. In the main menu, click on View and select Point List. Move the stage to an area of the chamber that shows a monolayer of cells.Click on Mark Point in the Point List window. Move the stage to select 25 to 30 points without any overlap to avoid overexposing the cells and image each field during each time course. In the Point List window, select Calibrate All to set ultimate focus for each point.Under the Run tab, save the file to the appropriate destination on the computer. On the Run Experiment window, select the Play button to start the movie. Open the Fiji software.Open the DIC and mCherry channels. Obtain a single maximum intensity projection of the mCherry channel by clicking on Image, followed by Stacks and Z Project and select Max Intensity from the dropdown menu. To merge the DIC and mCherry channels in one image, click on Image, Color, and select Merge Channels.Follow a single cell through meiosis. After meiosis II completion, record the number of DNA masses. In wild type cells with the anchor away background, there are typically four DNA masses at the end of meiosis II, representing the four products of meiosis.In a small fraction of the cells, only three masses are visible after meiosis II.When Ctf19-FRB is anchored away at the time of release of prophase I, approximately 47%of cells display more than four DNA masses upon the completion of meiosis, suggesting a defect in the attachment of kinetochores and microtubules. With anchoring away of Ctf19-FRB, either after kinetochore assembly but before meiosis I, or after meiosis II, approximately 16%of cells display additional DNA masses. One of the most important parts of this protocol is to add the supernatant dropwise to your chamber to avoid disrupting your monolayer.Additionally, note the time required to prepare for imaging to consider it in your analysis. By changing fluorescently packed proteins, this procedure can be used to answer the questions regarding cell cycle duration, chromosome segregation, protein abundance, and protein localization.

Research

JoVE Journal

Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Monitoring Actin Disassembly with Time-lapse Microscopy

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

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Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

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Flow Cytometry-based Purification of S. cerevisiae Zygotes

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Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...